Gut 1996; 38: 846-852

Changes in the enterocyte cytoskeleton innewborn rats exposed to ethanol in utero

J F Montes, G Estrada, M D L6pez-Tejero, J Garcia-Valero

Departament deBioquinmica i

Fisiologia, Facultat deBiologia, Universitatde Barcelona,Barcelona, SpainJ F MontesG EstradaM D L6pez-TejeroJ Garcia-Valero

Correspondence to:Dr Jose Garcia-Valero,Unitat de Biologia Cellular,Departament de Bioquimicai Fisiologia, FacultatBiologia, Avda Diagonal,645, 08028 Barcelona,Spain.Accepted for publication13 December 1995

AbstractBackground-Cytoskeletal changes afterlongterm exposure to ethanol have beendescribed in a number ofcell types in adultrat and humans. These changes can play akey part in the impairment of nutrientassimilation and postnatal growth retarda-tion after prenatal damage ofthe intestinalepithelium produced by ethanol intake.Aims-To determine, in the newborn rat,which cytoskeletal proteins are affected bylongterm ethanol exposure in utero and towhat extent.Animals-The offspring of two experi-mental groups of female Wistar rats:ethanol treated group receiving up to 25%(wlv) of ethanol in the drinking fluid andcontrol group receiving water as drinkingfluid.Methods-Single and double electronmicroscopy immunolocalisation and labeldensity estimation of cytoskeletal proteinson sections of proximal small intestineincubated with monoclonal antibodiesagainst actin, a-tubulin, cytokeratin(polypeptides 1, 5, 6, 7, 8, 10, 11, and 18),and with a polyclonal antibody anti-31,4-galactosyl transferase as trans golgi(TG) or trans golgi network (TGN)marker, or both. SDS-PAGE techniquewas also performed on cytoskeletalenriched fractions from small intestine.Western blotting analysis was carried outby incubation with the same antibodiesused for immunolocalisation.Results-Intestinal epithelium ofnewbornrats from the ethanol treated groupshowed an overexpression of cytoskeletalpolypeptides ranging from 39 to 54 kDa,affecting actin and some cytokeratins, butnot tubulin. Furthermore, a cytokeratinrelated polypeptide of28-29 kDa was iden-tified together with an increase in freeubiquitin in the same group. It was note-worthy that actin and cytokeratin wereabnormally located in the TG or the TGN,or both.Conclusions-Longterm exposure toethanol in utero causes severe dysfunctionin the cytoskeleton of the developingintestinal epithelium. Actin and cytoker-atins, which are involved in cytoskeletonanchoring to plasma membrane and celladhesion, are particularly affected, show-ing overexpression, impaired proteolysis,and mislocalisation.(Gut 1996; 38: 846-852)

Keywords: actin, cytokeratin, fetal alcohol syndrome,trans golgi network, tubulin, ubiquitin.

Longterm exposure to ethanol in utero resultsin postnatal growth retardation in the rat,'which could be related to prenatal damage2 3induced by the high ethanol concentrationfound in the fetal gastrointestinal lumen.4However, the mechanism by which ethanolimpairs nutrient assimilation in the neonates isnot well characterised. Enteral nutrition iscritically dependent on the functioning ofintestinal epithelium, based on (a) the organi-sation ofmembrane domains, (b) the transportpathways across these membranes, and (c) themaintenance of epithelial sheet integrity by celladhesion. Appropriate nutrient transport inepithelial cells requires the development andmaintenance of precise and polarised distribu-tions of proteins and lipids, as well as theorganisation and maintenance of specialisedcell-cell and cell-substratum contacts.5-7

Ethanol and its metabolic derivatives interactwith proteins and lipids, modifying their func-tional properties. Several ethanol inducedmolecular and cellular phenomena have beendescribed in alcoholic adults and cell cultures,such as decreased membrane bound enzymeactivities,8 9 intracellular accumulation of secre-tion proteins,9 10 changed gene expression,10-13inhibition of proteolysis,14 covalent bindingwith lipids15-17 and proteins,'8 19 and inhibitionof cell-cell adhesion.20 As cytoskeletal integrityis a prerequisite for many of these cell func-tions, several authors have investigated theinteractions between ethanol and the cyto-skeletal proteins.21 22 Pinazo-Duran et ai23found ethanol induced changes in cytoskeletalproteins such as GFAP in astrocytes, withoccasional disorganisation of the entire cyto-skeleton. Reddy et al 8 reported changes in actinassociated proteins such as myosin and Adickeset al 24 described cytoskeletal dysgenesis inmyocytes. So far, the ethanol induced changesseen in actin cytoskeleton have been related toacetaldehyde, the main metabolite of ethanoloxidation, as studies carried out in vitro byXu et al'8 reported that G actin forms stablecovalent adducts with acetaldehyde.

Given the significance of the cytoskeletonfor enterocyte function at birth, and its impor-tance to postnatal nutrition in mammals, theaim of this study is to determine in the new-born rat which cytoskeletal proteins areaffected by longterm ethanol exposure in uteroand to what extent.

Methods

Animals and treatmentsThe experiments were performed on the off-spring of female Wistar rats from our own

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Ethanol induced cytoskeletal changes

Figure 1: Actin immunolocalisation in the microvillus domain of control (A, X58 000)and prenatally ethanol exposed enterocytes (B, X 52 000). Tubulin immunolocalisation inthe cytoplasm of control (C, X 52 000) and prenatally ethanol exposed enterocytes(D, X54 000). Bars=5 ,um.

colony, maintained at 25°C, 75%/o of relativehumidity, and 12 hour light-dark cycles. Themothers, weighing 150-160 g, were placed inplastic cages distributed in groups of three ratsper cage and were fed ad libitum with standardrodent chow (Panlab, Barcelona, Spain). Fourweeks before pregnancy, the animals were

divided into two experimental groups and sub-jected to different nutritional treatments25:ethanol treated group received chow and 10%,15%, 20%, and 25% (w/v) of ethanol in thedrinking fluid in successive weeks beforefecundation (ethanol adaptation period). Aftermating with untreated males, pregnant rats

were kept on chow and 25% ethanol in drink-ing fluid until delivery (gestational period).The control group were handled in the same

way as the ethanol treated group, fed adlibitum with the normal standard diet andwater as drinking fluid. All rats were allowed to

deliver spontaneously. Immediately after birth,pups were removed from their mothers andkept in a thermostatically controlled humiditychamber at 37°C until death.

Specimen preparation and immunocytochemistryThree neonates from different litters in eachexperimental group were killed by decapitation

and the total small intestine (from the pylorusto the ileocaecal junction) was perfused gently,in situ, with cold NaCl 090/o solution. Theproximal small intestine (duodenum andproximal jejunum) were cut into small blocks oftissue and were fixed in 4% (w/v) paraformalde-hyde and 0.1% (v/v) glutaraldehyde in 041 Mphosphate buffered saline (PBS), pH 7.4, fortwo hours at 4°C. The samples were then cryo-protected by infusion with 2.1 M sucrose inPBS, mounted, and frozen in liquid nitrogen.Ultrathin cryosections were obtained using acryo system (Reichert FC-40) and transferredto grids in a drop of 2.3 M sucrose in PBS.

Before labelling, sections were rinsed twicewith 0.1 M glycine in PBS (gly PBS) for 10minutes and incubated with 2% ovalbumin inPBS for 20 minutes. For single immuno-labelling,26 the grids were incubated during 30minutes with monoclonal antibodies (mAbs)against cx-tubulin (Amersham, ArlingtonHeights, IL), cytokeratin (polypeptides 1, 5, 6,7, and 8 of the basic and 10, 11, and 18 of theacidic cytokeratins; ICN Biomedicals, CostaMesa, CA), actin (Amersham), and with apolyclonal anti-,B 1,4-galactosyl transferaseserum (gift ofDr S Vilaro, Facultat de Biologia,Barcelona). After washing three times with glyPBS for 15 minutes, bound mAbs were visu-alised with 10 nm colloidal gold conjugatedgoat antimouse Ig (Amersham) and boundanti-,8 1,4-galactosyl transferase serum wasdetected with 10 nm protein A gold (pAg)(Sigma Chemical, St Louis, MO). For doubleimmunolabelling,27 sections were first labelledfor anti-,B 1,4-galactosyl transferase serum with10 nm pAg, then stabilised with 1% glutaralde-hyde in PBS for five minutes, and incubatedwith the mAbs against actin or cytokeratinfollowed by unlabelled rabbit antimouse Ig(Amersham) and detected with 16 nm pAg(Sigma). The ultrathin cryosections wererinsed with PBS and double-distilled waterbefore staining with uranyl acetate andembedding in methyl cellulose.28 Observationswere carried out with a Hitachi H-600 ABtransmission electron microscope.

Quantitative evaluationLabel density was estimated as the number ofgold particles either per area of compartment

Label density expressed as number ofgold particles (gp) perunit length or area for a-tubulin, actin, and cytokeratin ofseveral cell compartment

Ethanol treatedControl group group

TubulinCytoplasm (gp/pLm2) 7-63 (1-10) 11-46 (0.74)

ActinMicrovilli (gp/lpm2) 95-22 (10-24) 121-43 (7.30)Lateral interdigitations

(gp/p.m) 2-48 (0.24) 4-76 (0.83)**Vesicles (gp/p.m) 1-75 (0.23) 12-13 (1-38)**

CytokeratinBundles (gp/4m-) 233-33 (27 60) 484-48 (36 20)**Vesicles (gp/,um) 0.79 (0.19) 10-19 (0 99)***

Values are mean (SEM) from at least 30 measurementsperformed on three animals from each experimental group.The significance of differences between control and ethanoltreated groups was evaluated using the Student's t test.**p<.0 1 and ***p<0.001 comparing values in bothexperimental groups.

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Figure 2: Actin immunolocalisation in control (A and C) and prenatally ethanol exposedenterocytes (B and D). Vesicular elements from apical and lateral enterocyte cytoplasmshowed no labelling in controls (A, X 5 000), whereas homologous areas in the ethanolexposed group were strongly labelled (B, X 53 000). The lateral interdigitations betweenadjacent enterocytes were little developed and poorly labelled in controls (C, X 57 000),whereas in the ethanol exposed group they were highly developed and labelled(D, X62 000). Bars=5 ,um.

or per unit of membrane length.29 Both para-meters, area and length, were estimated bystereological methods30 and significance ofmean differences was tested by Student's t test.

Preparation of cytoskeletal-enriched fractions fromsmall intestineEnriched fractions of intestinal cell cytoskele-ton were obtained according to Quaroni et al.31Briefly, the samples were homogenised in a

glass dounce homogeniser (Anorsa, Spain)with 20 mM TRIS-HCI, pH 7.4, containing amixture of protease inhibitors. Extraction ofcytoskeletal proteins was carried out bycentrifugations at 11 000-50 000 g in non-

ionic detergents and buffers of both low andhigh salt concentration containing a proteaseinhibitor mixture. The high salt extractedcytoskeletal pellets were washed with PBS con-

taining 0.2 mM PMSF and 0O1 mM DTT, andstored at -35°C in PBS until use.

SDS-PAGE and western blottingSDS-PAGE was performed in a Protean I slab

cell (Bio-Rad Laboratories, Richmond, CA) in5-12% acrylamide gels under reducing condi-tions. Polypeptides were stained in the gelsusing Coomassie Blue R (Bio-Rad). Apparentmolecular masses of polypeptides were deter-mined from their relative mobilities comparedwith a standard of molecular mass protein(thyroglobulin, 330 kDa; bovine serum albu-min, 67 kDa; catalase, 60 kDa; lactatedehydrogenase, 36 kDa, and ferritin 18.5 kDa).

After electrophoresis, polypeptides fromSDS gels were equilibrated in 50 mM TRIS-HCG, pH 7.4, in 20% glycerol for one hour31and then transferred to nitrocellulose mem-branes32 in carbonate blot buffer (10 mMNaHCO3, 3 mM Na2CO3, pH, 9.9, in 20%MeOH). Transfer was achieved at 20 Vovernight at 4°C. Membranes were blockedwith 3% ovalbumin in 10 mM PBS for 30minutes and then incubated either with thesame mAb against cytokeratin used in theimmunocytochemical techniques (diluted1:20 000 in PBS with 1% ovalbumin) or withrabbit antiubiquitin serum (Sigma, diluted1:300) overnight at 4°C. After three washes in10 mM PBS 0.2% TWEEN 20 (Sigma) themembranes were incubated for two hours atroom temperature with peroxidase conjugatedrabbit antimouse (Ig or peroxidase conjugatedswine antirabbit Ig (Dako, Glostrup,Denmark) depending on the primary antibody.The membranes were developed in a substratesolution of 3,3'-diaminobenzidine tetrahydro-chloride (DAB, Sigma) as chromogen andrecorded on Technical Pan film (Kodak,Hemel Hempstead, UK).

ResultsCytoskeletal immunolocalisation was per-formed in duodenum and proximal jejunum ofboth control and prenatally ethanol exposednewborn rats. Within each experimentalgroup, absorptive cells from the duodenumwere structurally similar to those of the proxi-mal jejunum, and had the same organisationpattern as those of adults. However, prenatallyethanol exposed enterocytes showed greaterdevelopment of endomembranes around thenucleus than controls. These membranes,which appeared in a vesicular form and associ-ated with saccules of Golgi complex (see Figs2, 3, and 4), were identified as trans golgi ortrans golgi network (TGN) by specificlabelling with anti-p 1,4-galactosyl transferase(see Fig 4).Tubulin was similarly distributed through-

out the cytoplasm of control and prenatallyethanol exposed neonates, mainly in micro-tubular structures. No atypical intracellularlocalisation was seen such as label associationwith microvilli or TGN (Fig 1 (C) and (D)).The label density of the tubulin associated withcytoplasm showed no significant differencesbetween control and prenatally ethanolexposed neonates (Table).

Actin distribution in the apical domain ofthe enterocyte was similar in control and pre-natally ethanol exposed neonates (Fig 1 (A)and (B)), and actin label density in the

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3uP =t' i,.S'

Figure 3: Cytokeratin immunolocalisation in control (A and C) and prenatally ethanolexposed enterocytes (B and D). Golgi related vesicles were devoid of label in controls(A, X 63 000), whereas in the ethanol exposed group they showed strong labellingassociated with the innerface of vesicular membranes (B, X 45 000). Cytokeratin bundlesshowed little label in controls (C, X21 000), whereas in the ethanol exposed group theyshowed abundant label (D, X53 000). Bars (A, B, D) =5 ,um; bar (C) =2 ,um.

microvilli showed no significant differencesbetween experimental groups (Table).Likewise, the cytoplasmic bundles of actin fila-ments showed no quantitative or distributiondifferences between experimental groups (datanot shown). However, the interdigitations inthe lateral domain of plasma membrane were

more developed in prenatally ethanol exposedneonates than controls (Fig 2 (C) and (D)),and the actin label density associated withthese structures was significantly increased inethanol exposed enterocytes (Table). Regard-ing the vesicular compartment, abundant actinlabel density was associated with the internalface of vesicular membranes in prenatallyethanol exposed enterocytes, whereas controlvesicles were devoid of label, showing a labeldensity similar to background (Fig 2 (A) and(B); Table).

Cytokeratin localisation at the terminal weband apical cytoplasm showed no significantdifferences between control and prenatallyethanol exposed enterocytes (Fig 3 (C) and(D)). However, the cytokeratin bundles frommedial and basal cytoplasm showed a signifi-cant label density increase in prenatally ethanolexposed enterocytes, compared with controls

(Table). Furthermore, like actin, cytokeratinwas associated with the vesicular compartmentof prenatally ethanol exposed enterocytes, andabsent in control vesicles (Fig 3 (A) and (B)).

Characterisation of this vesicular compart-ment with anti-1 1,4-galactosyl transferase(Fig 4 (A)) showed that both actin (Fig 4 (B))and cytokeratin (Fig 4 (C)), but not tubulin,were associated with the trans golgi or TGNcompartments, or both, after prenatal ethanolexposure.The protein pattern of intestinal cytoskeletal

enriched fractions was similar in control andprenatally ethanol exposed neonates, as shownby SDS-PAGE analysis. However, a significantincrease in polypeptides of molecular massranging from 39 to 54 kDa was seen in pre-natally ethanol exposed neonates (Fig 5 (A)),compared with controls. This result supportsthe data of immunogold quantifications(Table) as the increased protein rangecoincides with the molecular mass of mostintestinal cytoskeletal proteins.

Western blotting of the intestinal cytoskele-tal enriched fractions and subsequent analysisof cytokeratins with the same mAb as usedfor immunolocalisation, showed a similarcytoskeletal pattern for the two experimentalgroups within the range of 45-53 kDa (Fig 5(B)). However, an additional band of anapparent molecular mass of 28-29 kDa wasdetected in the prenatally ethanol exposedneonates. It was noteworthy that none of thecytokeratins characterised so far in publishedreports have a molecular mass similar to 28-29kDa.The increased abundance of ubiquitin in

prenatally ethanol exposed neonates, com-pared with controls, was shown by westernblotting of the intestinal cytoskeletal enrichedfractions (Fig 6). This increase correspondsbasically to the free ubiquitin pool.

DiscussionThis study shows the effects of ethanol on themajor cytoskeletal proteins of the gut epithe-lium from rat neonates, which was exposed tohigher concentrations in the gastrointestinallumen than in other fetal compartments.4Ethanol crosses the placental barrier and is dis-tributed and accumulated in a concentrationgradient.4 33 However, reports of the passage ofacetaldehyde, a product of ethanol oxidation,across the placental barrier are contradic-tory.34-37 Although placenta and fetal livercontain little alcohol dehydrogenase,4 36 thereis evidence during longterm alcoholism ofother pathways of ethanol oxidation both frommaternal and from fetal tissues,38 39 whichcould interact with the cytoskeleton. Thesederivatives of ethanol, such as acetaldehydeand hydroxyethyl radicals, are probably pro-duced either by peroxisomal catalase or by themicrosomal ethanol inducible cytochromeP450.40 Furthermore, Lange4' reported theformation of ethyl esters by non-oxidative reac-tions associated with glutathione S-transferase.

Increased concentrations of actin and cyto-keratins, but not tubulin, were first suggested

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1 2 3

___.7 .3360 -- ._

;36.S

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1..Figure 4: Positive controlfor the immunolocalisation of ,B 1,4-galactosyl transferase, amarkerfor trans golgi and TGN (A, X58 000). Double immunostainingfor ,B 1,4-galactosyl transferase (10 nm gold) and either actin (15 nm gold) (B, X46 000) orcytokeratin (15 nm gold) (C, X 56 000). The colocalisation shows the association of bothactin and cytokeratin epitopes with the TGN membranes. Section C illustrates both thedilatation of cytokeratin containing TGN vesicles and cytokeratin association withdesmosomes (arrowheads). Bars=5 gtm.

by the labelling densities obtained inimmunolocalisations of prenatally ethanolexposed enterocytes, compared with controls.Furthermore, this increase was confirmed bythe increased protein abundance in the rangeof 39 to 54 kDa found in SDS-PAGE analysis,as nearly all cytoskeletal proteins fall in thisrange. This cytoskeletal increase could be pro-duced either by protein overexpression or byinhibition of protein degradation. With regardto the first possibility, Zern et all0 showed a

considerable increase in albumin synthesis inhepatocytes of rats fed for one year with a dietin which 36% of energy came from ethanol,compared with control rats. This proteinincrease was caused by an increase in thesteady state level of active albumin mRNA inthe adult ethanol exposed liver. With regard tothe small intestine of neonates, a 10-fold to 15-fold increase in the values of lactase andintestinal alkaline phosphatase mRNAs was

found after prenatal ethanol exposure.42However, this increase was not correlated witha similar increase in the protein mass.

The possibility of an ethanol induced inhibi-tion of protein degradation has been studied in

Figure 5: (A) SDS-PAGE analysis of intestinalcytoskeleton enrichedfractions (40 ,g/lane). Lane 1corresponds to standard mass proteins. Lane 2 shows basalprotein expression in control neonates. Lane 3 shows theoverexpression in polypeptides rangingfrom 39-54 kDainduced by prenatal ethanol exposure. (B) Western blotanalysis of specific cytokeratins performedfrom the previousSDS-PAGE as described in methods. Lane 1 shows controlneonates; lane 2, prenatally ethanol exposed neonates. Theblot confirms the overexpression of cytokeratins afterprenatal ethanol exposure and shows a unique peptide of28-29 kDa (arrowhead), which is a specific feature for theethanol group.

the liver of adult alcohol fed rats. 14 Thevolume densities of autophagosomes andautolysosomes in hepatocytes was lower inethanol treated rats than controls. Moreover,the rate of proteolysis was decreased by 30% inethanol treated rats, suggesting that ethanolfeeding inhibits proteolysis in the adult liver bypreventing the sequestration of protein intolysosomes. Western blotting of intestinalcytoskeletal enriched fractions showed asignificant increase in ubiquitin in prenatallyethanol exposed neonates. Most of theubiquitin increase can be attributed to theeffect that ethanol exposure induces anincreased gene expression of stress proteins.12

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Ethanol induced cytoskeletal changes 851

GOf 4>t

Ubq _

Figure 6: Western blotanalysis offree ubiquitin(Ubq) in intestinalcytoskeletal enrichedfractions.

On the other hand, Corsi et a143 showed thatthe cytoskeleton is covalently modified byubiquitination. Furthermore, Akamatsu et al44stated that ubiquitination is an importantprocess in the degradation of intermediate fila-ment proteins in the cytoplasm. However, ourresults showed that the increase in the cytoker-atin ubiquinitation was not correlated with anincrease in the cytokeratin degradation.Moreover, the results obtained from theethanol treated group - that is, the increase incytokeratin amount and the presence of thecytokeratin related polypeptide of 28-29 kDa -suggest a defective degradation of the proteins.The increased reactivity that several cytoskele-tal proteins exhibit for ethanol derivatives,especially aldehydes, could block the specificbinding of ubiquitin, as both bind to lysineresidues.'8 45 46

Furthermore, we observed that enterocytesexposed to ethanol in utero showed deficienciesin cell-cell and cell-basal lamina adhesion, withoccasional detachment of the epithelial sheet(data not shown). This was in agreement withdata of neural cell experiments in which addi-tion of ethanol to the culture induced celldetachment.20 Ethanol in the culture mediuminteracted with the LI and N-CAM moleculesof neurones resulting in inhibition of celladhesion. In our study, the increased cyto-skeletal components are actin and cytokeratins,which happen to be the elements that interactwith the cell adhesion molecules of plasmamembrane. Tubulin, on the other hand, is notincreased after prenatal ethanol exposure, but ithappens to be a cytoskeletal element unrelatedto cell adhesion. We hypothesise that thepresence of a high concentration of ethanol (anaverage of 30 mM) in the fetal gastrointestinallumen4 would first result in covalent modifi-cations of the surface adhesion molecules ofenterocytes. Secondly, in response to loss ofadhesiveness, feed back mechanisms wouldincrease the expression of cell adhesion mole-cules, which might cause the overexpressionand rearrangement of cytoskeletal elements.The atypical localisation of actin and cyto-

keratin in the trans golgi or the TGN, or both,can only be explained by the molecular modifi-cations caused by ethanol or its derivatives, orboth, in both the cytoskeletal elements and themembrane of these compartments. Ethanol is asubstrate for phospholipase D,16 an enzymethat catalyses the hydrolysis of phospholipids,such as phosphatidylcholine, to phosphatidicacids. However, this enzyme also utilisesethanol and several short chain alcohols assubstrates, resulting in the formation ofspecific phosphatidylalcohols, such as phos-phatidylethanol. Phosphatidylethanol inducesan increase in the fluidity of artificial andnatural bilayers, considerably modifying thephysicochemical properties of membranes.15Moreover, the hydrophobic head group ofphosphatidylethanol might change the mem-brane architecture by disrupting the ionic bondlattice extending over the surface of the mem-brane.47 These molecular modifications ofcellular membranes might facilitate the non-specific entrance of altered proteins, actin, and

cytokeratins, in the trans golgi or TGN, orboth.

In conclusion, our findings suggest thatethanol in utero exerts severe changes in thecytoskeleton of the developing intestinalepithelium. Actin and cytokeratin are twoaffected components showing increasedexpression and atypical intracellular localisa-tion. In contrast, tubulin is not affected.Therefore, the effects of ethanol in utero arespecific for the cytoskeletal elements relatedwith the plasma membrane and thus, affectingcell adhesion. These findings may contributeto understanding the cellular and molecularbasis of intestinal dysfunctions found in theoffspring of alcoholic mothers, causing pre andpostnatal growth retardation.The authors are grateful to Dr Senen Vilar6 (Unitat de BiologiaCellular, Facultat de Biologia) for the serum against 1B 1,4-galactosyl transferase, to the staff of Serveis Cientifico-Tecnics(Universitat de Barcelona), and to Mr Robin Rycroft and MsMireia Martin for linguistic advice.

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