868 OLSON 13..AL

19. Dickinson KtJ. Nahorski SK. Willcocks AL 108 1 Serotonin recognition s~tcs 24. Reilly FD. McCuskey PA. McCuskey RS 1978 intrahcpatic distribution of are labcllcd In cerebral cortex b) the M-adrenoreceptor antagonist '"I- nerves in the rat. Anat Rec 191:55-68 hydroxybcnzylpindolol. Rr J Pharmacol 72:165P 25. Forssman WG, Ito S I977 Hepatocyte inncrvat~onin primates. J Ccll Biol 20. McMillian MK. Schanbcrg SM. Kuhn C'M 1983 Ontogeny of rat hepatic 74:299-3 13 adrcnorcceptors. J Pharmacol Exp Ther 227: 18 1-1 8(> 26. Hartman H, Beckh K. Jungermann K 1982 Direct control of glycogen metab- 2 I. Noguchi A 1983 Nor~nalontogeny of <,,adrencrgic receptor in rat liver is olisrn in the pcrfused rat liver by the syrnpathet~cinnervation. Eur J Biochem thyroid hormone dependent. Endocrinology 113572-676 123:52 1-526 22. Noguch~A. Jctt I'A. Gold .4H CAMP indcpendcnt stimulation of glycogen 27. Blazqucs E, Rubalcava B. Montesano R, Orci L, Unger RH 1976 Development pho~phorylasein newborn rat hcpatoc)tes. Am J Phgs~ol248:E560-E566 of insulin and glucagon b~ndingand thc adcnylatc cyclasc rcsponsc in liver 23. Met7 W. Forssman WG 1980 lnnervat~onof the livcr in guinea pig and rat. membranes of prenatal, postnatal, and adult rat: cvidcnce of glucagon Anat Embryo1 l60:239-252 "resistance." Endocrinology 98: 10 14- 1023

003 1 -39981851 1908-0868$02.00/0 PEDIATRIC RESEARCH Vol. 19. No. 8, 1985 Copyright a. 1985 International Pediatric Rcsearch Foundation. Inc Printed it7 C' X.4.

Differential Toxicity of RCAll (Ricin) on Rabbit Intestinal Epithelium in Relation to Postnatal Maturation'

ALLAN D OLSON. THEODORE J PYSI-IER, ALFRED0 LARROSA-HARO, AKHTAR MAHMOOD. AhD RAMOh TORRES-PINFDO C'n/~,cr..c//!c~/'Olilulroiiicr Ilcul111 S(.icwc,esCotitct., Dc;i)ur.~inen/of'1~ediutrcc.s und Depur/inr>nlofPu'arhologj., Seciion ofPc~diuir.ic,G'crclrocnto.o/0,~1~ uiid "V~~~lrili~n,Okl~/~oinu Cil!:, Ok/~/?oinu73/26

ABSTRACT. The purpose of this work was to assess the Abbreviations toxic lectin ricin (RCA,,) as a probe for the study of intestinal permeability in the developing small bowel. Je- RCAI,, ricin junal explants from suckling and adult rabbits were ex- PBS, phosphate-buffered saline posed to varying dosages of RCAll for 30 min at 25" C and then cultured in toxin-free medium. The RCA,, dose re- quired to inhibit protein synthesis during 6 h of culture increased from 0.1 pg/ml in 4-day-old rabbits to 25 pg/ml in weanling rabbits. KCA,, cytotoxicity was almost com- pletely blocked by 0.1 M lactulose in all age groups. The The early part of the suckling period in rodents is characterized kinetics of '251-RCAIIbinding to purified microvillus mem- by a high level of receptor-mediated responses in small intestinal branes were determined by incubating a fixed concentration epithelium. This has been clearly shown for the jejunal transport of membrane protein (30 pg) with increasing concentrations of breast milk iminunoglobulin (1, 2) and is also inferred from of labeled lectin (2-18 @g/ml).Binding attained saturation the high content of trophic factors in breast milk (3, 4). The with adult but not with suckling animal membranes. The surface characteristics underlying this high receptor responsive- latter yielded a curvilir~earrelationship in Scatchard plots, ness in the intestine of neonatal animals are poorly understood. suggesting either several classes of binding sites or negative A marked developmental change in surface reactivities to lectins cooperativity. RCA,, binding was confined to the delipi- has been described in the small intestine of suckling rats (5-7). dated fraction of the membranes and decreased by 42% In this animal species, the intestinal microvillus membrane from 6 days old to adult age. The extreme sensitivity of undergoes a progressive shift from sialylation to fi~cosylationof coiostral epithelium to RCA,, is probably related to the glycoproteins and glycolipids during postnatal development (7). high level of endocytosis exhibited by the immature mem- In the present work, we have examined the response of rabbit brane of suckling rabbits. The development of increasing intestinal epithelium to the toxic action of RCA,,. This toxin resistance to the toxin, and associated decrease in binding, inhibits protein synthesis in intact cells (8, 9) through a mecha- might be related to disappearance of saccharide sites in nism that requires terminal nonreducing galactosyl (or N-acetyl- productive surface receptors occurring in the derelop- galactosaminyl) residues (10) in a glycoprotein receptor- capable mental course of intestinal glycosylation. (Pediatr Res 19: of transmembrane signaling for toxin internalization (1 I). This 868-872, 1985) toxin, therefore, "mimics" the mechanism of interaction of trophic hormones (12). The RCA,, membrane receptors may be Reccivcd January 7. 1985: acccptcd March 26. 1985. part or be closely associated with cell surface components in- Rcquests for reprlnts Ramon Torrcs-Pincdo. M.D.. Professor of Pcdiati-ics. P.O. volved in cell-macromolecule and cell-cell recognition (I 3). The Box 26307. Oklahoma City. OK 73 126. Suppor-tcd b) Grant tID-13441 from the National lnstitlitc of Health probing of intestinal epithelium with RCA,, may provide insight ' 1'1-cscntcd in pat-t 211 thc annual mccting of [lie American Pcdi,itric Society and into the role of glycosylation on receptor-mediated responses in thc Socict? for Pcdlatric Kcscarch. Washington. D.C.. May 3-6. 1983 the developing small bowel. RCAj1 TOXICITY TO IN TESTINAL EPITHELIUM 86$1

MATERIALS AND METHODS containing "51-RCA11 and incubated for 30 min at 25" C in plastic tubes (Falcon). The incubation was ended by adding 2 ml Keugc.ni.s and rnu~criul.~.Ricin (RCA,o) was obtained com- of PBS (4" C) and immediately filtering through 0.45 p cellulatt: mercially (P-L Biochemicals, Inc, Milwaukee, WI). This protein filters (these filters were found to retain 100% of the microvillus was further purified by affinity chromatography on Sepharose protein used in the binding assays); the filters were then washedl 4B using 0.0 I M n-CalNAc in the eluting buffer (10). The protein twice with 4 ml of PBS at 4" C. The radioactivity retained by the gave a single band with a molecular weight of -65,000 on SDS- filters was counted in a Tri-Carb spectometer (Packard, Downer:; polyacrylamide gel electrophoresis. Lactose and lactulose (4-0- Grove, IL). Nonspecific binding was determined in the presence 0-r>-galactopyranosyl-n-fructofuranose) were purchased from of 0.2 M lactose and was less than 1 % of the total radioactivity Sigma (St. Louis, MO). [%I]-leucine (s.a. 59.2 Ci/mmol) and bound in all ages. Binding to the filter was less than 1 % of thc: [14C]-leucine(s.3. 54.5 n~Ci/mmol)were obtained from New total added radioactivity. England Nuclear (Boston. MA); lactoperoxidase beads from Prepurution (?ffi51-RCAI,. Iodination of about 0.5 mg of pur-. BioRad (Richmond. CA): cellutate filters (0.45 p pore size) from ified RCAll was performed using lactoperoxidase from BioRadl Millipore (Bedford, MA); organ culture dishes and stainless steel (Richmond. CA) as previously described (6). Each preparatiori organ culture screens from Falcon (Cockeysville, MD). New of labeled RCAllwas used within 2 wk. The specific activity was Zealand White rabbits were purchased locally and maintained about 5.5 x 10Qpm/mg protein (efficiency of "51 counting: on Purina rabbit Chow (St. Louis, MO). 49.4%). Prcpurarion and clrlflrrr c?/'.jeJzrnulexplunfs. Jejunal explants from New Zealand rabbits were obtained by two methods. In RESULTS anestliesized adult and suckling rabbits older than 4 days, the explants from proximal jejunum were cut and prepared as de- eflfizct (?fRCAI, on ruhhit ~eljunulexplunts. Figure 1 shows the: scribed by Browning and Trier (14). From birth to 4 days of age, time-dose relationships of the effect of RCAll on jejunal explants the animal was anesthetizcd with ether, the proximal jejunum from adult rabbits. The time required to reach 50% inhibition, was removed. washed with ice-saline, and sectioned into 2-3 mm long rings. The rings were opened with straight iris scissors and the whole tissue placed mucosal side up on a stainless steel grid. Thc thinness of the bowel in animals 1-4 days of age precluded the cutting of mucosal explants. The initial culture medium was serum-free RPMI 1640 with added glucose, insulin, and antibiotics. Trc~atmc~n/ofjejirrzul csp1unl.s ~t~irhRCAII. After equilibration in culture medium for 15 min, the tissue explants were treated with varying dosages of RCAll as follows: the grids with the mucosal explants attached were transferred !o glass Petri dishes and immersed in serum-free culture medium (4.5 ml/dish) con- taining RCAll or bovine serum albumin as controls. The dishes were placed in air-tight boxes. gassed with 95% O2and 5% C02, and shaken on a rotating platform (Fisher Rotator, Fisher Sci- entific Co. Dallas, TX) at 70 rpm for 30 min at 25" C. The grids were then removed, washed three times with 100 mM lactose in PBS and returned to cult~~redishes containing initial culture medium with added lactulose (5 mM) and 10% heat inactivated fetal calf serum. The culture times are indicated in the corre- sponding figures. Four hours prior to the end of the culture period, the medium was removed and replaced by Eagle's mini- mal essential medium without leucine to which 10% fetal calf serum. 1 niM lactulose, and 1 pCi/ml of ['HI- or [14C]-leucine had been added. After culture, the jejunal explants were removed from the grids, washed three times with 1 mM leucine in cold PBS and homogenized in warm 0.1 M KOH. Aliquots of the liomogenates were taken for protein determination and for pre- cipitation with cold 20% trichloroacetic acid. The precipitates were left at 4" C for 2 h and collected on Millipore filters, washed three times with 5% trichloroacetic acid and once with 95% I I I 8 I I ethanol, dried. and counted with 3 ml Aquasol in a Packard Tri- 0 4 8 12 16 20 24 Carb scintillation counter. Pz~r~/i~~u/ionof'rhc micarovillzr.srnc~mhrunrserum albumin as RCAII-treatedexplants (two cxplants per culture dish) from two animals. standard. Paired control (albumin treated) explants were included for each sct of '251-RCilllhiridinn.. In preliminary studies, we found that bind- RCAII-treatedexplants. Each control value represents mean + SD for 40 ing of "51-RCA11t~mic~ovillus membranes reached equilibrium explants fiom I0 animals. Bottom. the percentage of control values werc wchin 10 &at 25" C. In subsequent studies, microvillus calculated for each set of RCAII-treated sa~nplcswith rcspcct to thc: membrane protein (30 pg in 50 p1) was added to 150 of PBS corresponding set of albumin-treated samples. 870 OLSON E:'T AL of protein synthesis ranged from 4% 16 h for RCAll doses of 50 and I p/ml, respectively. lJsing this dosc range. we then assessed the effect of RCAI1on jejunal explants from suckling rabbits during a 6-h culture period. As shown in Figure 2, the toxin dose required to causc 50% inhibition of protein synthesis was 0.5 pg/ ml at birth, fell sharply to 0.1 pg/ml on days 4-7 after birth (p < 0.001), and then increased progessively to reach a value of 25 pglml on day 23. Extrapolation to 6 h in Figure 1 revealed the latter 50% inhibition dose to be similar in adult animals. Finally, we assesscd the specificity of the effect of RCAll by measuring the blocking action of lactulose-a sugar that is not hydrolyzed by intestinal mucosa. Figure 3 shows the toxin-hapten inhibitor relationships in mucosal explants from 6-day-old and adult rab- bits. In both animal groups, the toxic effect of 0.5 pg/ml RCAIl could be almost completely abrogated with 100 mM lactulose. In the next series of experiments, we compared the histological damage caused by RCA,, on mucosal explants of 6-day-old and weanling rabbits (Fig. 4). On light microscopy. control explants showed normal cellular morpohology, slightly shortened and broadened villi, and mild edema of the lamina propria. These changes were similar to those previously reported by Kagnoff el ul. (19). In 6-day-old pups. mucosal explants treated with RCAll showed marked stunting of the villi, as well as shortening and pyknotic degeneration of the epithelial cells. By contrast, jejunal explants from weanling animals similarly treated showed only slight pyknotic changes and retained a near-normal cell shape. LACTULOSE , mM Binding of '"I-luhc~lrd RCA,, to the n~icrovill~rsrncrni~rtinc~s. 'The binding of "51-labeled RCA,, to the microvillus membranes Fig. 3. Inhibition of RCAll cytotoxicity by lactulose. Explants from 6-day-old (0)and adult (m) rabbits were pulsed with 0.5 lrg/ml RCAll (30 min, 25" C) in the presence of variable concentrations of lactulose (0-100 mM). The tissues were then removed, washed with 100 mM lactose in phosphate-saline buffer pH 7.38. and cultured for 24 h as described in "Materials and methods." Control samples in which bovine serum albumin substituted for RCAll were also cultured for 24 h.

of suckling animals was higher than those of adult animals over a wide range of lectin concentrations (Fig. 5). When the results were represented according to the Scatchard equation (20) (Fig. 5, inset), the plot from adult rabbit membranes yielded a straight relationship at concentrations above 2 pg/ml of free lectin (n = 23 pg mg-I). In contrast, the membranes from suckling animals exhibited a curvilinear relationship throughout the plot, suggest- ing either the existence of several classes of binding sites of decreasing affinities. or the presence of additional nonsugar interactions. To assess the approximate distribution of RCAll binding sites between glycoproteins and glycolipids, about one-half of the membrane preparations (4-5 mg protein) was delipidated (Table 1). Binding to intact and delipidated membranes was then com- pared within the same binding assay, using equal amounts of membrane protein and labeled lectin. RCAI1 binding was almost identical in both membrane preparations. indicating that most of the receptor sites were contained in the glycoprotein fraction

0.014 , , , , , , , , , , , + of the membrane. Under the assay conditions, from early suck- 0 5 10 15 20 25 30 35 40 45 50 55 60 ling to adult ages, RCA11 binding to delipidated membranes AGE OF AElIMAL (DAYS) decreased by 42%.

Fig. 2. Developmental change in sensitivity to the toxic effect of DISCUSSION RCAIi in jejunal explants from suckling rabbits. Each poi/?/ represents the dosc of KC'AIIrequired to give 50% inhibition of protein synthesis The experimental approach used in these studies-a short after a 30-min pulse of RCAI, followcd by 6 h in culture. Triplicates of pulse of RCAll followed by organ culture in toxin-free medium organ culture grids each containing three explants were exposed to in the presence of the blocking sugar-allowed us to define varying doses of RCA,, or bovine serum albumin as described in "Ma- accurately the cytotoxic effect of RCA,, on rabbit jejunal mucosa terials and methods." The dosc of RCAll which gavc 50% inhibition of 1n vltro. The intestinal epithelium was highly sensitive to the protein synthesis was determined for cach experiment from semi-log effect of RCAll during the colostral period, and became increas- plots of RCAIl concentration against percentage inhibition of protein ingly resistant with growth and maturation. The cytotoxic effect synthesis. Each polt~l represents a single animal from different litters of RCAII could be completely abrogated by lactulose in both (cxccpt for day I. where three animals gave results within the dose range colostral and weanling animals, indicating that a receptor-me- covered by the syn~hol). diated process was required throughout development for toxin RCA,, TOXICITY TO INTESTINAL EPITHELIUM 87 1 penetration into the epithelial cells. high level of endocytosis characteristic of this period (1, 2, 26). The results of our protein inhibition and histological studies The marked decrease in the binding of RCAll to delipidatecl indicated a ~narkedage-related difference in the susceptibility of membranes of weanling animals suggested either the diappear- the intestinal villus cells to RCAll cytotoxicity. The decrease in ance of specific glycoproteins from the membrane or a change sensitivity of these cells to the effect of RCA,, was accompanied in the glycosidic structure of certain glycopeptides. Conceivably, by both qualitative and quantitative changes in RCA,, binding the specific glycopeptide involved in transmembrane signaling to the microvillus membrane. The kinetic characteristics of and internalization of RCAll (1 1) might be affected by such RCAll binding to membranes from suckling animals could be change in glycosylation. Similar to what we previously described explained through the presence of several classes of binding sites in the rat (7), the microvillus membrane of suckling rabbits of decreasing affinities; however, an alternative explanation could experiences a decrease in sialic acid and increase in fucost: be negative cooperativity induced by nonsugar interactions. A content during postnatal development (unpublished observa- similiar phenomenon has been observed in the differential tox- tions). This leads to a complete reversal of the sialic acid to icity of RCA, toward chick fibroblasts in relation to the stage of fucose molar ratios in glycoproteins and glycolipids at weaning. embryo development (21). The kinetic behavior of the RCAll It remains to be determined if this shift from sialylation to interaction with immature membranes might be related to the fucosylation plays a role in decreasing the accessibility of RCA,, unique molecular organization of the intestinal apical membrane to the receptor saccharide sites in the membrane. of suckling animals (22, 23). In this regard, it is interesting that In summary, the intestinal epithelium of suckling rabbit:; the binding of 1?51-choleratoxin to the microvillus membrane of exhibits high sensitivity to the toxic effect of RCA,, during tht: suckling and adult rabbits exhibited kinetic differences similar colostral period. At this time, the lectin-receptor interaction to those shown herein for RCAII (24). There is increasing evi- might be influenced by membrane structures primarily involved dence (25) that lectins and bacterial toxins share common prop- in enhanced endocytosis during the rabbit neonatal period. The erties in their ability to mimic hormonal interactions with the epithelium loses considerable sensitivity to the toxin with age- membrane to gain access into cells. The increased mucosal related maturation. This might be related to disappearance of uptake of RCA,, during the rabbit colostral period might occur saccharide binding sites in a productive membrane receptor, a:$ through interaction with membrane structures involved in the a consequence of developmental changes in glycosylation.

Fig. 4. Appearance of explants after 30-min exposure to albumin (controls) or RCAIIfollowed by 8-h culture. A, Adult rabbit intestine exposed to albumin. U, adult rabbit intestine exposed to RCA,, . C, 8-day-old rabbit intestine exposed to albumin. D, 8-day-old rabbit intestine exposed to RCAII. Note that albumin-exposed explants tolerated in 1,iIi.o conditions well. RCAll exposure resulted in both focal necrosis and alterations in surviving cells. the lattcr manifested principally by intercellular edema in cxplants from adult bowel, but marked rounding of cells and nuclei in explants from sucklirlg bowel. Hcmatoxylin and eosin. x500. 872 OLSON

Table 1. Spcc~jic"'I-RCall hiding io nulive and dcl/i~>il/aird wricicivill~~sinonhvancs,/iorn .rztckiirrg and wean/ing rlihhii inlc.vfine Rabbit age '"~~LRCA~,binding?

(day) Membrane preparation* ~ --(@g/mg protein) ~ ~ ------. 6 Native 16.4 * 2.7 Delipidated 16.1 + 2.0 40 Native 8.9 i- 2.53: Delipidated 9.4 i 3.l:L -- .- - "In comparison lo the whole rnucosal hornogcnates, thc purified microvillus membranes showed tiic following enrichmer~tin thc insoluble fraction of alkaline phosphatase: 6 day old, 23-fold (? 1): 40 day old. 2 1- fold (k2). Membranes were delipidated by a slight modification of the mcthod of Svennerholm and Frednian (27). In brief, to 3 vol or mem- brane suspension (3--5 mg protein) 8 vol of nicthanol and 4 vol of chloroform were added with mixing. After stirring for 30 min at room temperature. the mixture was centrifuged (15.000 x g 15 min) and the pellet resuspended in water-methanol-chlodorln (3:8:4) and centrifuged as above. Thc pellet was washed with absolute ethanoi, which was rcmoved by Pyophili~ation.The lr~oistdelipidated material was finally dried under reduced pressure and suspended in 0.05 M sodium maleatc pH 6.5 to a protein concentration of 1 .O-2.0 mg/ml. Protein recoveries were 50-7096. t The binding assay mixture contained 1.8 pg of ix51RCAII (sp. act. 5.5 x liph cpm/mg protcin) and 30 pg of microvillus membrane protcin in a total of 200 Values were nican ? SD of 8-10 binding assays from four pairs of animals. Statistical significance of differences bctwcen ages. n551-~~~ll FREE (~glrnl) $ p < 0.05.

Fig. 5. Spccific binding of i"i-labcled RCAll to intestinal inicrovillus mcmhrancs of 6-dia?-old (a)and adult (Bj rabbits. The binding assays mouse lymphoma cell lines. J Suprarnol Struc 8235-245 were performed as dcscribcd In "Materials and methods." Each pori?i 12. Carpenter 6. Cohcn S 1977 Influence of lectins on the bindlng of "'I-labeled ECF to hurnan fibroblasts. Biocheln Biophys Rcs Comrnun 70.545-552 rcprcscnts the mean k of cight to I0 binding assays with microvillus SD 13, Hughes RC I978 Lcctin receptors and ce:l surfacc recognition. in: Marchesi rnernhrancs from thrce animals. Specific acli~it)of i251-RCA . 3.3- - x 103 VT, C;insSurg V. Robbins PW and Fox CF (cds) Progress in Clinical and DD cpm/pg protein. Iti\r>i. Scatclial-d plots of data from rnaira body of thc Bioloeical~ - Research. Vol 23. Alan R. Liss. Inc. New York. . . 657-668 figurc. li. '25P-RCAIIbound in micrograms per ~nllligramof microvillus 14. Browning TH. Trier JS 1969 Organ culturc of niucosal biopsies of human small intestine. J Cl~nInvest 48:i433-1432 menahl-a~ieprotcin: I.'. frcc concentration of' "'I-RCA,, in micrograms IS. Schm~t! JS. Presier 14. Maestracci D. Ghosh BK. Ccrda JJ. Crane RK 1973 Dcr milliliter. Purification of the human intcstiiaa! brush border membrane. Biochim Biophys Acta 323:98-1 12 16. Ohsawa K, Kano A. Hoshi 7 1979 Purification of intestinal brush border mcrnbrane vesicles by the use of conrrolled-pore glass-beads column. Life Sci 24:669-678 I Abl-ohamson DR. I'ouers A. Rodcwald R 1'179 l~rtcstinalabsorprion of im- 17. Dahlqvlst A 1964 Method for assay of intestinal disaccharidascs. Anal Biochem munc complc\e\ h! neonatal rill\: a rourc of;~nligenlransfcr from rnotllcr 7: 18-25 to houng. Sciencc 206:567-56'1 18. Lowry OH. Rosehroirgll NJ. Fair AL, Randall RJ 195 1 Proteln measurement 2. Rodcwald R 1973 Irrtcstinal trnn\port of antibodies in the newborn rat. J Ccll with the fohn phcnol reagent. .I Blol Chem 193:265-275 13101 58: i 80-2 I 1 19. Kagnofl' MF. Donaldson RM. Trier JS I972 Organ culturc of rabbit small 3. Kccnan I W. Patton S 1975 The m~lkhi globule membrane. Bioch~rnBioph!, intest~ne:prolonged III i'~irosieady state protcin synti~esisand sccret~onand Acta 4 15273-300 secrctory IgA secretion. Gastroenterology 63:54P-55 1 4. Carpcnler G 1980 1;pidcrrnal gso\i.tli Paelor is a major gromth-promot~ngagent 20. Scatchard G 1949 Attractions of proran for small molecules and ions. Ann in human nlilk. Scleircc 210PlOX-i99 NY Acad Sci 5 1 :660-672 5. Elilcr ME. Bi-ansli-ntor %fL I070 in: Ciba l'oundation Symposium 70 (new 21. Bernard R. Auber? M. Bourriilon R 1979 Changes In the sensitii~t)of chick scr~cs).Fxccrptn Mcdica. Anisleidam. pp 5 1 -OX fibroblasts io ricinus leclin (RCA))toxiclty In relation to the stagc of embryo 6. Mahinood A. I~orrcs-l'incdo K !'I83 Postnatal changes in lccti~ibinding lo development. Biochcrn J 182.13-38 ~iiicro~ilius~i~eiiihranes lion] r-al rnlcstinc. Bioclicni Biop1i)s Res Comrnun 22. Bouhours D. Bouhou!-s JF 1983 Dcvcloprncntal changes in hemalosldc of rat 1 13:JOO-406 srnail ~ntestine.Post~raial hydroxylation of fatty acrdr and sialic acid. J Biol 7. Icirrcs-l'inedo R. Mahmood A 1'184 Postn;stal changes In biosyi~rt~esisof Chcnt 258299-304 arn~crovillu>mcntbrarrc glycans of rat srnall intesi~ne.I. E\.idence of a devel- 23. Moog F 1979 Differentiation and rcd~lTerentiationof the intestinal epithelium onnrental \hill ii.0111 terminal sial~lationto I'iicor\larion. Bic~clrenrRionh\\ . . snd 11s brush bordcr ~mcn?branc.In: Developn~entand Mammalian Absorp- Re\ Conirn 125:54h-553 tlve Processes. Ciba Foundation Syniposiurn 70 (nciv series). Exccrpta Mcd- 8. Bcnwn S. Ol~nc\S. Pilil A 1075 On rhc mechanism of protein-synthesis lea. Amsterdam, pp 31-50 inh~bitlonh! abr~iiand I-icin. Inhibition of the CiTP-hydrolysis sile on the 24. Bressoli JL. Pang KY. Walker WA 1984 M~crovillusmembrane differentiation: 60-S sibo:,oninl siibunli, Ewr J Biochcrn 59:573-5XO quantitative d~ffcrencesIn cholera toxin binding to the intestinal surface of '1. Olsncs S. P~hlh 1074 D~fFcrcnthlological propertics of the t~oconst~tuents newborn and adult rabbits. Pediarr Rer l 8:984-987 peptitle churn\ ofr~cln.a toxic protein inhibiting protein s?ntlre\is. Biochem- 25. Hughes RC 1979 How do toxins pcnctrate cells? Nature 281:526-327. ist~-).12.3 12 1-3 116 26. Udali JN. Pang K. Fri1r.e L. Kleiman R. Walker WA 1981 Dcvciopmenl of 10. Xicol\on GL 1974 ('ilaractcri/;itroii of tiso plant lectins iioii1 Ricinus com- gastrointestinal rnucosal barrier. I. Tl~ceffect of age on intestinal permeability I~ILIIII\and tlicis~luailtitiiliv~intc~-;iction with ;I in~iriiiely~nph~iii;i Biochcrr- io macromolcculcs. Pediatr Res 1524 1-244 1~11-y13: 196-204 27. Sbcnncrholrn I., Frcdman P 1980 A procedure for the quantitati\e ~\olationof 13 Nrrtrlson CL. Postc