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Laboratory of Experimental Cancerology, Department of Radiotherapy and Nuclear Medicine, Ghent University Hospital, B-9000 Gent; and
* Department of Anatomy, Embryology and Histology, Ghent University, B-9000 Gent, Belgium
1Correspondence: Laboratory of Experimental Cancerology, Department of Radiotherapy and Nuclear Medicine, Ghent University Hospital, De Pintelaan 185, B-9000 Gent, Belgium. E-mail: marc.mareel{at}rug.ac.be
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: invasion • transepithelial electrical resistance • paracellular permeability • ZO proteins • amebiasis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. A major role in the pathogenesis of invasive
amebiasis has been attributed to the multifunctional
Gal/GalNAc-specific amebic lectin, which also mediates trophozoite
adhesion and is transferred from the trophozoites to the enterocytes
(2
3
4)
. Clinical manifestations of the disease range from
mild diarrhea to severe dysentery, due mainly to loss of the barrier
function of the intestinal mucosa. The latter may result from changes
in the activity of the ion pumps and channels or from disturbance of
the tight junction complex. In a later stage of the disease, the
manifestations are aggravated by the cytotoxic effects of the parasite.
The tight junction complex constitutes, after the mucus, the first
barrier against the paracellular penetration of intestinal
microorganisms. This intercellular barrier is formed by the plasma
membrane-spanning proteins claudins (5)
and occludin
(6)
. Both proteins associate at their cytoplasmic part
with the peripheral plasma membrane protein ZO-1. Occludin (60–80 kDa)
binds directly with its carboxyl terminus to ZO-1 (220 kDa)
(7)
and associates with ZO-3 (130 kDa) (8)
.
ZO-1 in turn associates with ZO-2 (160 kDa) (9)
and AF-6
(180 and 195 kDa) (10)
. The ZO proteins are members of the
membrane-associated guanylate kinase (MAGUK) protein family
characterized by one or three PDZ domains, an SH3 domain, and a region
of homology with the enzyme guanylate kinase. Cingulin (140 kDa)
(11)
, 7H6 (155 kDa) (12)
, symplekin (126.5
kDa) (13)
, and ZA-1TJ (14)
colocalize with
the other tight junction molecules but their binding interaction and
function are not known. Phosphorylation (15)
of occludin
(16)
is on serine and weakly on threonine, of ZO-1 on
tyrosine and serine, of ZO-2 (17)
on tyrosine, and of
cingulin (18)
on serine. Tight junction complexes are
linked to the actin cytoskeleton (19)
.
Selective disturbance of tight junction complexes by trophozoites is
suggested by the rapid decrease of transepithelial electrical
resistance (TER) of epithelial cell layers in vitro, which
is caused by an increased paracellular permeability
(20
21
22)
.
The present experiments address the question whether or not functional
disturbance by trophozoites is associated with molecular changes in the
tight junction complex. For Vibrio cholerae
(23)
and Clostridium difficile
(24)
, selective molecular disturbance of epithelial tight
junctions has been reported. Therefore, we have seeded trophozoites on
human enteric T84 cell layers established on filters in two-compartment
culture chambers. In this model we have kinetically analyzed tight
junction complexes by immunocytochemistry, immunoprecipitation, and
Western blotting using antibodies against occludin, ZO-1, ZO-2, and
cingulin.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(ECACC, Salisbury, U.K.) were maintained at 37°C in a 1:1 mixture of
Dulbecco’s modified Eagle medium (DMEM) and Ham’s F-12 medium (Life
Technologies, Inc. Europe, Ghent, Belgium) supplemented with 100
µg/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine
serum under an atmosphere of 90% air and 10%
CO2. Trophozoites of E. histolytica
strain HM1:IMSS (4)
were grown axenically in TYI-S-33
medium (26)
. Trophozoites were harvested at the end of the
logarithmic growth phase (72 h cultures) by chilling at 4°C. They
were concentrated at 200 g for 5 min, washed in culture
medium without serum, and used immediately.
Cocultures in two-compartment chambers
T84 cells were grown either on the top or the bottom of 0.33
cm2 Transwell polycarbonate filters (5 µm pore
size, Costar Corp., Cambridge, Mass.) in 24-well cell culture dishes.
Trophozoites
(1x104-1x105) were
applied mostly onto the apical side of the enteric cell layer
established on the top of the filter (Fig. 1a
). In some experiments trophozoites were applied onto the
bottom of the 24-well culture dish (Fig. 1c
) without direct
contact with the enteric layer, which was established on the bottom of
the filter and thus faced the trophozoites. Sonicates of trophozoites
were prepared by intermittent treatment (Vibra Cell VC50; Sonics &
Materials, Danbury, Conn.) of 1 x 105 live
trophozoites for a total of 45 s. To evaluate cell death, live
cocultures were stained with fluorescein diacetate and propidium iodide
for examination by fluorescence microscopy (27)
. To test
amebic cytotoxicity, we also used the 51Cr
release assay (21)
. T84 cells grown on top of 24-well
filters were incubated with 60 µCi of 51Cr in
800 µl DMEM/HAMF12 with serum in the lower chamber for 2 h. The
monolayers were washed four times on the basal side and two times on
the apical side with unlabeled DMEM/HAMF12 with serum. After incubation
with trophozoites or control medium, the media in the apical and
basolateral wells were recovered separately. The T84 cells on the
filter were solubilized with 1 M NaOH; radioactivity was measured in a
gamma counter. Release was expressed as the percentage of the total
radioactivity released into the medium compared with the total
radioactivity recovered, i. e., apical well plus basolateral well plus
cellular radioactivity. Three cultures were used per experiment and
matched TER measurements were recorded.
|
Measurement of TER and paracellular tracer flux
Functional integrity of tight junctions in cell layers
established on filter inserts was assessed by measuring TER using a
Millicel ERS Volt-ohm meter (Millipore Corp., Bedford, Mass.). T84
cells cultured during 4 days develop a TER around 330 Ohm x
cm2. Values of TER are expressed as percentage of
the initial resistance. Flux assays were performed as described by
Madara et al. (28)
on T84 cell layers established on
filters and cocultured or not with 1 x 103,
1 x 104, or 1 x
105 trophozoites.
[3H]Mannitol (50 µCi/ml; ICN Biomedicals,
Costa Mesa, Calif.) was added to the upper chamber and radioactivity
was measured in the lower chamber after 5 h.
Histology and immunofluorescence microscopy
Invasion of trophozoites through enteric T84 cell layers was
evaluated on histological sections from cocultures on filters.
Cocultures were embedded in Technovit 8100 and processed in accordance
with the manufacturers instructions (Kulzer and Co, GmBH, Wehrlein,
Germany). For immunocytochemistry, cocultures with T84 cell layers
established on filters or on plastic substrate in Lab-Tek 8-Chamber
Slides (Nunc, Naperville, Ill.) were fixed in 3% paraformaldehyde for
20 min or for 3 min (to reveal ZO-2), quenched with 50 mmol/l
NH4Cl for 10 min, and permeabilized with 0.2%
Triton X-100 for 5 min, all at room temperature. After a preincubation
in 5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS)
for 30 min, the cocultures were incubated with the following
antibodies: rat monoclonal antibody against occludin (MOC37, kindly
provided by M. Furuse, Kyoto, Japan), mouse monoclonal antibody against
a Gal/GalNAc-specific amebic lectin (CD6) (29)
, rabbit
polyclonal antibodies against ZO-1 or ZO-2 (Zymed Laboratories, San
Francisco, Calif.), and rabbit polyclonal antibody against cingulin
(11)
. Secondary antibodies coupled to fluorescein
isothiocyanate (FITC) or biotin, followed by streptavidin-Texas red,
were obtained from Amersham (Buckinghamshire, U.K.). Finally, the
cocultures were mounted on a slide with Gelvatol (Dako, Glostrup,
Denmark). Examination was done by epifluorescence microscopy (Dialux
20; Leitz, Wetzlar, Germany).
Immunoprecipitation and Western blotting
To study phosphorylation/dephosphorylation equilibria of tight
junction proteins in coculture, T84 cells were metabolically labeled
for 3 h with 2.5 µCi/ml
[32P]orthophosphate (ICN Biomedicals) in
phosphate-free DMEM (Life Technologies, Inc.) and trophozoites were
added to the cell layers during the last h of labeling. For
immunoprecipitation with antibodies against ZO-1, ZO-2, occludin, or
E-cadherin, cocultures were washed in PBS and lysed with 100 µl urea
lysis buffer (9 mol/l urea, 50 mmol/l Tris-HCl, 1 mol/l NaCl, 1%
Triton X-100, 5 mmol/l EDTA.Na2) containing the
following protease and phosphatase inhibitors: 5 mmol/l iodoacetamide,
2 mmol/l phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10
µg/ml aprotinin, 0.2 mmol/l
trans-epoxysuccinyl-L-leucylamido(4-guanidino) butane (E64),
1 mmol/l NaVO3, and for metabolically labeled
cocultures the phosphatase inhibitors
Na4P2O7
(10 mmol/l) and NaF (10 mmol/l). After incubation for 30 min at 37°C,
these samples were diluted 10 times with the same lysis buffer
containing the same inhibitors but without urea. Control experiments
were done to evaluate breakdown of tight junction proteins by proteases
released from the trophozoites during the preparation of the lysate
(colysis), as described by Moll et al. (30)
. For these
colysis experiments, we mixed lysates of enteric T84 cell layers,
[32P]-radiolabeled or not, with lysates of
trophozoites. Both lysates were made with the urea lysis buffer,
containing the same inhibitors but with 0.9 mol/l instead of 9 mol/l
urea. Immunoprecipitations were performed as described by van Hengel et
al. (31)
. Precipitated proteins were separated by 7.5%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and transferred onto Immobilon-P membranes (Millipore Corp.).
Transferred proteins were revealed by autoradiography. Levels of
phosphorylation of ZO-1 and ZO-2 were quantified with the aid of a
PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.) and expressed as
percentage of the level of untreated T84 cells, labeled during the same
period as the treated ones. For immunostaining, blots were prewashed at
room temperature in 3% BSA and 0.5% Tween 20 dissolved in PBS. After
1 h, the blots were stained with rat anti-occludin (MOC37), rabbit
antibodies against ZO-2, ZO-1 (Zymed Laboratories), and mouse
antibodies against E-cadherin (HECD-1). Blots were then washed three
times (in 0.5% Tween 20), incubated overnight in alkaline
phosphatase-labeled secondary antibodies, washed again, and developed
with a bromochloroindolyl phosphate/nitro blue tetrazolium substrate.
Quantitation of Western blots from at least three separate experiments was done by Quanti Scan (1.5; Biosoft, U.K.).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Kinetic analysis revealed that the
decrease of TER is more pronounced, with higher numbers of trophozoites
as is obvious from the slope of the curves shown in Fig. 1b
.
For the lower number of trophozoites (1.0x104),
a decrease of TER necessitated at least 3 h of coculture. In
cocultures where trophozoites were seeded on the bottom of the well,
without making contact with the enterocytes seeded on the bottom of the
filter, the TER remained unaltered for at least 3 h. When
sonicated trophozoites were used instead of living ones, no decrease of
TER was observed (Fig. 1c
). We found an increase in
[3H]mannitol flux for TER values lower than 150
Ohm x cm2 all obtained from cocultures with
trophozoites (data not shown). This indicates that at least for the
lower TER values, decrease of TER is due to an increase in paracellular
permeability.
Loss of TER precedes lysis of the enteric cell layer
Trophozoites attach to the apical side of the enteric T84 cell
layers immediately after seeding. The decrease of TER was first
recorded after 15 to 30 min of coculture (data not shown). By contrast,
holes in the enteric cell layers caused by trophozoites became visible
under the phase contrast microscope and on histological sections only
after 3 to 6 h of coculture (data not shown). Moreover, 1 x
104 trophozoites caused a decrease of TER after
3 h (see Fig. 1
). In such cocultures, the number of dead enteric
cells (less than 100) detected by vital staining with fluorescein
diacetate and propidium iodide was similar to that of control cultures
without trophozoites and without decrease of TER. Release of
51Cr (less than 4%) in cocultures with
trophozoites, showing a clear-cut decrease in TER, was not different
from that in control cultures without trophozoites and with no decrease
in TER (data not shown).
Decrease of TER is associated with release of ZO-1 from ZO-2
Enteric T84 cell layers were challenged for 15, 30, and 60
min with trophozoites and banding patterns of different junctional
molecules were analyzed (Fig. 2
). Immunoprecipitates separated by SDS-PAGE, electroblotted, and
subsequently stained with the same antibodies as used for
immunoprecipitation did not reveal differences in the banding pattern
of occludin or E-cadherin. A slight decrease in intensity of ZO-2 bands
was observed in some experiments: a decrease to 68% (Fig. 3a
) or to 82% (Fig. 3b
) as compared to enteric
cell layers without trophozoites. This decrease was not observed in the
experiment shown in Fig. 2
. The ZO-1 bands decreased in intensity
within 15 min of coculture from 54% of the control over 46%, after 30
min to 26% after 60 min of coculture. Electroblots from
immunoprecipitates with an antibody against ZO-2 were immunostained
with an antibody against ZO-1. These blots showed the double band of
ZO-1 around 220 kDa that initially coimmunoprecipitated with ZO-2. The
intensity of this coimmunoprecipitated double band decreased or
disappeared almost completely after 1 h of coculture. Moreover,
the amount of ZO-1 in ZO-2/ZO-1 complex is smaller than after
immunoprecipitation with antibody against ZO-1 (Fig. 2)
. These results
suggest a release of ZO-1 from ZO-2 before it is degraded.
Phosphorylation of ZO-1 as well as of ZO-2 (Fig. 3a
) was
reduced, as evidenced by autoradiographs quantified with the aid of a
PhosphorImager. For ZO-1, reduction of phosphorylation (36%)
corresponded with protein reduction (42%), whereas for ZO-2
dephosphorylation (30%) was stronger than the protein reduction
(68%). Release of ZO-1 from ZO-2, decrease of ZO-1 protein level, and
dephosphorylation of ZO-2 in presence of trophozoites were found also
in extracts made of cocultures established on filters in which a
concomitant decrease of TER was registered (data not shown). To see
whether dephosphorylation of ZO-2 was due to amebic enzymes released
during detergent lysis of the coculture, we performed experiments as
described by Moll et al. (30)
. In such colysis experiments
with mixed lysates, the ZO-2 protein levels as well as ZO-2
phosphorylation (101%) were not lower than the ones in lysates from
cell layers that had not been in contact with trophozoites (Fig. 3b
). Immunocytochemistry was used to find out whether the
failure of ZO-1 to coimmunoprecipitate with ZO-2 was due to delicate
changes in the molecular organization of the tight junction complex or
to gross displacement of proteins. Control enteric T84 cell layers on
solid substrate revealed occludin, ZO-1, ZO-2, and cingulin as fine
networks at the cell–cell borders (Fig. 4
). We did not find immunocytochemical changes of the tight junction
molecules of T84 cell layers challenged with trophozoites.
Immunocytochemical observations made with cultures on filters were
similar to these on solid substrate, but the less well-spread cell
layers in the former system were less readable. Reorganization of the
actin cytoskeleton was observed after at least 1 h of coculture
(data not shown), namely, in the retraction ring around the
trophozoite-associated holes described by us previously
(32)
. By contrast with trophozoites, incubation of the
cell layers in Ca2+-free S-MEM resulted in a
dramatic reorganization of the tight junction molecules, as evidenced
by the granular dot-like patterns of ZO-1 (Fig. 4g
) and of
occludin (data not shown).
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The transferred amebic Gal/GalNAc-specific lectin binds basally to
tight junctions
In a previous study (4)
we found that the
amebic 170 kDa subunit of the Gal/GalNAc-specific lectin was
transferred to the enteric cell layer and concentrated at enteric
cell–cell borders as evidenced by confocal laser scanning after
immunostaining with the monoclonal antibody CD6. To find out whether or
not tight junction proteins may serve as binding sites for the
transferred lectin, we did double immunostaining with antibodies
against molecules of the tight junction complex and with the CD6
antibody (data not shown). It is clear that the tight junction complex
is localized apically from the CD6 signal that reveals the transferred
lectin. The latter immunosignal rather matches the honeycomb-like
staining pattern of E-cadherin, the transmembrane molecule of the
adherens junction complex situated basally from the tight junction
complex as illustrated previously (4)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A dose- and time-dependent, trophozoite-induced decrease of TER of
enteric cell layers established on filters in two-compartment chambers
was also observed by others (20
, 21)
, but a molecular
explanation had, so far, not been provided. Decrease of TER was
ascribed to an increase in paracellular rather than in transcellular
permeability. The arguments lie in the relationship between
[3H]mannitol flux and TER of enteric T84 cell
layers in our and in the others’ experiments and the lack of changes
in short-circuit current (21)
. A similar interpretation
was given to the effect of the actin-disturbing agent cytochalasin D
and of low Ca2+ concentration on tight junctions,
respectively, in mucosal preparations (28)
and in MDCK
cell layers (33)
.
Trophozoite-induced increase of paracellular permeability may be due to
aspecific cytotoxicity, e.g., mediated by amoebapores and cysteine
proteinases (34
, 35)
, or to selective disturbance of tight
junctions. The lack of cell death with lower amebic inocula or their
appearance more than 1 h after decrease of TER with higher inocula
argues against cytotoxicity. Moreover, cytotoxicity as evidenced by
51Cr release was not associated with a decrease
of TER. Differences between our kinetic analysis of TER decrease as
compared to 51Cr release and the results obtained
by others (21
, 36)
may be ascribed to differences in
target cell type, technique, and time of coculturing or to decreased
virulence of trophozoites maintained in axenic culture.
Sonicated trophozoites failed to induce a decrease of TER, indicating
that the enhancement of paracellular permeability was not due to the
release of proteinases or other cytotoxic molecules into the
microenvironment. Arguments in favor of a selective amebic attack on
tight junction complexes are the molecular alterations of the
above-mentioned elements of the complex and the lack of degradation of
other elements such as occludin or the adherens junction protein
E-cadherin. Mixing lysates of T84 enteric cells with lysates of
trophozoites did not mimic the effect of live trophozoites in
coculture. This observation indicates that the trophozoite-induced
molecular changes do not occur during detergent lysis of the
cocultures, as it was described by Moll et al. (30)
for
the in vitro degradation of endothelial catenins by a
neutrophil protease.
Lack of light microscopical immunocytochemical changes in the
localization and organization of the tight junction complex does not
exclude functional changes as demonstrated in the present cocultures of
E. histolytica and T84 cells. We also observed this
phenomenon when treating T84 cells with tyrosine phosphatase
inhibitors, such as phenylarsine oxide and
NH4VO3 (our unpublished
results). These inhibitors caused a decrease of
TER and immunocytochemistry with the antiphosphotyrosine antibody PY20
of treated cell layers revealed an increase in tyrosine
phosphorylation, but no changes were observed in the distribution of
the molecules of the junction complexes, in agreement with data from
others (17)
. Furthermore, a significant decrease of TER in
Xenopus kidney epithelial cell layers treated with a
synthetic peptide, homologous to the second extracellular domain of
occludin, was associated with only limited changes in the
immunocytochemical staining pattern of occludin and no changes of ZO-1,
cingulin, or E-cadherin (37)
. In contrast, at low
Ca2+ concentration the function of tight
junctions is altered as well as the immunocytochemical pattern, but in
immunoprecipitates the composition of the complex remained unchanged.
We can only speculate about the molecular pathways followed by signals
from trophozoites to tight junctions. It is the opinion of Li et al.
(21)
that the loss of the apical brush border in the
region of contact between the trophozoites and epithelial cells, as
also demonstrated in others (20)
and our (unpublished
results) transmission electron micrographs, reflects a more generalized
disruption of the cytoskeleton. The latter might implicate disturbance
of the tight junction complex as it is connected to the actin
cytoskeleton (38)
. Alternatively, trophozoites might
provoke a signal transduction pathway on direct contact with the apical
surface of the enterocytes resulting in destabilization and dysfunction
of the tight junction complex, as already described for bacteria
(39)
. However, the elements of this putative amebic
pathway remain to be determined. Moreover, we were unable to prevent
trophozoite-induced loss of TER by inhibitors of the most common signal
transduction pathways, such as the tyrosine kinase inhibitors
herbimycin A at 0.01 mg/ml, genistein at 2.5 x
10-5 M, the phosphatidylinositol-3 kinase
inhibitor wortmannin at 1 x 10-6 M, or the
inhibitor of trimeric G proteins pertussis toxin at 200 ng/ml (our
unpublished results). Dephosphorylation of tight junction proteins and
a decrease of TER obtained with trophozoites resemble these induced by
ATP depletion in MDCK cells (15)
. In the latter
experiments, ATP repletion led to TER recovery and rephosphorylation of
the proteins, and both were inhibited by genistein, possibly explaining
why genistein did not prevent trophozoite-induced loss of TER in our
experiments.
Neither could the effect of trophozoites on TER be neutralized by
treatment with any of the following protease inhibitors at
concentrations nontoxic for trophozoites (4)
and not
affecting TER in control cultures: N-ethylmaleimide at 20 µM;
aprotinin at 1 µg/ml; E-64 at 200 µM; soybean trypsin inhibitor at
1 mg/ml (our unpublished results). These results and the intact
patterns of occludin in immunoblots argue against specific proteolytic
attack as described for the Der p1 allergen from fecal pellets of the
house dust mite (40)
. Is there a place for the
Gal/GalNAc-specific lectin in such pathway? This multifunctional amebic
lectin is also involved in attachment of the trophozoites to the
enteric cells (41)
and it is transferred to areas of
contact between enteric cell (4)
. The enteric receptor of
this lectin, however, is still unknown. As for trophozoite-induced
decrease of TER, transfer of the lectin to the area of intercellular
contact necessitates direct contact between trophozoites and
enterocytes. Transfer is focally visible within 5 min and increases
with the time of coculture. Despite this positive correlation between
decrease of TER, transfer of the lectin and molecular rearrangements in
the tight junction complex, occludin is not a good candidate receptor
for the amebic lectin because of the poor matching in their respective
immunostaining patterns. A third possibility is suggested by the
observation that the alkyl lysophosphocholine ET-18-OCH3, known to
increase the fluidity of the plasma membrane, decreases the TER of T84
cell layers (A. Leroy, G. K. P. De Bruyne, L. C. J. M. Oomen, and M. M. Mareel, unpublished results). This
finding may be interpreted in the frame of the new aspect of cell
membrane structure where sphingolipid-cholesterol rafts serving as
platforms for the attachment of proteins move in the fluid bilayer
(42)
. We are currently examining whether trophozoites
affect rafts and, if so, whether this has consequences for the
organization of the transmembrane molecules of the tight junctions
claudins and occludin that may explain the molecular rearrangements of
the cytoplasmic ZO proteins and the dysfunction of the complex.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-catenin-negative human colon cancer cell variants via induction of desmosomes. J. Cell Biol. 137,1103-1116This article has been cited by other articles:
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|
 |
|
 J. Yoo, A. Nichols, J. Mammen, I. Calvo, J. C. Song, R. T. Worrell, K. Matlin, and J. B. Matthews Bryostatin-1 enhances barrier function in T84 epithelia through PKC-dependent regulation of tight junction proteins Am J Physiol Cell Physiol, August 1, 2003; 285(2): C300 - C309. [Abstract] [Full Text] [PDF]
|
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 |
|
 T. Lauwaet, M. J. Oliveira, B. Callewaert, G. De Bruyne, X. Saelens, S. Ankri, P. Vandenabeele, D. Mirelman, M. Mareel, and A. Leroy Proteolysis of Enteric Cell Villin by Entamoeba histolytica Cysteine Proteinases J. Biol. Chem., June 13, 2003; 278(25): 22650 - 22656. [Abstract] [Full Text] [PDF]
|
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|
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P. D. Ward, R. R. Klein, M. D. Troutman, S. Desai, and D. R. Thakker Phospholipase C-gamma Modulates Epithelial Tight Junction Permeability through Hyperphosphorylation of Tight Junction Proteins J. Biol. Chem., September 13, 2002; 277(38): 35760 - 35765. [Abstract] [Full Text] [PDF]
|
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|
X.-J. Cheng, M. A. Hughes, C. D. Huston, B. Loftus, C. A. Gilchrist, L. A. Lockhart, S. Ghosh, V. Miller-Sims, B. J. Mann, W. A. Petri Jr., et al. Intermediate Subunit of the Gal/GalNAc Lectin of Entamoeba histolytica Is a Member of a Gene Family Containing Multiple CXXC Sequence Motifs Infect. Immun., September 1, 2001; 69(9): 5892 - 5898. [Abstract] [Full Text] [PDF]
|
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|
 S. L. Stanley Jr. and S. L. Reed Microbes and Microbial Toxins: Paradigms for Microbial-Mucosal Interactions: VI. Entamoeba histolytica: parasite-host interactions Am J Physiol Gastrointest Liver Physiol, June 1, 2001; 280(6): G1049 - G1054. [Abstract] [Full Text] [PDF]
|
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|
|
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L. H. Kasper and D. Buzoni-Gatel Ups and Downs of Mucosal Cellular Immunity against Protozoan Parasites Infect. Immun., January 1, 2001; 69(1): 1 - 8. [Full Text] [PDF]
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