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Article| December 01 1997
Epithelial Cell Adhesion Molecule (Ep-CAM) Modulates Cell–Cell Interactions Mediated by Classic Cadherins
Sergey V. Litvinov,
Sergey V. Litvinov
Department of Pathology, Leiden University, Leiden 2300 RC, The Netherlands
Maarten Balzar,
Maarten Balzar
Manon J. Winter,
Manon J. Winter
Hellen A.M. Bakker,
Hellen A.M. Bakker
Inge H. Briaire-de Bruijn,
Inge H. Briaire-de Bruijn
Frans Prins,
Frans Prins
Gert Jan Fleuren,
Gert Jan Fleuren
Sven O. Warnaar
Address all correspondence to S.V. Litvinov, Department of Pathology, Leiden University, Rijnsburgerweg 10, Building 1, LI-Q, P.O. Box 9600, 2300RC Leiden, The Netherlands. Tel.: 31.71.524.8158. Fax: 31.71.526. 6628. E-mail: slitvinov@pathology.medfac.leidenuniv.nl
Received: September 16 1996
Revision Received: September 25 1997
J Cell Biol (1997) 139 (5): 1337–1348.
https://doi.org/10.1083/jcb.139.5.1337
Revision Received:
Sergey V. Litvinov, Maarten Balzar, Manon J. Winter, Hellen A.M. Bakker, Inge H. Briaire-de Bruijn, Frans Prins, Gert Jan Fleuren, Sven O. Warnaar; Epithelial Cell Adhesion Molecule (Ep-CAM) Modulates Cell–Cell Interactions Mediated by Classic Cadherins . J Cell Biol 1 December 1997; 139 (5): 1337–1348. doi: https://doi.org/10.1083/jcb.139.5.1337
The contribution of noncadherin-type, Ca2+-independent cell–cell adhesion molecules to the organization of epithelial tissues is, as yet, unclear. A homophilic, epithelial Ca2+-independent adhesion molecule (Ep-CAM) is expressed in most epithelia, benign or malignant proliferative lesions, or during embryogenesis. Here we demonstrate that ectopic Ep-CAM, when expressed in cells interconnected by classic cadherins (E- or N-cadherin), induces segregation of the transfectants from the parental cell type in coaggregation assays and in cultured mixed aggregates, respectively. In the latter assay, Ep-CAM–positive transfectants behave like cells with a decreased strength of cell–cell adhesion as compared to the parental cells. Using transfectants with an inducible Ep-CAM–cDNA construct, we demonstrate that increasing expression of Ep-CAM in cadherin-positive cells leads to the gradual abrogation of adherens junctions. Overexpression of Ep-CAM has no influence on the total amount of cellular cadherin, but affects the interaction of cadherins with the cytoskeleton since a substantial decrease in the detergent-insoluble fraction of cadherin molecules was observed. Similarly, the detergent-insoluble fractions of α- and β-catenins decreased in cells overexpressing Ep-CAM. While the total β-catenin content remains unchanged, a reduction in total cellular α-catenin is observed as Ep-CAM expression increases. As the cadherin-mediated cell–cell adhesions diminish, Ep-CAM–mediated intercellular connections become predominant. An adhesion-defective mutant of Ep-CAM lacking the cytoplasmic domain has no effect on the cadherin-mediated cell–cell adhesions. The ability of Ep-CAM to modulate the cadherin-mediated cell–cell interactions, as demonstrated in the present study, suggests a role for this molecule in development of the proliferative, and probably malignant, phenotype of epithelial cells, since an increase of Ep-CAM expression was observed in vivo in association with hyperplastic and malignant proliferation of epithelial cells.
Tissue and organ morphogenesis can be viewed as the result of interactions of various cell populations. One important type of intercellular interaction involved in the processes of tissue morphogenesis, morphogenetic movements of cells, and segregation of cell types, are adhesions mediated by cell adhesion molecules (Steinberg and Pool, 1982; Edelman, 1986; Cunningham, 1995; Takeichi, 1995; Gumbiner, 1996). Except for their direct mechanical role as interconnectors of cells and connectors of cells to substrates, cell adhesion molecules are also believed to be responsible for a variety of dynamic processes including cell locomotion, proliferation, and differentiation. There is also evidence that the adhesion systems within a cell may act as regulators of other cell adhesions, thereby offering a means of signaling that is relevant for rearrangements in cell or tissue organization (Edelman, 1993; Rosales et al., 1995; Gumbiner, 1996).
In many tissues, a critical role in the maintenance of multicellular structures is assigned to cadherins, a family of Ca2+-dependent, homophilic cell–cell adhesion molecules (Takeichi, 1991, 1995; Gumbiner, 1996). In epithelia this critical role belongs to E-cadherin, which is crucial for the establishment and maintenance of epithelial cell polarity (McNeil et al., 1990; Näthke et al., 1993), morphogenesis of epithelial tissues (Wheelock and Jensen, 1992; Larue et al., 1996), and regulation of cell proliferation and programmed cell death (Hermiston and Gordon, 1995; Hermiston et al., 1996; Takahashi and Suzuki, 1996; Wilding et al., 1996; Zhu and Watt, 1996). Expression of different types of classic cadherin molecules (Nose et al., 1988; Friedlander et al., 1989; Daniel et al., 1995), and even quantitative differences in the levels of the same type of cadherin (Steinberg and Takeichi, 1994), may be responsible for segregation of cell types in epithelial tissues. The phenotype of epithelial cells may be modulated by expression of combinations of different types of cadherins (Marrs et al., 1995; Islam et al., 1996). However, cadherins represent only one of the intercellular adhesion systems that are present in epithelia, along with adhesion molecules of the immunoglobulin superfamily, such as carcinoembryonic antigen (Benchimol et al., 1989), and others. The actual contribution of Ca2+-independent nonjunctional adhesion molecules to the formation and maintenance of the epithelial tissue architecture and epithelial cell morphology is not clear.
We have recently demonstrated that a 40-kD epithelial glycoprotein, which we have designated epithelial cell adhesion molecule (Ep-CAM)1 (Litvinov et al., 1994a), may perform as a homophilic, Ca2+-independent intercellular adhesion molecule, capable of mediating cell aggregation, preventing cell scattering, and directing cell segregation. This type I transmembrane glycoprotein consists of two EGF-like domains followed by a cysteine-poor region, a transmembrane domain, and a short (26-amino acid) cytoplasmic tail, and is not structurally related to the four major types of CAMs, such as cadherins, integrins, selectins, and the immunoglobulin superfamily (for review see Litvinov, 1995). Ep-CAM demonstrates adhesion properties when introduced into cell systems that are deficient in intercellular adhesive interactions (Litvinov et al., 1994a). However, the participation of the Ep-CAM molecule in supporting cell–cell interactions of epithelial cells was not evident (Litvinov et al., 1994b).
Most epithelial cell types coexpress E-cadherin (and sometimes other classic cadherins) and Ep-CAM (for review see Litvinov, 1995) during some stage of embryogenesis. In adult squamous epithelia, which are Ep-CAM negative, de novo expression of this molecule is associated with metaplastic or neoplastic changes. Thus, in ectocervical epithelia, expression of Ep-CAM occurs in early preneoplastic lesions (Litvinov et al., 1996); most squamous carcinomas of the head and neck region are Ep-CAM positive (Quak et al., 1990), and basal cell carcinomas are Ep-CAM positive in contrast to the normal epidermis (Tsubura et al., 1992).
In many tumors that express Ep-CAM heterogeneously, an Ep-CAM–positive cell population may be found within an Ep-CAM–negative cell population, with both cell types expressing approximately equal levels of cadherins, as illustrated in Fig. 1,A by a case of basal cell carcinoma. In glandular tissues such as gastric epithelium, which are low/ negative for Ep-CAM, expression of Ep-CAM is related to the development of early stages of intestinal metaplasia (our unpublished observation). Even in tissues with relatively high Ep-CAM expression, such as colon, the development of polyps is accompanied by an increase in Ep-CAM expression (Salem et al., 1993). In intestinal metaplasia one may observe Ep-CAM–positive cells bordering morphologically identical normal cells that are Ep-CAM–negative (as illustrated in Fig. 1 B) Ep-CAM–positive cells bordering Ep-CAM–negative epithelial cells may also be found in some normal tissues such as hair follicles (Tsubura et al., 1992).
From the examples presented, an increased or de novo expression of Ep-CAM is often observed in epithelial tissues in vivo. Expression of an additional molecule that may participate in cell adhesion in the context of other adhesion systems may have certain effects on the cell–cell interactions. Therefore, we have investigated whether the increased/de novo expression of Ep-CAM in epithelial cells (a) has any impact on interactions of positive cells with the parental Ep-CAM–negative cells, and (b) modulates in any way intercellular adhesive interactions of cells interconnected by E-cadherin, which is the major morphoregulatory molecule in epithelia.
Here we demonstrate that expression of Ep-CAM by some cells in a mixed cell population expressing classical cadherins induces segregation of the Ep-CAM–positive cells from the parental cell population due to a negative effect on cadherin junctions caused by expression of Ep-CAM. The cadherin-modulating properties observed for Ep-CAM suggest a role for this molecule in the development of a proliferative and metaplastic cell phenotype, and probably in the development and progression of malignancies.
DNA Constructs
The SmaI–BglII fragment of human Ep-CAM cDNA was used for the wild-type Ep-CAM expression constructs, as reported earlier (Litvinov et al., 1994a). Mutant Ep-CAM with a truncated cytoplasmic tail (Mu1) was generated by PCR amplification of the fragment of Ep-CAM cDNA corresponding to amino acids 1–289. The wild-type and mutant Ep-CAM cDNAs were cloned into pCEP4 and pMEP4 vectors (Invitrogen BV, Leek, The Netherlands) under the control of cytomegalovirus or metallothionein II promotors, respectively. Both vectors used contain the Epstein-Barr virus's origin of replication and the EBNA-1 gene, which allow both episomal replication and self-support of the plasmid to copy large numbers of human cells. In murine cells, both vectors integrate into the cellular DNA.
Cells and Transfections
The murine E–cadherin transfected L (LEC) cells (clone LUN.6), and the HCA clonal cell line isolated from the SV-40 immortalized normal mammary epithelial cell line HBL-100 were recloned in our laboratory before transfections (provided by J. Hilkens, The Netherlands Cancer Institute, Amsterdam, The Netherlands). L cells (L929), colon carcinoma cell line (LS180), mammary carcinoma cells (MCF-7), and the pancreatic carcinoma cell line (CAPAN) were obtained from American Type Culture Collection (Rockville, MD). The normal human mammary epithelium-derived cell line RC-6 and the squamous carcinoma cell line (U2) were cultured in DME with 10% FCS (provided by E. Schuuring, Leiden University, Leiden, The Netherlands) as were all cell lines. Cells were transfected using the DOTAP reagent (Boehringer Mannheim Corp., Mannheim, Germany) as described earlier (Litvinov et al., 1994a). For murine cells, the stable clones obtained were either grown as isolated clones or were pooled; human transfected cell lines were continuously cultured in the presence of the selection marker (hygromycin, 1 mg/ml; Boehringer Mannheim Corp.). 48 h before the experiments, cells were passaged into medium without hygromycin. To induce the expression of constructs under the control of the metallothionein promotor (pMEP4 vector), CdCl2 was added to the culture medium at concentrations of 2–50 μM depending on the cell line.
Cell Aggregation Assay
Cell aggregation assays were performed as described earlier (Litvinov et al., 1994a). The cells were detached with either TC treatment (Hank's buffer with 0.01% trypsin and 1 mM CaCl2), or by 0.2% EDTA in Hank's buffer. The degree of cell aggregation was calculated as D = (No − Nt)/No, where Nt is the number of remaining particles at the incubation time point t, and No is the initial number of particles corresponding to the total number of cells.
Labeling of Cells and Cell Sorting Experiments
For cell sorting experiments, the cells were labeled according to the manufacturer's protocol with fluorescent dyes (PKH-2 [green fluorochrome] or PKH-26 [red fluorochrome]; Zynaxis Cell Science Inc., Malvern, PA), that incorporate the membrane's lipid bilayer. These fluorochromes provide a stable labeling of living cells and do not interfere with either cell surface proteins or with the cell's behavior and interactions (Horan et al., 1990; Litvinov et al., 1994a). Cell sorting/patterning experiments were performed as described elsewhere (Nose et al., 1988; Litvinov et al., 1994). Briefly, cells were dissociated with TC, washed three times in Dulbecco's PBS, labeled with one of the fluorochromes, washed three times in 50% FCS in DME, resuspended in DME containing 0.8% FCS and 1 mg/ml DNase (Boehringer Mannheim Corp.), mixed at various ratios depending on the experiment, and allowed to aggregate as described for the aggregation assay. After 1–2 h of aggregation, the suspension of aggregates was analyzed under a confocal microscope (model BRC-600; Bio-Rad Laboratories, Carlsbad, CA). Images from different areas of the preparation were taken, and the number of cells of each color in the aggregates was determined. To study the segregation/patterning of cells in aggregates, the two cell types labeled with different fluorescent dyes were mixed at equal ratios, spun down, and allowed to aggregate in the pellet during the next 2 h at 37°C. The large aggregate formed was mechanically dispersed into smaller fragments, which were further cultured in suspension on a rotating platform (at 140 rpm). After 30 min and 24 h of culture, respectively, samples of the aggregates were fixed with 4% formaldehyde in PBS/1 mM CaCl2, and analyzed with a confocal microscope.
Anti–Ep-CAM antibodies were 323/A3 (against human Ep-CAM; Litvinov et al., 1994) and G8.8 (against murine Ep-CAM; Nelson et al., 1996). The antibodies against epitopes in extracellular domains of human E-cadherin and human P-cadherin were obtained from Thamer Diagnostica BV (clones HECD-1 and NCC-CAD-299, respectively; Uithoorn, The Netherlands). A mAb against an epitope in the extracellular domain of N-cadherin (clone GC-4) was obtained from Sigma Chemical Co. (St. Louis, MO). Antibody to the cytoplasmic domain of classical cadherins (clone CH-19; Sigma Chemical Co.), strongly reactive with N-cadherin and weak with other cadherin types, was used for immunoblotting experiments. Two mAbs against murine E-cadherin were used: one reactive with an epitope in the cytoplasmic domain (clone 36; Transduction Laboratories, Lexington, KY), and one reactive with the extracellular domain of the molecule (ECCD-2; Takara Shuzo Co., Shiga, Japan). Antibodies to α-catenin (clone αCAT-7A4; Zymed Laboratories, South San Francisco, CA) and β-catenin (clone 14; Transduction Laboratories) both had cross-species reactivity. For immunoblotting experiments on immunoprecipitates, a polyclonal rabbit antiserum to α-catenin was used (gift of J. Behrens, Max Delbrueck Center for Molecular Medicine, Berlin, Germany).
Immunofluorescence Microscopy
Cells growing on either glass slides or in multiwell chamber slides (Nunc, Naperville, IL) were fixed for 10 min in −20°C MeOH, rinsed quickly in −20°C acetone, and allowed to dry. The preparations were blocked in 5% skim milk solution in PBS for 30 min at 37°C, and indirect immunofluorescent staining was performed using a specific mAb and a goat anti–mouse IgG–FITC conjugate (Southern Biotechnologies, Birmingham, AL). The preparations were analyzed with a confocal microscope (model BRC-600; Bio-Rad Laboratories).
Reflection Contrast Microscopy and Electron Microscopy
Both were performed as described (Prins et al., 1993). Cell aggregates were fixed with 2% paraformaldehyde/1.25% glutaraldehyde, postfixed with 1% osmium tetroxide, embedded into Epon, and ultrathin sectioned. The preparations were examined, respectively, with a microscope equipped for epi-illumination (model Orthoplan; Leithz, Wetzlar, Germany), and an electron microscope (model CM10; Philips Electron Optics, Eindhoven, The Netherlands).
The expression of Ep-CAM was detected on living cells by using F(ab) fragments of mAb 323/A3 directly conjugated with 5(6)-Carboxy-fluorescein-N-hydroxysuccinimide ester (FLUOS), a FITC-like fluorochrome. The conjugation was performed according to the manufacturer's protocol (Boehringer Mannheim Corp.). To detect the cell surface expression of murine E-cadherin or human N-cadherin the mAbs ECCD-2 and GC-4 were used. The bound mAb was detected with an appropriate species-specific anti–IgG-FITC conjugate (Southern Biotechnologies).
Cell Lysis and Cell Extraction with Detergents
Cells of various transfected cell lines were seeded at equal density on 10-cm Petri dishes 48 h before lysis and cultured during the last 24 h in either the presence or absence of Cd2+ cations in the medium. To prepare total cell lysates, cells on dishes were rinsed twice with ice-cold PBS (pH 7.4), and lysed in 1 ml of hot (100°C) 1% SDS/10 mM EDTA. The extraction of detergent-soluble cadherins and catenins was performed as described by Hinck et al. (1994). Cells were rinsed three times with cold PBS, and 2 ml of cold extraction buffer (50 mM Tris/HCl, pH 7.0, 50 mM NaCl, 3 mM MgCl2, 0.5% Triton X-100, 300 mM sucrose, and protease inhibitor [complete; Boehringer Mannheim Corp.]) were added to the cells. Cells were incubated for 45 min on a shaker at 4°C, detached with a scraper, collected, and spun down in a centrifuge for 10 min at 15,000 rpm (5415C; Eppendorf Scientific, Inc., Hamburg, Germany). The pellet was lysed with hot (100°C) 1% SDS, 10 mM EDTA and then boiled for 5 min. The lysates obtained with hot SDS were spun through a spin column (QiaShredder; QUIAGEN Inc., Hilden, Germany) to reduce the viscosity caused by DNA, and the preparations obtained were used for gel electrophoresis. The protein content was determined for each sample by measuring the optical density at 224 nm of a sample aliquot prediluted with 4% SDS.
Immunoprecipitation and Immunoblotting
Cells were lysed with extraction buffer (as described above), the lysates were clarified by centrifugation in a centrifuge for 10 min at 15,000 rpm (5415C; Eppendorf Scientific, Inc.), and used for immunoprecipitation. 5 μg of a specific mAb was added to a lysate from 5 × 106 cells in 1 ml and incubated at 4°C in an end-to-end rotator for 1 h (RKIOVS; Emergo BV, Landsmeer, The Netherlands). 100 μl of 50% Protein G–Sepharose slurry (Pharmacia Biotech, Inc., Uppsala, Sweden) was then added to each tube, and the tubes were further incubated for 1 h. The immunoadsorbent beads were washed four times with 1 ml of the extraction buffer, and the precipitates were dissolved in Laemmli's sample buffer (1% SDS, 10% glycerol, 10 mM EDTA, 125 mM Tris-HCl, pH 6.8) containing 2% β-mercaptoethanol, boiled for 10 min, and used for further immunoblotting experiments. Immunoblotting was performed as described earlier (Litvinov et al., 1994a), using the Alkaline-Phosphatase Protoblot System (Promega Corp., Madison, WI), or the enhanced chemiluminescent detection system (Amersham Intl., Little Chalfont, UK).
Ep-CAM Directs Segregation within E-cadherin–positive Cells
To investigate to what extent the expression of Ep-CAM in epithelia modulates the interactions of Ep-CAM–positive cells with neighboring Ep-CAM–negative cells, we have established a simple model: murine fibroblast L cells transfected with murine E-cadherin, and a derived cell line additionally supertransfected with Ep-CAM. The murine L cell fibroblasts have previously been used to demonstrate the adhesion properties of both E-cadherin (Nagafuchi et al., 1987), and Ep-CAM (Litvinov et al., 1994); these cells are able to support the functional activity of both molecules and do not express endogenous cadherins or murine Ep-CAM (as was tested with a mAb specific to murine Ep-CAM; data not shown).
LEC cells demonstrated all morphological changes related to E-cadherin expression reported for other E-cadherin transfectants of L cells (Nagafuchi et al., 1987; Chen and Öbrink, 1991; Wesseling et al., 1996). LEC cells were supertransfected with either Ep-CAM cDNA under the control of the constitutive cytomegalovirus promotor (LEC-Ep cells), or with the blank pCEP4 expression vector (LEC-C cells). These transfected cell lines were established without clonal isolation and represented a mix of >200 individual clones from each transfection. Additionally, L cells transfected only with the Ep-CAM cDNA were established (LEp cells).
When mixed in suspension, neither LEC nor LEp cells interacted with the parental L cells. The LEC and LEp cells also did not interact with one another, as was tested in coaggregation assays performed to exclude possible heterotypic interactions between Ep-CAM and E-cadherin (Fig. 2). LEC-C and LEC-Ep cells, when mixed, showed segregation in suspension coaggregation assays (Fig. 2). Although some aggregates contained cells of both types, and the segregation could be described as partial only, the two cell types did show a clear preference for independent aggregation.
Immunoblotting revealed that LEC-C and LEC-Ep cells expressed approximately equal levels of E-cadherin molecules (Fig. 2 B). Since even relatively minor differences in the levels of cadherin expression may affect cell–cell interaction (Steinberg and Takeichi, 1994), we repeated this experiment with five pairs of cell lines obtained from several independent transfections. The degree of cell segregation varied (as estimated by the relative proportion of mixed aggregates formed), and correlated positively with the level of Ep-CAM expression at the surface of double transfectants (not shown).
When mixed as monocellular suspensions and sedimented together, LEC-C and LEC-Ep cells were able to establish connections in the pellet. The resulting large aggregate, formed by randomly distributed cells of both types, was mechanically dispersed into a number of smaller aggregates. After 24 h of culturing these aggregates in suspension, it was found that the LEC-C cells formed the tight core of the aggregates, with LEC-Ep cells forming the external layer. This structure was observed for all aggregates irrespective of their size, with the latter ranging from 100 to more than 1,000 cells (Fig. 3). When either LEC-C or LEC-Ep cells were mixed with the differentially labeled cells of self-type, no cell patterning was observed, indicating that segregation was unrelated to the labeling and other experimental procedures.
The positioning of cells in mixed structures (to the inner or outer layer, respectively) is determined by the relative strength of intercellular connections between the cells of each type (Steinberg and Pool, 1982; Foty et al., 1996). In this respect, the Ep-CAM transfectants of E-cadherin– positive cells interacted with the parental cells as did cells with a relatively decreased strength of cell–cell adhesion. In contrast to what could be expected from the expression of an additional intercellular adhesion molecule, the overall strength of intercellular adhesion among LEC cells seemed to become reduced upon the expression of Ep-CAM. Since Ep-CAM is a relatively weak adhesion molecule compared to E-cadherin (Litvinov et al., 1994a), it is highly suggestive that the E-cadherin–mediated cell–cell adhesions in LEC-Ep cells were negatively affected because of expression of Ep-CAM.
Expression of Ep-CAM Suppresses the E-Cadherin–mediated Cell Aggregation
Ep-CAM–mediated aggregation in suspension is rather slow, with ∼40% of aggregation reached in 120 min for L cells with high levels of Ep-CAM expression (Litvinov et al., 1994a). In contrast, E-cadherin–mediated cell aggregation is relatively fast, as L cell transfectants expressing E-cadherin reach the plateau level of aggregation (50-80%) within 30 min, at which time the aggregation mediated by Ep-CAM is hardly noticeable (not shown). If Ep-CAM is indeed able to negatively affect the E-cadherin–mediated cell–cell adhesion, the aggregation rates of E-cadherin/Ep-CAM transfectants should inversely correlate with the levels of Ep-CAM expression.
To investigate this, LEC cells with inducible Ep-CAM expression (LEC-MEp) were established by introducing the Ep-CAM cDNA under the control of an inducible metallothionein promotor. This promotor can give high levels of expression upon induction, but is leaky, with the construct being expressed to a certain level without induction with heavy metal ions. We selected two individual clones (LEC-MEp.6 and LEC-MEp.2) with different basal levels of Ep-CAM expression. Both the total number of Ep-CAM molecules (Fig. 4,A), as well as Ep-CAM expressed at the cell surface (Fig. 4 B), could be gradually induced in cells of isolated clones by increasing the Cd2+ concentrations in the medium ⩽10 μM. A control cell line was prepared by transfecting a blank pMEP vector into LEC cells (LEC-MC cells).
After being cultured for 24 h at different concentrations of Cd2+ , the cells were detached with EDTA and subjected to the aggregation assay in the presence of Ca2+ to allow both Ep-CAM and E-cadherin adhesion systems to participate in cell aggregation. The results showed that prior culturing in the presence of CdCl2 had no effect on the aggregation rate of the control LEC-MC cells, whereas the aggregation of the Ep-CAM transfectants decreased in direct proportion to the levels of Ep-CAM expressed (Fig. 4 C). The differences in aggregation were reproducible in independent experiments and were highly significant (P < 0.01, Student's t test).
Not only were the aggregation rates for cells expressing Ep-CAM reduced, but also the morphology of the aggregates was different from those formed by the control cells. Thus, most of the LEC-MC cells produced tight, large aggregates, whereas LEC-MEp.6 cells mainly formed small aggregates (Fig. 4 D). This was clearly related to the levels of Ep-CAM expressed by the cells, as the aggregates formed by LEC-MEp.6 cells after induction of Ep-CAM expression were loosely interconnected and even smaller than those formed by the noninduced cells.
Over time (⩽24 h of suspension culture), even the induced LEC-MEp.6 cells were able to form some larger multicellular aggregates. However, the internal structure of these aggregates differed substantially as compared to aggregates of LEC-MC cells, with the latter consisting of tightly interconnected cells, whereas the cells in LEC-MEp.6 aggregates had only sporadic connections (Fig. 5). This argues against the hypothesis that the expression of Ep-CAM simply delayed, rather than inhibited, the E-cadherin–mediated cell aggregation, as the loose interconnection of cells in LEC-Mep.6 aggregates clearly points to a reduction in the number of the E-cadherin–mediated intercellular adhesions.
As was revealed by immunoblotting, the total number of cadherin molecules in transfectants was not affected by Ep-CAM expression or CdCl2 treatment, remaining approximately at the same level in LEC-MC and LEC-MEp cells of both clones. Also, the surface expression of E-cadherin, as measured by flow cytometry, was almost identical (Fig. 6).
Ep-CAM Affects the Morphology of E-cadherin–transfected L Cells
When seeded at low density, both Ep-CAM–positive clones LEC-MEp.2 and LEC-MEp.6 did not differ in morphology from the parental or control LEC-MC cells. However, when the cells were seeded at high density to form a monolayer culture, some morphological differences, which were especially pronounced between LEC-MC and LEC-MEp.6 cells, appeared after 24 h. By that time, >50% of all control cells had acquired an epithelioid morphology, with a clear interconnection of the lateral domains of neighboring cells (Fig. 7). In contrast, in cultures of LEC-Mp.6 cells, when cultured at the same high density in the absence of Cd2+, <10% of all cells had the morphology observed in control cultures (as was estimated from direct counting). When cultured in the presence of 10 μM CdCl2, no cells with epithelial-like morphology were observed in LEC-MEp.6 cultures (Fig. 7). Interactions among the cells seemed to be reduced to sporadic contacts, mainly involving filopodia-like structures.
The presence of Cd2+ in the culture medium had no effect on the morphology of the control LEC-MC cells. The LEC-MEp.2 cells demonstrated a somewhat intermediate morphology when compared to the other two cell lines (not shown).
Expression of Ep-CAM Disturbs the Association of E-cadherin with the Cytoskeleton
The data presented so far suggest that expression of Ep-CAM does not affect the number of E-cadherin molecules in cells but does affect the ability of these molecules to form stable adhesions.
As the differences between LEC-MC and LEC-MEp.6 cell lines were most clearly pronounced, we further investigated the changes of cadherin junctions in LEC-MEP.6 cells upon expression of Ep-CAM. Immunofluorescent staining revealed that in dense cultures of LEC-MC cells, most of the cell surface E-cadherin molecules were present in typical cadherin-type intercellular junctions and were absent from the free domains of the cell membrane (Fig. 8). However, at the basal level of Ep-CAM expression in LEC-MEp.6 cells, only a fraction of the E-cadherin molecules was present in junctions, with some E-cadherin being located on the free domains of the cell membrane. Induction of Ep-CAM for 24 h resulted in redistribution of E-cadherin, which was now found all over the cell surface, and in a substantial reduction of structurally recognizable E-cadherin junctions (Fig. 8). As was previously shown (Fig. 6) by flow cytometry analysis and immunoblotting, both the total number of E-cadherin molecules and the cell surface fraction of E-cadherin were not changed upon induction of Ep-CAM, suggesting that the latter did not cause either internalization or enhanced degradation of the E-cadherin molecules, or suppression of their transportation to the cell surface. Rather, the observed changes in subcellular distribution of E-cadherin suggest some disturbance in the association of the E-cadherin molecules with the cytoskeleton, an association required for the E-cadherin junction formation (Nagafuchi and Takeichi, 1988). Indeed, the cytoskeleton-anchored (Triton X-100– insoluble) fraction of cadherins was decreased upon the induction of Ep-CAM in LEC-MEp.6 cells, as can be seen in immunoblotting for E-cadherin in detergent-extracted cells (Fig. 9 A).
Interaction of E-cadherin molecules with the cytoskeleton is mediated by β- and α-catenins (Ozawa and Kemler, 1992; Jou et al., 1995), which are absent in L cells but appear upon the transfection of E-cadherin (Nagafuchi et al., 1994). We compared the total content and detergent solubility of both catenins in LEC-MC and LEC-MEp.6 cells. Approximately similar levels of β-catenin were observed in control cells, and in LEC-MEp.6 cells regardless of the level of Ep-CAM expression in the latter. The detergent-insoluble fraction of β-catenin was, however, reduced in the induced LEC-MEp.6 cells.
In contrast to β-catenin, a reduction in both detergent-insoluble and total cellular fractions of α-catenin molecules was observed in induced LEC-MEp.6 cells (Fig. 9 A).
Apparently, the expression of Ep-CAM leads to a decrease in the cytoskeleton-anchored fraction of E-cadherin molecules. To investigate whether some catenins were still associated with the detergent-soluble fraction of the E-cadherin molecules, the induced and noninduced LEC-MEp.6 cells were extracted with 0.5% Triton X-100, and immunoprecipitations were performed from the extracts using antibodies specific to E-cadherin or β-catenin (Fig. 9,B). Upon the induction of Ep-CAM in LEC-MEp.6 cells, there was a clear increase in the amount of β-catenin coprecipitating with the detergent-soluble E-cadherin molecules (Fig. 9,B). The reverse precipitations with anti–β-catenin mAb produced similar results, with increased coprecipitation of E-cadherin upon induction of Ep-CAM. However, when the immunoprecipitates of soluble E-cadherin were probed with anti–α-catenin antibody, no increased presence of α-catenin associated with this fraction of E-cadherin molecules was observed. The latter result was consistent with the observation of the decrease in total α-catenin content in the cells. Additionally, a certain increase in α-catenin molecules coprecipitating with β-catenin was observed upon the induction of Ep-CAM (Fig. 9 B).
These results show that expression of Ep-CAM in cells results in accumulation of non–cytoskeleton-anchored E-cadherin/β-catenin complexes and in a reduction of the α-catenin molecules available for the formation of E-cadherin junctions. Since the observations were made after induction of Ep-CAM for 24 h, these soluble E-cadherin/ β-catenin complexes may originate from either (a) dissociation of the preexisting cadherin junctional complexes, or (b) may represent newly produced E-cadherin molecules that had formed a complex with β-catenin, but for which further anchorage to the cytoskeleton was not possible because of the nonavailability of α-catenin. Experiments with short-time induction of Ep-CAM in LEC-Mep.6 cells (2,4, and 6 h) provided no clear support to either of these options, since the levels of cellular Ep-CAM after 6 h of induction were only slightly higher than in uninduced cells, and were substantially lower than in cells induced for 24 h. After the short-time induction, no substantial changes in either solubility of cadherins or in the level of cellular α-catenin were observed (data not shown).
Ep-CAM Affects the Cadherin-mediated Junctions in Epithelial Cells
To investigate whether the expression of Ep-CAM in human epithelial cells affects cadherin junctions in a way similar to what has been observed for E-cadherin transfectants of L cells, we have chosen the immortalized mammary epithelial cell line HBL-100. This cell line expresses low levels of E- and P-cadherin, and relatively high levels of N-cadherin (as was tested in immunoblotting and flow cytometry with mAbs specific to each cadherin), and, in contrast to all other cell lines of epithelial origin tested, contains no endogenous Ep-CAM. Transfectants of a clonal cell line derived from HBL-100 origin, HCA, were obtained using episomal vectors that do not integrate into the genome and support themselves in transfected cells at high copy numbers. The results obtained with HCA/C (blank pCEP4 vector transfectants) and HCA/CEp (pCEP4/Ep-CAM transfectants) cells in coaggregation assays were similar to those obtained with LEC cell transfectants, with the HCA/CEp segregating partially from the parental cells. In mixed two-cell type aggregates, the cell patterning was similar to that observed in the LEC cell model (not shown).
Transfection with Ep-CAM affected the aggregation from monocellular suspensions of HCA cells. After 30 min of aggregation in the presence of Ca2+ , 50 ± 4% aggregation was reached by HCA/C cells, and only 24 ± 3% by HCA/CEp cells. However, after 90 min the degree of aggregation for both cell lines was approximately equal, reaching 55%. This may be due to the formation of Ep-CAM–mediated cell–cell interconnections, since after 90 min in the absence of Ca2+ (allowing mainly the Ep-CAM–mediated cell aggregation), the degree of aggregation for HCA/CEp cells was approximately the same, 47 ± 4% (only 8.3% for the control HCA/C cells).
Despite the fact that after 90 min no substantial differences in aggregation extent were observed between the control and the Ep-CAM transfectants, the structure of the aggregates formed by these two cell lines differed substantially. As can be seen on cross sections, the HCA/C aggregates were composed of tightly interconnected cells, in contrast to HCA/CEp aggregates, which were formed by loosely interconnected cells (Fig. 10). In the internal layers of the HCA/C aggregates, the membranes of the cells were interconnected by multiple adherence junctions (Fig. 10), whereas in HCA/CEp cell aggregates the cell–cell contacts were rare and often did not contain morphologically distinguishable adherens junctions. The membranes of neighboring cells in these aggregates were mainly not in close proximity. In these intercellular spaces, microvilli were often observed (Fig. 10), which were present in HCA/C aggregates at the outer surface of the external layer of cells only.
To further investigate the effect of Ep-CAM on cell–cell contacts of HCA cells, the cells were also transfected with Ep-CAM cDNA under the control of the metallothionein promotor (Wt cells; Wt indicates wild-type Ep-CAM molecule). This regulated the levels of Ep-CAM expression from ∼50% to 350% of the levels observed in HCA/CEp transfectants (estimated by flow cytometry on living cells, data not shown).
At the basal level of Ep-CAM expression, the morphology of Ep-CAM transfectants in cell culture was quite similar to the control cells transfected with the blank vector. However, induction of high levels of Ep-CAM resulted in loss of the polygonal morphology and in a more scattered phenotype, although the cells still remained attached to one another (Fig. 11). This change in morphology was observed for the entire cell population only at the highest (50 μM) concentration of Cd2+ tested, although some of these changes could be observed for some cells or groups of cells at lower concentrations of Cd2+ (2–5 μM), with changes increasing proportionally to the Ep-CAM levels. The effect was reversible, and within 3 d of culture in the absence of the Cd2+ in the medium, the cells regained their original morphology, consistent with the decrease of Ep-CAM levels back to that of noninduced cells (flow cytometry data, not shown).
Induction of Ep-CAM in cells did not affect either the total level of cell cadherins (as was detected with immunoblotting with pancadherin antibody, (see Fig. 13), or the surface expression of N-cadherin (as was detected with flow cytometry, not shown). The surface expression of E- and P-cadherins in HCA cells was too low to be measured by flow cytometry. However, upon induction of Ep-CAM, the subcellular distribution of cadherins was changed: the typical junctional structures, including cadherins that were present in HCA cells (or control transfectants), were absent in induced (Fig. 11) but not in noninduced Ep-CAM transfectants. The disturbance of cadherin's subcellular localization suggested that the effect of Ep-CAM was similar to that observed for LEC cells. Indeed, the detergent solubility of the cadherins in HCA cells was increased after 24 h of Ep-CAM (see Fig. 13). Induction of Ep-CAM had no effect on total level of cellular β-catenin, but it negatively affected the level of total α-catenin (Fig. 12); the detergent-insoluble fraction for both molecules was reduced in relation to the expression of Ep-CAM (Fig. 12). All observations are based on 24-h induction of Ep-CAM.
The Cytoplasmic Domain of Ep-CAM Molecule Is Required to Affect the Cadherin-mediated Junctions
In both L and HCA cell models, we observed a decrease in cadherin-mediated adhesion and a reduction in cellular α-catenin upon the expression of Ep-CAM. Such changes in adherens junctions suggest that Ep-CAM induced certain intracellular effects, and we therefore investigated whether an Ep-CAM mutant lacking the cytoplasmic domain would be able to negatively affect cadherin-mediated adhesions. This mutated form of Ep-CAM (Mu1) was generated as shown in Fig. 13. When transfected into L or HCA cells, Mu1 was expressed at the cell surface, similar to the wild-type Ep-CAM, and was then capable of mediating weak homotypic binding, but was not able to form stable adhesions (investigated in more detail in Balzar M., H.A.M. Bakker, I.H. Briaire, G.J. Fleuren, S.O. Warnaar, and S.V. Litvinov, manuscript submitted for publication). No effects produced by the wild-type Ep-CAM were observed in LEC cells transfected with Mu1 with respect to cell morphology, cell aggregation, and the number and extractability of the cadherin and catenin molecules (not shown). However, the transfections of Mu1 construct into LEC cells never gave rise to a clonal cell line with the levels of Mu1 expression comparable to the wild-type Ep-CAM expression in LEC-MEp.6 cells, although the expression levels comparable to LEC-MEp.2 cells were achieved. This makes strict interpretation of the effects of Mu1 on E-cadherin in LEC cells difficult.
In contrast, when transfected into HCA cells under the control of an inducible promotor, Mu1 expression could reach levels similar to those of wild-type Ep-CAM expression in these cells (Fig. 13). Nevertheless, no effects on cell morphology nor on the internal structure of aggregates formed by the transfectants were observed upon expression of Mu1 in HCA cells, as compared to nontransfected cells or cells transfected with the blank pMEP4 vector (HMC cells). Expression of Mu1 did not affect the total level of cadherins in HCA cells or their solubility, as did the wild-type molecule expressed at the same level (Fig. 13).
Ca2+-independent cell–cell adhesion molecules are present in epithelial and carcinoma cells along with the classic cadherins, and, although the data is scarce, it suggests a possible influence of the noncadherin cell–cell adhesion molecules on cadherin mediated cell–cell adhesion. Thus, the expression of noncadherin adhesion molecules, such as protein Zero (Doyle et al., 1995) or N-CAM (Michalides et al., 1994), may cause an augmentation of the cadherin-mediated adhesions.
In contrast to these observations, we demonstrate here that expression of Ep-CAM in cells interconnected by classic cadherins results in abrogation of the cadherin-mediated junctions in a direct correlation to the levels of Ep-CAM expressed. Despite the fact that Ep-CAM, being an adhesion molecule, provides additional intercellular connections to the cells, its expression leads to an overall decrease in the strength of intercellular interactions for cells interconnected by the classic E- and N-cadherins, as shown in this study. In modulating cadherin adhesions, Ep-CAM acted to some extent as an antiadhesion molecule, if the overall strength of the intercellular adhesion in transfected cells is considered. This effect, however, differs from the previously demonstrated antiadhesion effects of N-CAM– bearing polysialic acid (Rutishauser, 1992; Yang et al., 1992), since polysialylated N-CAM does not bear an adhesion function, and its action is more similar to the antiadhesion effects of some other sialoglycoproteins, such as episialin (Wesseling et al., 1996) or CD43 (Ardman et al., 1992), which inhibit intercellular adhesion by not allowing the membranes of interacting cells to reach a proximity necessary to form adhesions. Ep-CAM not only weakens/ abrogates the cadherin-mediated adhesions, but replaces them with Ep-CAM adhesions becoming predominant (as was seen with HCA/CEp cell aggregation) to some extent.
The observed effects more likely originate from a possible existing coordination between the two adhesion systems rather than a simple antiadhesion effect of Ep-CAM. In epithelial cells a certain coordination between different adhesion systems was demonstrated for keratinocytes, where an increase in E-cadherin induces downregulation of β1-integrin (Hodivala and Watt, 1994). A similar coordination may exist between other systems involved in intercellular adhesion, such as Ep-CAM and cadherins, with E-cadherin in particular.
The molecular mechanism behind the observed effect is not quite clear. Expression of Ep-CAM did not affect the expression nor the number of the cadherin molecules but did affect the formation of the cytoplasmic junctional complex of the cadherin molecules. In both murine fibroblast and in human epithelial cells tested, a reduction in the total level of the cellular α-catenin and an increased detergent solubility of cadherins were seen upon the expression of Ep-CAM. Cadherins that lost their connection to the cytoskeleton were mainly present as complexes with β-catenin. This argues against involvement of β-catenin in the modulating effect of Ep-CAM on cadherins, in contrast to the role suggested for β-catenin in regulation of cadherin junctions (for review see Gumbiner et al., 1993). However, v-src expression may shift cell–cell adhesion mediated by cadherins from a strong to a weak state without involvement of β-catenin (Takeda et al., 1995), which suggests the existence of an alternative mechanism for regulating the cadherin junctions. Our observations indicate that the modulation of cadherin adhesions by Ep-CAM involves α-catenin, since the observed dissociation of cadherin junctions would require dissociation of α-catenin from its connections within the complex, anchoring E-cadherin molecule to the actin cytoskeleton. α-Catenin was shown to be a link between the E-cadherin–β-catenin complex (Nagafuchi et al., 1991; Hirano et al., 1992; Hinck et al., 1994) and actin filaments, interacting with β-catenin on one side and with α-actinin (Knudsen et al., 1995) and probably actin filaments on the other side (Rimm et al., 1995).
We have recently demonstrated that Ep-CAM interacts with the actin based cytoskeleton via α-actinin without involvement of α- or β-catenins (Balzar et al., manuscript submitted for publication). A mutant Ep-CAM lacking its cytoplasmic domain did not affect the cadherin junctions. Therefore, the connection between Ep-CAM and α-actinin seems to be related to the negative effect on cadherin junctions. It is conceivable that overexpression of Ep-CAM may result in redistribution towards Ep-CAM of α-actinin molecules involved in the formation of cadherin junctions. It is not clear, however, to what extent or how the availability of α-actinin regulates the cellular levels of α-catenin, which become reduced upon induction of Ep-CAM. An alternative way by which E-cadherin junctions may be affected is through active signaling via the cytoplasmic domain of Ep-CAM, leading to downregulation of α-catenin since the connection of Ep-CAM to cytoskeleton via α-actinin is very similar to that of ICAM-1 (Carpen et al., 1992). The signal transduction via ICAM-1, resulting in phosphorylation of cortactin, was recently demonstrated (Durieu-Trautmann et al., 1994).
E-cadherin is involved in regulation of epithelial cell polarity (McNeil et al., 1990; Näthke et al., 1993), in morphogenesis of epithelial tissues (Wheelock and Jensen, 1992; Larue et al., 1996), and in regulation of cell proliferation and programmed cell death in epithelia (Hermiston and Gordon, 1995; Hermiston et al., 1996; Takahashi and Suzuki, 1996; Wilding et al., 1996; Zhu and Watt, 1996). In carcinomas, this molecule clearly acts as a suppressor of carcinoma cell invasion (Behrens et al., 1989; Chen and Öbrink, 1991; Frixen et al., 1991; Vlemnickx et al., 1991). The cadherin-modulating properties of Ep-CAM, as reported here, may play a role in epithelial cell proliferation, and possibly in tumor progression. This hypothesis is supported by the observed association of Ep-CAM expression with actively proliferating or premalignant cell populations, respectively, such as proliferating cells in hair follicles (Tsubura et al., 1992), cells of preneoplastic lesions in the uteral cervix (Litvinov et al., 1996), colonic polyps (Salem et al., 1993), or in intestinal metaplasia in gastric mucosa (this article). The previously reported association of Ep-CAM expression with a poor prognosis in breast carcinoma patients (Tandon et al., 1990), and our recent similar observations in patients with head and neck cancer (Takes et al., 1997) as well as the increase in Ep-CAM expression from low to high grade bladder carcinomas (Zorzos et al., 1995), indicate a possible role for Ep-CAM in tumor progression.
The data presented in this paper demonstrate an interaction between the Ep-CAM and classic cadherin-based adhesion systems and suggest a cross-talk between adhesion systems as a mechanism through which the strength of the intercellular adhesion among epithelial cells may be regulated.
This study was supported by a grant from the Dutch Cancer Foundation (RUL 95-1107), and a research grant from Centocor Inc. (Malvern, PA).
Abbreviations used in this paper
Ep-CAM
epithelial cell adhesion molecule
murine E-cadherin-transfected L cells
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Examples of Ep-CAM expression by some cells within the E-cadherin–positive cell population. (A) Heterogeneous expression of Ep-CAM in a basal cell carcinoma, as detected by immunofluorescent staining with mAb 323/A3 to Ep-CAM (green fluorescence); the red fluorescence indicates the expression of E-cadherin (mAb HECD-1). (B) The de novo expression of Ep-CAM in gastric mucosa in relation to the development of intestinal metaplasia; immunohistochemical staining with mAb 323/A3. Note the bordering Ep-CAM–positive and –negative cells. Bars, 30 μM.
Cell segregation directed by Ep-CAM. L cells were transfected with cDNA for E-cadherin (LEC) or Ep-CAM (LEp), and the E-cadherin transfectants were additionally supertransfected with either Ep-CAM cDNA (LEC-Ep) or the blank vector (LEC-C). (A) Pairs of transfected cells were tested in coaggregation assays: dispersed cells of two types (Type1 + Type2), each labeled with a different fluorescent dye, were mixed at equal concentrations. After 2 h of culturing in suspension, cell aggregates consisting of >10 cells were analyzed for the presence of cells of each type. The data is presented as percentage of aggregates (y-axis) containing the respective percentage of the Type 2 cells (x-axis). (B) Expression of E-cadherin and Ep-CAM in the transfectants, as determined by immunoblotting in total cell lysates using antibodies to E-cadherin (36) and to human Ep-CAM (323/A3), respectively.
Segregation of Ep-CAM–positive LEC cell transfectants from the parental cells in multicellular aggregates. LEC-C and LEC-Ep cells, labeled with fluorescent dyes PKH-26 and PKH-2, respectively, were mixed at a 1:1 ratio, sedimented, and allowed to form an aggregate. This aggregate, in which both cell types were represented in a random pattern, was mechanically dispersed, and the smaller aggregates obtained were further cultured in suspension for 24 h, fixed, and analyzed. The micrographs present optical cross sections at the equatorial area of the aggregates after 24 h, as seen with a confocal microscope. The artificial colors were assigned to the cells depending on the color of the fluorochrome and the cell type: LEC-C (red); LEC-Ep (white) cells. The figures show similar cell patterning in different size aggregates in the range of <100 to ∼1,000 cells. A dark area in the middle of some aggregates is an optically nontransparent zone.
Effect of increasing expression of Ep-CAM on cell–cell interactions in L cell E-cadherin transfectants (LEC). LEC cells were supertransfected with Ep-CAM cDNA under the control of the metallothionein promotor (clones LEC-MEp.2 and LEC-MEp.6), or with blank vector (LEC-MC). Induction of Ep-CAM expression with CdCl2 for 24 h resulted in an increased total Ep-CAM (A, immunoblot of total cell lysates) and in an increased presence of Ep-CAM molecules at the cell surface (B, flow cytometry with anti–Ep-CAM F(ab)-FLUOS conjugate). Cells detached with TC treatment were allowed to aggregate in suspension for 30 min in the presence of Ca2+, and the degree of cell aggregation was determined (C). The statistical significance of the observed differences in aggregation rates was determined using the Student's t-test (p). Where indicated, cells were cultured for 24 h before the assay in the presence of CdCl2 in culture medium. (D) The morphology of aggregates formed in 30 min by LEC-MC and LEC-MEp.6 cells (the latter noninduced and induced with 10 μM CdCl2).
The internal structure of the large aggregates formed by LEC-MC and LEC-MEp.6 cells in 24 h of suspension culture. The expression of Ep-CAM in LEC-MEp.6 cells was induced with 10 μM CdCl2 for 24 h before the aggregation assay (+). The aggregates were fixed with 2% paraformaldehyde/1.25% glutaraldehyde, embedded into Epon, and ultrathin sectioned. Sections were analyzed by reflection contrast microscopy. Bar, 25 μm.
Expression of E-cadherin in blank vector (−MC) and Ep-CAM transfectants (clones −MEp.2 and −MEp.6) of LEC cells. The cells were cultured for 24 h in the presence or absence of 10 μM CdCl2 in culture medium. Cells were either (A) detached from the substrate with EDTA and analyzed in flow cytometry after staining with mAb ECCD-2 to murine E-cadherin, or (B) lysed with 1% SDS, and E-cadherin was detected in total cell lysates (5 μg protein/sample) by immunoblotting with mAb 36.
The morphology of LEC-MC and LEC-MEp.6 cells (the latter shown after induction with 10 μM CdCl2, marked by +). Note the groups of the epithelial-like cells interconnected along their lateral domains in the culture of LEC-MC cells, which are absent in the LEC-MEp.6 cell culture.
E-cadherin and catenins in LEC cells and the derived Ep-CAM transfectants (MEp-6). (A) Cells were cultured in the absence (−) or presence (+) of CdCl2 for 24 h, and lysed in 1% SDS either directly (Total lysate), or after extraction with 0.5% Triton X-100 (Insoluble fraction). Samples of cell lysates, equalized by protein content, were analyzed by immunoblotting with antibodies to E-cadherin (mAb 36), β-catenin (mAb 14), or α-catenin (mAb 7A4). 5× more protein was taken for the extracted cell preparations. (B) LEC-MEp.6 cells cultured in the absence (−) or presence (+) of CdCl2 were lysed in buffer with 0.5% Triton X-100, and immunoprecipitations were performed from the soluble fraction. The immunoprecipitates were analyzed in Western blotting with antibodies to E-cadherin (E) or catenins (α- and β-, respectively). A rabbit polyclonal serum was used for α-catenin detection. The precipitations were performed from an excess of the lysate; therefore, the total amount of an antigen precipitated by an antibody remains equal.
Subcellular localization of E-cadherin in LEC-MC cells and in LEC-MEp.6 cells. Ep-CAM in LEC-MEp.6 was induced by 10 mM CdCl2 for 24 h before cell fixation. Immunofluorescent staining of methanol-fixed cells was performed as described in Materials and Methods using mAb ECCD-2 to murine E-cadherin. Bar, 10 μm.
Ultrastructure of the aggregates formed by HCA/C and HCA/CEp cells. Reflection contrast micrographs of cross sections through the aggregates formed in 90 min by each cell type reveals a tight organization of the HCA/C aggregates and loosely interconnected cells forming the HCA/CEp aggregates. (A) Electron microscopy on the preparations shows the abundant presence of the adherens junctions between HCA/C cells (arrows). In contrast, microvilli (B, arrow) were found at the intercellular space between the cells of internal layers of HCA/CEp cells. In HCA/C cells, microvilli were present almost exclusively at the apical membrane of the outer layer of cells in the aggregate and not on the surface of the cells from internal layers. Bars: (left) 10 μm; (right) 0.25 μm.
Changes in subcellular distribution of cadherins in HCA cells upon expression of Ep-CAM. HCA cells transfected with inducible pMEp4 vector, either blank (HMC) or containing the Ep-CAM cDNA (Wt), were induced with 50 μM CdCl2 24 h before fixation, fixed, and stained with anti-pan cadherin mAb CH-19 (reacts mainly with N-cadherin, the major cadherin of HCA cells). Note the redistribution of cadherins from adherens junctions to free domains of cell membrane in Wt Ep-CAM transfectants, and a general shift of the cell phenotype to a more scattered one. Bar, 5 μm.
Detergent extractability of catenins in HCA cells and Wt Ep-CAM transfectants. Immunoblotting was performed on total cell lysates and on lysates of cells preextracted with 0.5% Triton X-100. The + indicates induction of cells with 50 μM CdCl2 for 24 h before lysis.
The role of the cytoplasmic domain in the effect of Ep-CAM on cadherins. (A) The structural maps of the wild-type (wild-type) and mutant (Mu1) Ep-CAM molecules. The leader peptide (L), EGF-like domains (EGF-I, EGF-II) cysteine-poor region (CPR), the transmembrane domain (TM), and cytoplasmic domain (CYT) are marked. The cytoplasmic domain is deleted in the Mu1 molecule. (B) HCA cells were transfected with either blank vector (HMC), or the wild-type Ep-CAM (Wt), or Mu1, under the control of a metallothionein promotor. Where indicated (+Cd), the cells were cultured in the presence of 50 μm CdCl2 for 24h before lysis. Aliquots of total cell lysates equalized by protein were probed in immunoblotting using the Ep-CAM–specific mAB. Content of N-cadherin in whole cell lysates and after the extraction of cells with 0.5% Triton X-100 was analyzed with anti-pan cadherin mAb CH-19. Note that expression of Wt Ep-CAM, but not of an approximately equal level of Mu1, affects the detergent solubility of cadherins.
KSA Antigen Ep-CAM Mediates Cell–Cell Adhesion of Pancreatic Epithelial Cells: Morphoregulatory Roles in Pancreatic Islet Development
Ep-CAM: a human epithelial antigen is a homophilic cell-cell adhesion molecule.
Neural Cell Adhesion Molecule (N-CAM) Is Required for Cell Type Segregation and Normal Ultrastructure in Pancreatic Islets
About JCB


2013 (Freshman): Redshirted…Atlantic Sun Conference All-Academic Scholar.
High School: Played at the Christian Academy of Knoxville … District Champions Baseball in three seasons from 2010-12 and Regional Champions Baseball in 2012 … All-State in two seasons 2011-12, All-Region for two seasons 2011-12, All-District for three seasons 2010-12 and captain for 2012 seasons…Team MVP for two seasons from 2011-12…Four-year letterwinner in baseball and basketball… 1,000-point scorer in basketball, all-time leading rebounder in school history and 2nd all-time leading scorer…National Honor Society Member, Mu Alpha Theta Member and French and Business Club Member…National Distinguished Christian Student Award, CAK Leadership Award, Jonny Mauer Award and FCA Heart of a Champion Award…Tennessee All Two-Way Team 2011 and District Pitcher of the Year 2011…Warrior Award, AGAPE Award (Highest honor at CAK) and Boys and Girls Club Student Athlete Award.
Personal: Son of Greg and Jill Keck…One brother (Dylan) and two sisters (Marie-Michele and Yollande)…Father played basketball at King College and Maryville College…Uncle Jeff Browning played at Middle Tennessee as a pitcher…Uncle Paul Cochran played at Walters State as a catcher.


Almost everything I need to know I learned in kindergarten, to paraphrase Robert Fulghum. Kindergarten was when I learned to share, play fair, and to wonder unapologetically. But there was one lesson I didn't learn until I was 18 years old and starting a degree in anthropology here at Portland State – a lesson that, three years later, I'm still upset I didn't learn earlier.
The lesson was this: Race is not a biological reality.
Today, we're lucky enough to not only have an impressive archaeological record containing fossils of our ancestors reaching back millions of years, but also to have the capability to sequence entire human genomes. These databases of knowledge support the model of human evolution beginning with humans in Africa 200,000 years ago and migrating into Europe around 60,000 years ago. Change in skin pigmentation was a result of the increasingly limited UV radiation those migrants were exposed to, and was merely a surface-level change – both phenotypically and genotypically. Because it was an adaptation to different environments, loss of pigmentation occurred at varying levels.
This history of early man means two big things for modern man: 1) The range in human skin color we see today only started evolving in the last fourth of our history! 2) There's no biological way to validate racial categories, because human variation exists on a spectrum, with no places to draw distinct divisions between populations.
Race is very much a social reality. In no way do I intend to take away from its cultural significance. But it is a much too common misconception that there is more than a social justification for dividing humanity into discrete units. We need to celebrate the remarkable phenotypic and cultural diversity among us. But, now more than ever, we should also be taking Robert Fulghum's advice to "hold hands and stick together" – by recognizing and teaching that we're more alike than different.


text:
Chile: New Mexico's Ambassador Crop
As you know, the Bogle ranch is located in New Mexico. While we're primarily cattle ranchers, we are big fans of other items raised and grown in our state, including one of the most popular items - chile. Did you know they're the official state vegetable of New Mexico? Their massive increase in popularity over the years has led to a chile shortage, however, putting pressure on producers.
Chile farming requires hand picking each chile, which adds an additional labor expense, and has partially resulted in a decrease in chile production in the past year. And many consumers are forced to import chile from other countries to meet the demand. Even despite this challenge, however, chile remains New Mexico's ambassador crop to the rest of the world.
Albuquerque Business First shared that New Mexico has exported more chile outside of the United States this year than in the previous year, as reported by the state department of agriculture. The increase was worth over $500,000 in value, showing that the stateside chile shortage isn't having a negative effect on international demand.
While we aren't chile farmers at Bogle Ltd, we can still appreciate the effect that this ambassador crop has on spreading the good word about quality products coming from New Mexico. We do think we're well positioned in the market with our cattle and hay pursuits. Dreaming New Mexico states that after dairy, the beef industry brings in the highest amount of cash receipts for the state.
As supporters of the rural, farming lifestyle, we're happy to be part of an important industry in our state. When you think New Mexico, think chiles - and Bogle Ltd!

redpajama_set_name: RedPajamaCommonCrawl


text:
Scientists ID Clue in Effort to Contain Herpes Virus
THURSDAY, Nov. 7, 2019 (HealthDay News) -- An inflammatory protein may play a part against the spread of sexually transmitted genital herpes virus in the nervous system, a new study says.
The findings could help lead to improved treatment of herpes, according to the researchers at the University of Arizona College of Medicine-Phoenix.
The protein, called IL-36y, was identified 20 years ago. Since then, research has shown that it plays an important role in chronic inflammatory diseases, but only recently have investigators started to examine its role in infectious diseases such as herpes.
After initial vaginal infection with genital herpes simplex virus 2 (HSV-2), the virus spreads through a woman's nervous system and goes into "hiding." It remains in hiding for a lifetime and periodically reactivates and causes lesions, the researchers explained.
"We were the first group to detect IL-36y in the female reproductive tract and have been extending this work to evaluate the extent that this molecule can protect against, or contribute to, genital infection," study senior author Melissa Herbst-Kralovetz said in a university news release. She's an associate professor of basic medical sciences.
Study leader Jameson Gardner said the researchers "really were excited to see that after intravaginal HSV-2 infection, the virus was able to spread systemically throughout the nervous system and to the brain in those lacking IL-36y."
He said the control group, which had the gene intact, was significantly more protected against HSV-2 spread in the nervous system.
Gardner is a graduate research student in the lab of Herbst-Kralovetz.
"We've shown that IL-36y appears to function in a previously uncharacterized manner to protect against virus neuroinvasion [an infection that affects the nervous system]," Gardner said in the release.
However, it's not yet known how IL-36y does this, the study authors said.
Despite the use of antiviral medications to treat genital herpes, more than 260 million women worldwide have the lifelong infection that can be transmitted to others, according to the authors.
Researchers have worked for decades to develop a vaccine to protect against genital herpes, but none has succeeded so far, Gardner said.
"Our understanding of how IL-36y functions can aid in the development of novel therapeutics to protect against -- and hopefully decrease -- the burden of [sexually transmitted infections] around the world," he added.
The findings are in the October issue of the Journal of Immunology.
The American College of Obstetricians and Gynecologists has more on genital herpes.
SOURCE: University of Arizona College of Medicine-Phoenix, news release, Oct. 28, 2019
As U.S. Measles Outbreaks Spread, Why Does 'Anti-Vax' Movement Persist?
Attention, Seniors: Drink More Water and Head Off Disease


text:
Q: Check if user input matches answer to sum of 2 random Numbers
Hi Im trying to generate 2 random numbers, add them together and have the user input match the answer, it works for lower numbers in the range(0-50) but not for higher numbers. Im relatively new to programming so any help would be great. Thanks in advance
static int randomNum(){
Random numR = new Random();
int randomNum = numR.nextInt(51);

return randomNum;

}
public static void main(String[] args) {
    // TODO code application logic here

    Scanner sc = new Scanner(System.in);
    int num1 =randomNum();
    int num2 =randomNum();
    int answer =num1+num2;

    System.out.println("What is "+num1+"+"+num2+" equal to?");
    if (sc.hasNextInt(answer))
        System.out.println("Correct");
    else
        System.out.println("Wrong");

}
}

A: hasNextInt(someInt), which is what you're invoking, treats the argument as the radix, totally not what you want. Even if it did what you wanted, it wouldn't consume the input - it's hasNextInt, not consumeNextInt.
You want to call sc.nextInt(), and then compare what that returned with answer.

redpajama_set_name: RedPajamaStackExchange


text:
How Margaret's gift will change the lives of children
November 26, 2020 BY Rita Mu CHILDFUND AUSTRALIA CATEGORY Supporter Stories
Supporter Spotlight: Russell Spencer and Ursula Groves– champions for children's education in Vietnam
CATEGORY Supporter Stories
Meet a grandmother who trekked across Tasmania's Tarkine for children in need
Make-up artist brings beauty and joy to children in need
Leave a lasting legacy. You can leave a gift in your Will and give children the support they need.
Margaret Moloney was brilliant at many things, most notably tennis, Irish dancing and teaching. She was also a loyal footy fan, attending countless games to cheer on her beloved Tigers, had a great sense of fashion and a unique ability to see the potential beauty in things that had been discarded.
But if there are a few things that stand out more than anything else about Margaret, it is her kindness and willingness to help those less fortunate than herself.
Margaret, or "Margie" as she was affectionately remembered by her family and friends, was always conscious of the needs of others.
She carried this quality throughout her life, right until her last years when she left a gift in her Will to ChildFund Australia to help children in need around the world.
Lessons of love
The youngest of four girls, Margaret stood out from her sisters Patricia, Maureen and Bernadette; she was creative, followed her passions, and had enormous empathy for people.
Margaret, with her mother Merna, would regularly visit her uncle Jack when he had lost his wife and moved into a nursing home. After Jack passed away, Margaret insisted she and her mother continue visiting the home, taking afternoon tea for the residents to enjoy. "It was so typical of her thoughtfulness and kindness," Patricia says.
She was a single mother and dedicated her life to making sure her daughter, Katherine (pictured above with Margaret), who had a disability, had the tools and support she needed to reach her full potential.
"Margie was completely selfless about Katherine," Bernadette says. "She catered for Katherine's needs and created opportunities for her to achieve. Katherine drove a car and lived independently, thanks to her beautiful mother."
"She taught us many things and we hope that in her passing that we are vigilant and responsive to others less fortunate."
Bernadette, margaret's sister
Margaret trained as a teacher and taught in several primary schools. She wanted to devote most of her time to Katherine and eventually resigned from teaching. She studied a master's degree in family therapy and became a parent advocate for students with disabilities.
In 2001, Margaret's contribution to Australian society was officially recognised when she was awarded the Centenary Medal for her service to "the support of students at extreme education risk and their families".
When Katherine passed away at the age of 31, Margaret channelled her grief into writing a film about the challenges and achievements of a middle-aged single mother who had lost her beloved daughter.
Margaret devoted most of her life to looking after her daughter Katherine (pictured above in pink), and supporting children in need overseas.
Margaret spent the last four years of her life writing the 116-page film script, titled Lessons of Love.
For Margaret, it was a "labour of love", says Patricia. "The document is now in good hands and, hopefully in time, it will be given the exposure and credit it deserves."
Helping children and families in need
Among helping children with a disability and families navigate the challenges of life, Margaret's generosity and kindness shone through her support for ChildFund Australia's work. Over 27 years, Margaret sponsored disadvantaged children around the world through ChildFund, helping to ensure these children had access to healthcare, an education and opportunities to reach their potential.
Today, Margaret's gift in her Will is helping to change the lives of children and their families in Papua New Guinea, Timor-Leste and Cambodia.
In Papua New Guinea, two health facilities in remote and rural areas – where the majority of the country's population live – are being built with the help of the Margaret Moloney Estate. The health facilities will ensure mothers and their children in remote communities no longer need to travel up to two hours for medical care and services. Margaret's gift will also help provide equipment for the health facilities, and train health workers on caring for newborns, vaccine management, paediatric tuberculosis, and HIV/AIDS.
In Cambodia, two early childhood centres are being built to ensure young children living in poverty can learn and play, and transition smoothly to primary school. The centres will include fully-equipped, child-friendly classrooms and libraries with modern technology. They will also include clean water and sanitation facilities to help keep children safe and healthy.
Margaret (far right) with (from left) her sisters Maureen, Patricia, her mother Merna, and her sister Bernadette.
In Timor-Leste, Margaret's gift is helping to train teachers on creative and inclusive teaching methods to improve the quality of learning – particularly for children with disability – in 12 primary schools. Teachers will also be trained on how to identify and refer children with disability to health services.
For many disadvantaged children in Papua New Guinea, Timor-Leste and Cambodia, Margaret leaves a legacy that will transform their lives.
Closer to home, Margaret leaves behind some important lessons of love.
Her generosity and kindness inspired her family and friends. "Margie had a rare gift for making and sustaining friendships, and enjoyed a very large and diverse group of friends," Maureen says.
"Every single one I spoke with said she was one very special lady."
Margaret, says Patricia, enriched the lives of her family and friends. "She made us better people."
She was her own person, creative, supportive, kind and generous, loyal and courageous. Above all, though, she was empathetic, says Bernadette.
Margaret's gift in her will to ChildFund will help to change the lives of disadvantaged children around the world.
REGION Asia Pacific COUNTRY Australia


The lady was last seen in Tamil with Udhayanidhi Stalin's Gethu and is awaiting her next release Theri with Vijay soon. Meanwhile, she is also working in Shankar directorial 2.0 with superstar Rajinikanth.
Last weekend being the Easter weekend, Amy decided to get back home and surprise her folks. She flew to the UK to surprise her family and friends and spend the weekend with them on this beautiful, special holiday.
After wrapping her 2.0 test shoot in Bolivia, she dashed off to Bangkok for her brand shoot and then has been in Mumbai to kick start her shooting schedule for Sohail Khan's next opposite Nawazuddin Siddiqui. Post the pack up of her Mumbai film shoot on Thursday, she headed straight to the airport to fly out and be with her family and friends for the weekend enjoying the Easter festivities. That's a good balancing act Amy!
People looking for online information on Amy Jackson, Easter weekend will find this news story useful.


text:
Utah skier describes chilling experience being buried alive in avalanche
By Cat Ellis
Travis Haussener was swept away after accidentally triggering an avalanche while backcountry skiing
(Image credit: Getty)
A skier has described the experience of being buried by several feet of snow after accidentally triggering an avalanche last weekh. Travis Haussener is a regular skier who has been enjoying the sport for around 12 years, but ran into trouble while skiing in Utah last week when he suddenly heard the tell-tale sound of shifting snow.
"I heard the big 'wumpf', felt like the ground shake, and then all of a sudden there's this wall of snow coming down on me that envelops me," Haussener told Fox 13 Now (opens in new tab). "And then I thought, that was it. I thought, you know, my life was over."
Haussner said that he usually kept himself safe by avoiding areas where avalanches are known to happen, but on this occasion he pushed it too far,
Once the snow settled, he found himself buried with only one arm free, and struggled to dig himself out. "In between digs, I started yelling for help, in, you know, the hopes of some miracle," he said.
Luckily for Haussner, that miracle arrived in the form of an off-duty EMT, who hard his cries and summoned rescue services. It took eight hours to get Haussner off the mountain, by which time he was suffering from hypothermia in addition to a punctured lung, broken ribs, and dislocated shoulder.
"I want to keep being in the mountains, keep doing what I love," he said while recovering. "I'll be maybe a little bit more careful next time."
Avalanches often happen after heavy snowfall, and can happen due to the sheer gradient of a mountain, but most are caused by human activity,
Last month, a 19-year-old had a lucky escape after accidentally triggering an avalanche in Colorado. The youngster was carried about 40ft, but escaped without injury and was able to walk away after emergency responders provided him with snowshoes.
Always check for avalanche warnings before heading out, and regularly check for warnings signs including cracks appearing under your feet or skis. A cracking or 'whumphing' sound is also a sign that snow is shifting, and you may be in danger.
For more advice, see our guide avalanche safety: an introduction to the risks and warning signs of snow slides.
Best ski gloves: for warm and cozy hands in the depths of winter
Cat Ellis
Cat is the editor of Advnture, She's been a journalist for 13 years, and was fitness and wellbeing editor on TechRadar before joining the Advnture team in 2022. She's a UK Athletics qualified run leader, and in her spare time enjoys nothing more than lacing up her shoes and hitting the roads and trails (the muddier, the better).
How skiing can reduce back pain – yes, really, says osteopath
What makes Mount Baldy so dangerous?
Hopeless Yellowstone tourist demonstrates the worst way to photograph bears
Best 4-season sleeping bags 2023: snug sleeping bags for all-year camping
Coros Apex 2 review: a lightweight GPS watch that gets better the more you use it


Cover up in the radiant Miraclesuit Tunisian Tunic Coverup, #C68993, designed to minimize the backside and thighs while slenderizing your silhouette. The magnificent appeal of the casual sleeves and mid-thigh length combined with crisp white stitching adds a treat to the eye. Match this solid stunner overtop any of our slimming swimsuits. Made with LYCRA® XTRA LIFE™ Spandex for a long lasting fit.


While it may seem foreign to many physicians that they have a choice when it comes to entering into third party payment arrangements, the reality is that traditionally the doctor patient relationship was a two-party contract. That is quite different from today's market where we almost exclusively look to third parties to pay for the care we render to patients.
There is usually an incentive offered to participate in a health care plan, such as Medicare. The flip side is that you usually work at a greatly reduced rate, and the patient may still be your responsibility after the plan declares bankruptcy. Medicare is an interesting paradigm because typically the seniors who are eligible are the most responsible and have saved up resources to cover such risks as paying for medical care.
Nevertheless, the Health and Human Services would like to enlist all physicians as "participants," in an effort to insure the health care of the population as a whole. Remember, ultimately its your choice, you can say "no" to participation in Medicare and any other plan and simply look to the patients for reimbursement.
Introduction: In the face of an impending 5.4% cut in the Medicare conversion factor, a number of physicians have inquired about their options with regard to Medicare billing arrangements. There are three options.
Physicians may sign a participation agreement and accept Medicare's allowed charge as payment in full.
They may elect to be a non-participating physician, which permits them to bill patients for somewhat more than the Medicare allowance.
They may become a private contracting physician agreeing to bill patients directly and forego any payment from Medicare either to the patient or the physician.
Physicians who wish to change their status from participating (PAR) to nonparticipating (Non-PAR) or vice versa must do so annually. Once made, the decision is irrevocable except where the physician's practice situation has changed significantly, such as relocation to a different geographic area or a different group practice. To become a private contractor, physicians must give 30 days notice before the first day of the quarter the contract takes effect. Those considering a change in status should first determine that they are not bound by any contractual arrangements, which require them to be participating doctors.
Participation: Participating physicians must agree to take assignment on all Medicare claims, which means that they must accept Medicare's approved amount as payment in full for all covered services for the duration of the calendar year. The patient is still responsible for the 20% coinsurance but the physician cannot bill the patient for amounts in excess of the Medicare allowance.
The Medicare payment amount for participating physicians is 5% higher than the rate for non-par physicians.


BASRA: Iraqi religious leader Ayatollah Ali Sistani has called for a political shake-up in Baghdad and a halt to violence against demonstrators on Friday, after days of deadly protests tore through the main city in the south and shut the country's main seaport.
On Thursday, demonstrators escalated the unrest by shutting down Umm Qasr, the country's main seaport, 60 km south of Basra, which handles the vast majority of imports into a country dependent on food bought abroad to feed its 37 million citizens.
Iraq's politicians have so far failed to agree on a new government after an inconclusive election in May. The new Parliament finally met on Monday for the first time, but broke up after a day with no faction assembling enough votes to elect a speaker much less name the next prime minister.
In his Friday prayer sermon read out by an aide, Sistani demanded an end to the use of violence against "peaceful protests" and placed the blame squarely on politicians for corruption and unemployment.
Also on Friday, local security sources said protesters stormed the Iranian Consulate in Basra. The consulate is in the upscale neighborhood of Al-Barda'iya, southeast of the city center.
Iraqi factions mainly came together during a 2014-2017 war against terrorists who seized large swathes of the country's north.
Its two most influential allies, Washington and Tehran, also backed the Baghdad government despite their deep hostility toward each other.
But since Daesh was largely defeated last year, other divisions have re-emerged. Shiites in the south, where most of Iraq's oil wealth is produced, say Baghdad politicians have squandered state funds while leaving them desperate.
Moqtada Sadr,whose electoral bloc came first in May's election, said on Twitter that Prime Minister Haidar Abadi must release more funds for Basra to improve conditions there.
Sadr, the former leader of an anti-American sectarian militia who has reinvented himself as an anti-corruption campaigner, has allied himself with Abadi.
Their alliance is competing to form a government against a rival bloc backed by Abadi's predecessor Nuri Al-Maliki and the leader of an Iran-backed Shiite armed group, Hadi Al-Amiri.
Sadr called on Thursday for an emergency parliament session and for Abadi and other senior officials to attend. Abadi said he would do so.
Amid the political crisis and worsening protests in Basra, security forces have launched a search operation to determine the source of three mortar shells that landed inside Baghdad's heavily fortified Green Zone.
Iraq's newParliament faces the twin tasks of rebuilding the north of the country following the war against Daesh and rehabilitating services in the south, where severe water and electricity shortages have fueled protests.


If you are hunting for something that will be the perfect accompaniment to your carefully put-together ensemble, take heart: there are choices galore. Meet the accessory stars of the future.
Sonali Dalwani's latest collection of bags and shoes inspired by the butterfly is a riot of green, purple, blue, red, orange and yellow. Appliqués give the accessories a multi-dimensional feel while interwoven fabrics, sequins and tassels complete the look. "It is a lively, fun collection of duffels, clutches and totes embellished with butterfly motifs and semi-precious gem stones, footwear that comprises wedges for day and black Lurex with tassels and gold mesh for evening, and belts embellished with large gem stones," she says. We, at Vogue absolutely love the technicolor beach bag with butterflies on it.
–	Statement pieces like scarves that weld fabric, silver and semi-precious gems to give a new spin to accessories, showcased by Eina Ahluwalia at the Lakme Fashion Week Resort 2010, resulted in increased footfalls to her stall at the venue. Kolkata-based Eina is not your average designer; on completing her Masters in Business Administration, she worked in the corporate world for a few years. following which she switched tracks to the fashion industry. "I started off as a jewellery and accessories artist in 2003, after four boring years in the corporate world. One day I just figured I did not want to spend the rest of my life working my way up a corporate ladder… so I quit," she grins. "On an average I put in around 12 hours of work a day, and before Fashion Week, an exhibition or a showing, it can go up to 16 hours. But because I love what I do there's nothing else I'd rather be doing."
Stunning jewellery and accessories designed to thrill the contemporary woman form Suhani Pittie's latest collection titled "The Grunge Begum". From Kasulaperu coins, Nawabi insets and carved wood blocks on copper, silver and dull gold to fabrics embellished with vintage motifs, this Hyderabad-based Director and Creative Head of Zorya Fashions has used them all to design creative pieces guaranteed to make you belle, or rather 'begum', of the ball! We adore the exposed zippers with textured gold-plated silver balls and a choker of uncut crystals and chain on fabric.


text:
Over the last five years, UNICEF China has kept its commitment to children's rights at the centre of its work. We are proud of our impact. We have improved the health, development and protection of children, and we have always focused on the most vulnerable.
We have seen UNICEF's Barefoot Social Worker model develop from an experimental project into a programme that is being adapted and rolled out to every village in China. Our Conditional Cash Transfer programme, which provided subsidies on request to pregnant women and children under 1 year of age, saw an increase in the
uptake of 9 essential maternal and child health services – from 73 per cent in 2013 to almost 82 per cent in 2018. The most vulnerable groups benefited most from this programme.
Our success depends on our partnerships. UNICEF has productive relationships with key partners in the government who are working towards the same goals of upholding children's rights. We are also building our partnerships with academia, the private sector and with civil society, so that we can do more, and reach more children and youth.
We are committed to leaving no child behind – whether they are a girl or a boy, a child with a disability, or a child from a particular economic, ethnic, or religious background. We will build on what we have achieved so far, until all girls and boys in China have the opportunity to fulfil their potential.


text:
package de.bht.ebus.spotsome.security;

import java.io.IOException;
import java.util.Objects;

import javax.servlet.FilterChain;
import javax.servlet.ServletException;
import javax.servlet.http.HttpServletRequest;
import javax.servlet.http.HttpServletResponse;

import org.springframework.security.authentication.AuthenticationProvider;
import org.springframework.security.core.Authentication;
import org.springframework.security.core.AuthenticationException;
import org.springframework.security.core.context.SecurityContextHolder;
import org.springframework.security.web.AuthenticationEntryPoint;
import org.springframework.web.filter.OncePerRequestFilter;

public class AccessTokenAuthenticationFilter extends OncePerRequestFilter {

	// ~ Instance fields
	// ==============================================================

	private AuthenticationProvider authenticationProvider;
	private AuthenticationEntryPoint authenticationEntryPoint;

	public AccessTokenAuthenticationFilter(AuthenticationProvider authenticationProvider,
			AuthenticationEntryPoint authenticationEntryPoint) {
		this.authenticationProvider = Objects.requireNonNull(authenticationProvider);
		this.authenticationEntryPoint = Objects.requireNonNull(authenticationEntryPoint);
	}

	// ~ Methods
	// ==============================================================

	@Override
	protected void doFilterInternal(HttpServletRequest request,
			HttpServletResponse response, FilterChain filterChain)
			throws ServletException, IOException {
		final String token = request.getHeader("X-Access-Token");

		if (token == null) {
			logger.debug("\"X-Access-Token\" header field missing");
			filterChain.doFilter(request, response);
			return;
		}

		final AccessTokenAuthentication authRequest =
				new AccessTokenAuthentication(token);

		try {
			final Authentication authResult = authenticationProvider.authenticate(authRequest);
			SecurityContextHolder.getContext().setAuthentication(authResult);

			logger.debug("Authentication success");

		} catch (AuthenticationException failed) {
			logger.debug("Authentication request failed" + failed);

			authenticationEntryPoint.commence(request, response, failed);
			return;
		}

		filterChain.doFilter(request, response);
	}

}

redpajama_set_name: RedPajamaGithub


text:
Mercado Foot & Ankle Center
Tamarack Medical Center
24024 W. Brancaster Drive
Overlapping toes are characterized by one toe lying on top of an adjacent toe. The fifth toe is the most commonly affected. Overlapping toes may develop in the unborn fetus. Passive stretching and adhesive taping is most commonly used to correct overlapping toes in infants, but the deformity usually recurs. Sometimes they can be surgically corrected by releasing the tendon and soft tissues about the joint at the base of the fifth toe. In some extreme cases, a pin may be surgically inserted to hold the toe in a straightened position. The pin, which exits the tip of the toe, may be left in place for up to three weeks.
Underlapping toes usually involve the fourth and fifth toes. (A special form of underlapping toes is called congenital curly toes). The cause of underlapping toes is unknown. It is speculated that they may be caused by an imbalance in muscle strength of the small muscles of the foot. If deformed toes are flexible, a simple release of the tendon in the bottom of the toe will allow for them to straighten. If the deformity is rigid, surgery may be needed to remove a small portion of the bone in the toe.
Podiatrist - Mercado Foot & Ankle Clinic, Tamarack Medical Center 24024 W. Brancaster Drive Naperville, IL 60564 (630) 922-7722
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