Selective Disruption of Cadherin/Catenin Complexes by Oxidative Stress in Precision-Cut Mouse Liver Slices

Monika Schmelz*, Vanessa J. Schmid{dagger} and Alan R. Parrish{dagger},1

* Department of Pathology, College of Medicine, University of Arizona, Tucson, Arizona; and {dagger} Department of Medical Pharmacology and Toxicology, College of Medicine, Texas A&M University System Health Science Center, College Station, Texas

Received November 30, 2000; accepted February 20, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous work has shown that chemically induced oxidative stress disrupts the protein interactions of the E-cadherin/ß-catenin/{alpha}-catenin complex in precision-cut mouse liver slices (Parrish et al., 1999Go, Toxicol. Sci. 51, 80–86). Although these data suggest a role for oxidative stress in disruption of hepatic cadherin/catenin complexes, multiple complexes are co-expressed in the liver. Both E- and N- cadherin are co-expressed in hepatocytes, as well as ß-catenin and {gamma}-catenin; thus four distinct complexes mediate cell-cell adhesion in the liver: E-cadherin/ß-catenin/{alpha}-catenin, E-cadherin/{gamma}-catenin/{alpha}-catenin, N-cadherin/ß-catenin/{alpha}-catenin, and N-cadherin/{gamma}-catenin/{alpha}-catenin. Taking advantage of the retention of normal organ architecture and cellular heterogeneity offered by precision-cut mouse liver slices, the current study was designed to examine the impact of chemically induced oxidative stress on cadherin/catenin complexes. Precision-cut mouse liver slices were challenged with diamide (25–250 µM; 6 h) or tert-butylhydroperoxide (5–50 µM; 6 h). A polyclonal antibody against ß- or {gamma}-catenin was used to immunoprecipitate proteins prior to Western-blot analysis with monoclonal antibodies to E- or N-cadherin. Although a decrease in E-cadherin:ß-catenin co-immunoprecipitation was seen, interactions between ß-catenin and N-cadherin were not disrupted by chemical challenge. In addition, no effect on protein interactions of {gamma}-catenin with either cadherin was observed. Indirect immunofluorescence was used to co-localize catenins and cadherins following chemical challenge. Consistent with the biochemical observations, a heterogeneous reduction in co-localization of E-cadherin and ß-catenin was seen in precision-cut liver slices, but not other cadherin/catenin complexes. Taken together, these data suggest that oxidative stress selectively disrupts E-cadherin/ß-catenin complexes in the liver. This response is dictated, in part, by the protein composition of the cell-adhesion complex.

Key Words: cadherin; catenin; oxidative stress; precision-cut liver slices.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cadherin gene superfamily encodes for transmembrane proteins that regulate Ca++-dependent cell–cell adhesion (Wu and Maniatis, 1999Go). On the basis of sequence comparisons, the cadherin superfamily can be grouped into six subfamilies: (i) type-I (classical); (ii) type-II (atypical); (iii) desmocollins; (iv) desmogleins; (v) protocadherins; and (vi) flamingo cadherins (reviewed in Nollet et al., 2000). Type-I (classical) cadherins comprise a large family of genes which share significant structural conservation and include E-[epithelial (L-CAM, uvomorulin], N-[neuronal (A-CAM)], and P-[placental] cadherin (reviewed in Kemler, 1992). The extracellular domain contains 4 highly conserved Ca++-binding "cadherin repeats" (EC1-4) and one membrane-proximal extracellular domain (EC5). {alpha}-Catenin is linked to the cytoplasmic domain of cadherins via ß- or {gamma}-catenin; {alpha}-catenin does not directly bind to cadherin (Fig. 1Go). This finding is supported by studies demonstrating 2 mutually exclusive cadherin/catenin complexes in cells; ß-/{alpha}- or {gamma}-/{alpha}-catenin (Nathke et al., 1994Go). Due to homology with vinculin, it is suggested that {alpha}-catenin links the cadherin/catenin complex to the cytoskeleton (Tsukita et al., 1992Go). A more recently identified protein, p120ctn, binds to the cadherin cytoplasmic domain and shares sequence homology with ß- and {gamma}-catenin but does not bind {alpha}-catenin (Reynolds et al., 1994Go).



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FIG. 1. A schematic representation of the cadherin/catenin complex is shown. E- and N-cadherin are single-pass transmembrane proteins that mediate cell–cell adhesion in the liver via a homophilic, Ca++-dependent mechanism. The cadherins are linked to the actin cytoskeleton via the cytoplasmic binding of ß-catenin (or {gamma}-catenin), which is in turn bound by {alpha}-catenin. The function of a fourth catenin, p120, is unknown, but it may participate in the lateral interactions of cadherins.

 
E-cadherin was originally termed uvomorulin or L-CAM (liver cell adhesion molecule), thus suggesting that it is a major regulator of hepatocyte intercellular adhesion (Gallin et al., 1985Go; Ringwald et al., 1987Go). However, other members of the cadherin superfamily are expressed in the liver, most notably N-cadherin (Linnemann et al., 1994Go) and LI-cadherin (liver-intestine cadherin) (Berndorff et al., 1994Go). Interestingly, LI-cadherin is characterized by a truncated cytoplasmic domain that does not support interactions with the catenins (Kreft et al., 1997Go). As E- and N-cadherin are co-expressed in hepatocytes (Kozyraki et al., 1996Go), 4 distinct cadherin/catenin complexes are present in the liver; E-cadherin/ß-catenin/{alpha}-catenin; E-cadherin/{gamma}-catenin/{alpha}-catenin; N-cadherin/ß-catenin/{alpha}-catenin; and N-cadherin/{gamma}-catenin/{alpha}-catenin.

A common link between several liver diseases is the disruption of normal intracellular redox status, commonly referred to as oxidative stress (reviewed in Kaplowitz and Tsukamoto, 1996). Oxidative stress has been associated with hepatic bacterial and viral infections (Larrea et al., 1998Go; Sipowicz et al., 1997Go), diabetes (Traverso et al., 1999Go), xenobiotic-induced hepatotoxicity (Unemura et al., 1999Go), chronic ethanol consumption (Fataccioli et al., 1999Go), and hepatocellular carcinoma (Factor et al., 1998Go). Findings from animal models are supported by data from human patients demonstrating that the oxidized form of plasma ubiquinone is significantly increased and plasma ascorbate levels decreased in chronic active hepatitis, liver cirrhosis, and hepatocellular carcinoma (Yamamoto et al., 1998Go). Although oxidative stress has been extensively linked to hepatocyte death (Rosser and Gores, 1995Go), little is known about the potential impact of non-lethal oxidative stress on hepatocyte function. Disruption of cell–cell adhesion, however, is associated with the accumulation of inflammatory cells, an event associated with several hepatopathologies associated with oxidative stress (Kaplanski et al., 1997Go). Thus, intercellular adhesion may be disrupted by oxidative stress in the liver. This suggestion is supported by the finding that elevated circulating levels of adhesion molecules are associated with hepatitis, cirrhosis, and cancer (Katayama et al., 1994Go; Lucka et al., 1998Go).

Previous work has shown that chemically induced oxidative stress disrupts protein interactions of the ß-catenin with both E-cadherin and {alpha}-catenin (Parrish et al., 1999Go). However, the impact of oxidative stress on the N-cadherin and {gamma}-catenin complexes has not been investigated. In hepatocellular carcinomas, a significant loss of E-cadherin, but not N-cadherin, is seen (Kozyraki et al., 1996Go), suggesting that distinct complexes may have fundamental differences in respect to complex stability. The current studies were designed to examine the impact of non-cytolethal oxidative stress on the expression and protein interactions of each of the cadherin/catenin complexes present in the liver.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
Diamide and tert-butylhydroperoxide were purchased from Sigma Chemical Co. (St. Louis, MO). DMEM/F12 media, gentamycin, and antibiotic/antimycotic solution were also purchased from Sigma. Monoclonal antibodies to E- and N-cadherin, and {alpha}-, ß-, {gamma}- and p120 catenin were purchased from Transduction Laboratories (Lexington, KY); polyclonal antibodies to ß- and {gamma}-catenin were obtained from Santa Cruz (Santa Cruz, CA). Secondary antibodies (anti-mouse and anti-goat) conjugated to horseradish peroxidase were purchased from Sigma. Secondary antibodies used for immunofluorescence microscopy were Cy3- and FITC-conjugated goat or donkey antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) to immunoglobulins of mouse or goat, respectively. GammaBind plus Sepharose® was purchased from Pharmacia Biotech (Piscataway, NJ). ECL detection reagents and nitrocellulose membranes were obtained from Amersham (Arlington Heights, IL).

Precision-cut mouse liver slices.
Male CD-1 mice (20–25 g) were purchased from Charles River (Wilmington, MA). Following a 1-week acclimation period, the livers were excised and chilled in Krebs-bicarbonate buffer. Cylindrical cores (8-mm diameter) were cut and slices (275 µm thick) were produced using the Brendel/Vitron tissue slicer. The slices were incubated on mesh screen rollers with DMEM/F12 medium for 1 h at 37°C. At this time, slices were challenged with diamide (DA; 25 or 250 µM) or tert-butylhydroperoxide (tBHP; 5- or 50 µM) for 6 h. These concentrations are not associated with cytolethality as assessed by biochemical evaluation (Parrish et al., 1999Go). All experiments were performed within 3 weeks following the arrival of the mice.

Western blot/immunoprecipitation.
Slices were homogenized in 500 µl of lysis buffer (4% SDS) and 20 µg of protein separated on an 8% polyacrylamide gel. Proteins were transferred to nitrocellulose membranes and blocked overnight with a 5% non-fat milk solution. Monoclonal antibodies (Transduction Laboratories) against N-cadherin, {gamma}-catenin, and p120 (all 1:5000) were added for 2 h at room temperature. Membranes were washed and incubated with a secondary antibody conjugated to horseradish peroxidase (1:12500) prior to washing and detection via ECL.

For immunoprecipitation studies, slices were homogenized in immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl2, 10 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium orthovanadate, 0.2 mM PMSF, 0.5% NP-40) and 300 µg of protein incubated for 1 h with 3 µg of antibody (ß- and {gamma}-catenin, polyclonal, Santa Cruz; p120 monoclonal, Transduction Laboratories). GammaBind plus Sepharose® (20 µl) was added and the samples incubated overnight. The immunoprecipitates were collected by centrifugation and processed for Western-blot analysis.

Confocal microscopy.
Following chemical challenges, slices were snap-frozen in isopentane pre-chilled in an LN2 bath. Thin (5 µm) frozen sections were placed on slides. The slides were fixed for 10 min in –20°C acetone followed by air drying. Primary antibodies were applied for 30 min, followed by 3 5-min PBS washes, before application of secondary antibodies for 30 min. After secondary antibody binding, specimens were then washed again 3 x 5 min in PBS, rinsed briefly in water and then fixed in ethanol for 5 min, air-dried, and mounted.

Confocal laser scanning-immunofluorescence microscopy was done on a Zeiss LSM 410 UV (Carl Zeiss). For double-label fluorescence, an argon/krypton ion laser operating at 488 nm and 568 nm was used together with a long-pass filter 590 for visualization of Cy 3 fluorescence and a band-pass filter 515–540 for visualization of FITC and Cy 2 fluorescence, respectively. RGB images were taken in high-resolution mode using 1,024 x 1,024 image points (pixels) and 8-s scan time. Noise levels were reduced by line averaging of the scans.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Initial experiments were designed to determine the impact of chemical challenge on the total protein expression of cadherins and catenins in precision-cut mouse liver slices. Consistent with the previous results (Parrish et al., 1999Go), a 6-h challenge of precision-cut mouse liver slices with either DA or tBHP did not affect the protein expression of E- or N-cadherin, {alpha}-, ß- or {gamma}-catenin, or p120ctn (Fig. 2Go). As cleavage of cadherins or catenins is associated with cytotoxicity (Brancolini et al., 1997Go; Bush et al., 2000Go; Schmieser et al., 1998Go), the fact that the expression or integrity of the individual cadherins and catenins was not altered by DA or tBHP supports the conclusion that these conditions of chemically induced oxidative stress are not cytolethal.



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FIG. 2. The impact of chemically induced oxidative stress on cadherin/catenin protein expression in precision-cut liver slices. Following a 1 h pre-incubation, slices were challenged with diamide (D; 25 or 250 µM), or tert-butylhydroperoxide (T; 5 or 50 µM) for 6 h. Total cellular lysates were harvested and Western-blot analysis of E- or N-cadherin, {alpha}-, ß- or {gamma}-catenin and p120ctn performed as described. Similar results were seen in 5 independent experiments.

 
A series of experiments were designed to assess the stability of the cadherin/catenin complex using an immunoprecipitation/Western-blot strategy. A polyclonal antibody against ß-catenin was used to immunoprecipate proteins from precision-cut mouse liver slices, followed by Western-blot analysis with a monoclonal antibody against E- or N-cadherin. DA or tBHP dramatically reduced protein interactions of ß-catenin with E-cadherin (Fig. 3Go), consistent with our previous report (Parrish et al., 1999Go). However, similar conditions were not associated with a decrease in the co-immunoprecipitation of ß-catenin and N-cadherin (Fig. 3Go). These data suggest that distinct cadherin/catenin complexes differ with respect to their stability following chemical challenge. Using a similar experimental strategy, the impact of oxidative stress on {gamma}-catenin/cadherin complexes was examined. Chemically induced oxidative stress did not disrupt the E-cadherin/{gamma}-catenin complex, or the N-cadherin/{gamma}-catenin complex (Fig. 4Go), and in fact, a small increase in the interaction of {gamma}-catenin with the cadherins was observed. At this time, the significance of this observation is unclear. The finding that {alpha}-catenin was not immunoprecipitated with a polyclonal antibody against {gamma}-catenin in control precision-cut mouse liver slices suggests a lack of interaction between these proteins in hepatocytes (data not shown), although it is difficult to interpret this data given the low level of {alpha}-catenin expression in the liver. Interactions of p120ctn with either E- or N-cadherin were not altered by chemically induced oxidative stress (data not shown). Taken together, these data suggest that of the 4 cadherin/catenin complexes expressed in the liver, only the E-cadherin/ß-catenin/{alpha}-catenin complex is disrupted by chemically induced oxidative stress.



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FIG. 3. The impact of chemically induced oxidative stress on the protein interaction of the ß-catenin with E- and N-cadherin in precision-cut liver slices. Following a 1 h pre-incubation, slices were challenged with diamide (D; 25 or 250 µM) or tert-butylhydroperoxide (T; 5 or 50 µM) for 6 h. Total cell lysates were immunoprecipitated under native conditions with a polyclonal antibody against ß-catenin and Western-blot analysis performed as described. Similar results were seen in 3 independent experiments. Con, control slice; TCL, 20 µg of total cell lysate (protein).

 


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FIG. 4. The impact of chemically induced oxidative stress on the protein interactions of the {gamma}-catenin with E- or N-cadherin in precision-cut liver slices. Following a 1-h pre-incubation, slices were challenged with diamide (D; 25 or 250 µM) or tert-butylhydroperoxide (T; 5 or 50 µM) for 6 h. Total cell lysates were immunoprecipitated under native conditions with a polyclonal antibody against {gamma}-catenin and Western-blot analysis performed as described. Similar results were seen in 3 independent experiments. Con, control slice; TCL, 20 µg of total cell lysate (protein).

 
The impact of oxidative stress on cadherin/catenin complexes in precision-cut mouse liver slices was also examined by laser-scanning confocal immunofluorescent microscopy. Frozen sections of treated and untreated precision-cut mouse liver slices were processed for immunofluorescence evaluation of cadherin and catenin localization. Consistent with previous reports (Butz and Larue, 1995Go; Kozyraki et al., 1996Go), no zonal localization of cadherins or catenins was observed in the mouse liver slices. In untreated control slices, E-cadherin (Fig. 5AGo) and ß-catenin (Fig. 5BGo) were localized along the entire cell surface, where they showed co-localization in the junctional regions (Fig. 5EGo). This is consistent with expression of cadherins and catenins in hepatocytes (Ihara et al., 1996Go). However, following challenge with tBHP, a dramatic reduction in co-localization and the onset of some internalization were seen in certain areas (Fig. 5C and 5DGoGo), in which the distinct outline of the hepatocyte cell periphery disappeared. Similar results were seen following challenge with DA (data not shown). Interestingly, the E-cadherin/ß-catenin co-localization in the bile duct epithelium, which expresses E-cadherin but not N-cadherin (Kozyraki et al., 1996Go), was not affected by chemical challenge (Fig. 5CGo). The disruption of the E-cadherin ß-catenin complex was not observed homogenously throughout the liver slice. The internalization of cadherin and catenins is consistent with disruption of the complex as previously reported (Mandel et al., 1994Go; Kevil et al., 1998Go; Boterberg et al., 2000Go). In untreated control slices, N-cadherin (Fig. 6AGo) and {gamma}-catenin (Fig. 6CGo) showed a similar staining pattern to E-cadherin and ß-catenin. However, consistent with the biochemical analyses, no effect on N-cadherin or {gamma}-catenin localization was observed following chemical challenge (Fig. 6B and 6DGoGo). Precision-cut mouse liver slices provided a useful in vitro model to examine the stability of cadherin/catenin complexes. This interpretation is supported by the findings that disruption of the E-cadherin/ß-catenin complex was confined to hepatocytes (not bile duct epithelium), and that the response was heterogeneous throughout the liver.



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FIG. 5. The impact of chemically induced oxidative stress on the co-localization of E-cadherin and ß-catenin in precision-cut liver slices. Following a 1-h pre-incubation, slices were challenged with tert-butylhydroperoxide (tBHP, 50 µM) for 6 h. Liver slices were harvested and processed for immunofluorescence staining (laser scanning confocal microscopy) as described. The immunostaining for E-cadherin (A) and ß-catenin (B) in untreated control slices is shown. Double staining for E-cadherin (red) and ß-catenin (green) is shown for tBHP treated (C, D) and untreated (E) slices. Note the significant loss of cell-surface staining and co-localization in hepatocytes (C, D) compared to E (yellow-orange). However, bile duct epithelium (which express E-, but not N-cadherin) was not affected. The loss of co-localization of E-cadherin and ß-catenin was heterogeneous, and not seen in all hepatocytes in a section. Similar results were seen in 3 independent slices. B, bile duct; v, blood vessel. Arrow in (C) indicates area shown in (D) at higher magnification.

 


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FIG. 6. The localization of N-cadherin and {gamma}-catenin in control precision-cut mouse liver slices. Liver slices, untreated (A, C) and treated with tert-butylhydroperoxide (50 µM) for 6 h (B, D), were harvested for immunofluorescence staining (laser scanning confocal microscopy) as described. In both treated and untreated samples, N-cadherin (A, B) and {gamma}-catenin (C, D) were localized along cell–cell borders. No effect on N-cadherin and {gamma}-catenin localization was observed upon oxidative stress. Similar results were seen in 3 independent slices.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In conjunction with our previous work (Parrish et al., 1999Go), these data suggest that chemically induced oxidative stress selectively disrupts cadherin/catenin complexes in the liver. The level of protein expression of cadherins or catenins in precision-cut liver slices is not altered by either tBHP or DA; however, the E-cadherin/ß-catenin/{alpha}-catenin complex is disrupted by oxidative stress, while {gamma}-catenin complexes (both E- and N-cadherin) are not affected by chemical challenge. In addition, the N-cadherin/ß-catenin complex is not disrupted by oxidative stress, nor are complexes between p120ctn and E- or N-cadherin. The differential susceptibility of cadherin/catenin complexes following chemical challenge is supported by the fact that differences in complex stability have previously been reported in cells that co-express multiple cadherins. For example, the renal MDCK cell line expresses both E- and P-cadherin, and the anchorage of P-cadherin to the actin cytoskeleton was suggested to be weaker than that of E-cadherin (Wu et al., 1993Go). In addition, retinoic acid has been shown to differentially influence expression of desmosomal cadherins in cultured keratinocytes (Wanner et al., 1999Go).

The finding that oxidative stress disrupts the E-cadherin/ß-catenin complex is supported by several studies suggesting that oxidative stress disrupts either cell-cell adhesion or, specifically, the cadherin/catenin complex. Hydrogen peroxide induces tyrosine phosphorylation of a number of proteins similar in molecular weight to cadherins and catenins, and disrupts the barrier function of several intestinal cell lines (Rao et al., 1997Go). In addition, the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), an inducer of oxidative stress (Hu and Cotgreave, 1995Go), causes internalization of E-cadherin from the plasma membrane to the cytosol without decreasing the total amount of protein (Jansen et al., 1996Go). Exposure of endothelial cell monolayers to hydrogen peroxide is also associated with internalization of cadherins (Kevil et al., 1998Go). Thus, oxidative stress may be a common mechanism of disruption of the cadherin/catenin complex for a number of chemical or physiological stimuli.

Oxidative stress is an important epigenetic mechanism of hepatocarcinogenesis in a number of studies (reviewed in Klaunig et al., 1998). Disruption of gap junctional intercellular communication (GJIC) is a sensitive target of oxidative stress (Sai et al., 1998, reviewed in Trosko and Ruch et al., 1998). This is of interest given the relationship between the cadherin/catenin complex and GJIC. The cadherin/catenin complex is requisite for the formation of gap junctions, and intercellular communication (Jongen et al., 1991Go). This interpretation is supported by the finding that antibodies to cadherin inhibit gap junction formation (Frenzel and Johnson, 1996Go) and that disruption of cadherin expression or complex assembly correlates with loss of GJIC (Jansen et al., 1996Go). Thus, the effect of chemically-induced disruption of the E-cadherin/ß-catenin complex in hepatocytes on GJIC is an exciting area for future studies of epigenetic mechanisms of hepatocarcinogenesis.

The demonstration of both susceptible and non-susceptible complexes within hepatocyte populations offers exciting possibilities in the development/progression of hepatocellular carcinomas. In human hepatocellular carcinoma, a striking down-regulation of E-cadherin, but not N-cadherin has been reported (Kozyraki et al., 1996Go). This is supported by more recent data suggesting the decreased expression of E-cadherin, but not N-cadherin, precedes down-regulation of {alpha}- and {gamma}-catenin expression in human hepatocellular carcinomas (Nuruki et al., 1998Go). The findings that E-cadherin appears to be more sensitive to disruption is consistent with our results, which demonstrate that oxidative stress selectively disrupts the E-cadherin/ß-catenin complex in hepatocytes.

In summary, we have demonstrated a selective disruption of E-cadherin/ß-catenin complexes in precision-cut mouse liver slices. Although the molecular mechanism underlying the sensitivity of the E-cadherin/ß-catenin complex to chemically induced oxidative stress remains undefined, this pattern of disruption parallels that seen during the development of human hepatocellular carcinomas. These changes were observed in an integrated in vitro system that allows for examination of the influence of organ architecture and cellular heterogeneity on the cell adhesion complex. Given the increased awareness of the influence of xenobiotics on cell adhesion, these results provide the basis for further investigation of the role of oxidative stress on the cadherin/catenin complex.


    ACKNOWLEDGMENTS
 
The authors are grateful for the contributions of each author to the work: immunoprecipitation/Western-blot analysis (VJS, ARP); confocal microscopy (MS); manuscript preparation (MS, ARP). Supported by the Center for Environmental and Rural Health, Texas A&M University (ES09106) and the Department of Medical Pharmacology and Toxicology, Texas A&M University System Health Science Center.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (979) 845-0699. E-mail: Parrish{at}medicine.tamu.edu. Back


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 RESULTS
 DISCUSSION
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