1Anatomy and Physiology and 2Biochemistry, Kansas State University, Manhattan, Kansas 66506
Submitted 16 September 2002 ; accepted in final form 14 January 2004
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ABSTRACT |
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transepithelial resistance; cystic fibrosis; tight junction; epithelial barrier; amphipathic -helix
Tight junctions are complex, highly regulated, dynamic structures that are a barrier to movement of solutes between apical and basolateral compartments and form a "fence" that maintains cell membrane polarity (4, 37). Regulated openings of junctions occur in a variety of situations such as sperm maturation (18), extravasation of lymphocytes (8), and nutrient uptake in the intestine (23). Pathology associated with aberrant function and dysregulation of tight junctions includes cancer metastases (16), autoimmune dysfunction (20), and inflammatory bowel disease (12), to name just a few. Tight junctions are targets of bacterial toxins such as the Vibrio cholerae zonula occludens toxin (ZOT) and Clostridium difficile toxins TcdA and TcdB (36) in pathological, experimental, and perhaps therapeutic situations (9, 10). A mammalian homolog of ZOT has been identified and may be a primary physiological regulator of tight junctions in intestinal tissues (36). Additionally, cytokines and a number of second messengers are known to be involved in the modulation of tight junctions (8, 9, 14, 34, 37), although the mechanisms by which these processes occur remain to be elucidated.
A variety of treatments, including Ca2+ chelation, surfactants, fatty acids, and cationic polymers, have been used to experimentally modulate the paracellular pathway (17, 37). However, when applied in vivo, side effects of the chemical treatments used to modulate gTE can include hypersensitivity, asthma, anaphylaxis, and the sloughing of epithelial cells (9, 17, 37). Surfactants and detergents can cause cell lysis and sloughing, whereas Ca2+ chelators can cause cytoskeletal rearrangements and interfere with calcium signaling pathways (37). An additional drawback to these treatments is that, in general, there is little separation between the effective concentration and the cytotoxic concentration (37). Alternatively, ZOT, which lacks many undesirable side effects, has been used to modulate the epithelial barrier in an experimental therapeutic setting (10). The results are encouraging and provide a "proof of concept" indicating that intestinal tight junctions can be modulated to allow for the absorption of high-molecular-weight therapeutic compounds without apparent deleterious side effects (10). Other studies (5) have suggested that gene therapy might be augmented by transiently reducing epithelial tight junction integrity as well. Thus there is a great need to identify safe and efficient means by which to modulate the epithelial barrier function at the tight junction in a wider variety of epithelia.
The goal of the present study is to define the relationship between the apical exposure of epithelial cells to NC-1059, a channel-forming peptide, to changes in barrier function as typified by the increase in gTE and transepithelial solute permeability. Results suggest that the peptide provides a route for ion permeation across the cell membrane and modulates the paracellular pathway over a similar concentration range, although the time course of the two responses is dramatically different.
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MATERIALS AND METHODS |
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Cell culture.
MDCK cells were provided by Dr. Lawrence Sullivan (University of Kansas Medical Center, Kansas City, KS) and were maintained in culture as described previously (2). Briefly, the culture medium was a 1:1 mixture of DMEM and Ham's F-12 (GIBCO BRL, Rockville, MD) supplemented with 5% heat-inactivated FBS (BioWhittaker, Walkersville, MD) and 1% penicillin and streptomycin (GIBCO BRL). Cells were grown in plastic 25-cm2 culture flasks (Cellstar, Frickenhausen, Germany) in a humidified environment with 5% CO2 at 37°C. Confluent cultures were dissociated for subculture with PBS containing 2.6 mM EDTA and 0.25% trypsin. For permeation and flux experiments, cells were seeded on 1.13-cm2 permeable supports (Snapwell; Costar, Cambridge, MA) at a density of 1 x 106 cells/well and incubated in DMEM/F-12 supplemented with FBS and antibiotics (refreshed every other day) for 23 wk before cells were mounted in modified Ussing flux chambers for evaluation.
Electrical measurements.
Transepithelial ion transport was evaluated in modified Ussing chambers (Model DCV9; Navicyte, San Diego, CA). For typical electrical measurements of ion flux, cells were bathed in symmetrical Ringer solution (composition in mM: 120 NaCl, 25 NaHCO3, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 MgCl2, 1.2 CaCl2; 290 ± 2 mosmol/kgH2O). The diffusion chambers were maintained at 37°C and continuously bubbled with 5% CO2-95% O2 to maintain pH, provide aeration, and mix the fluid in the chambers. The transepithelial potential was clamped to zero, and Isc was measured continuously with a voltage-clamp apparatus (Model 558C; Department of Bioengineering, University of Iowa, Iowa City, IA). Data were acquired at 1 Hz with a Macintosh computer (Apple Computer, Cuppertino, CA) using Aqknowledge software (v. 3.2.6; BIOPAC Systems, Santa Barbara, CA) with an MP100A-CE interface. gTE was determined by exposing the epithelia to a 5-s, 1-mV, bipolar pulse at 100-s intervals. The recorded current deflections were used with Ohm's law to calculate gTE (gTE = I/
V).
Alternative apical bathing solutions that allowed for the imposition of defined transepithelial ion gradients were employed for one set of experiments. Three solutions of virtually identical osmolality (280290 mosmol/kgH2O) and total electrolyte strength to normal Ringer solution were formulated: nominally Na+-free [in mM: 120 N-methyl-D-glucamine (NMDG)·Cl, 25 choline-HCO3, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 CaCl2, 1.2 MgCl2], nominally Cl-free (in mM: 120 Na-gluconate, 25 NaHCO3, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 CaSO4, 1.2 MgSO4, 2.8 CaCl2), and nominally NaCl-free (in mM: 120 NMDG-gluconate, 25 choline-HCO3, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 CaSO4, 1.2 MgSO4, 2.8 CaCl2). CaCl2 was added to the gluconate-containing solutions to maintain the free Ca2+ concentration and to ensure adequate Cl for proper electrode function.
Xenopus oocyte isolation. Oocyte isolation was performed as described previously with minor modifications (15, 28, 30). Briefly, sexually mature, human chorionic gonadotropin-treated Xenopus laevis were purchased (Xenopus 1, Ann Arbor, MI) and individually maintained in aquaria in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited facility. Oocyte isolation was accomplished by using Institutional Animal Care and Use Committee-approved protocols in which Xenopus were anesthetized by exposure to MS-222 (Sigma, St. Louis, MO) and a laparoscopic approach was employed to isolate and remove the ovary. Oocytes were separated from follicular cells by incubation in nominally Ca2+-free ND-96 (in mM: 96 NaCl, 1 KCl, 1 MgCl2, 5 HEPES, pH 7.5) including 0.7 mg/ml collagenase (GIBCO BRL) and 0.1 mg/ml trypsin inhibitor (Sigma) on a low-speed rocker at room temperature for 3560 min. Oocytes were rinsed five times and incubated in K2HPO4 (100 mM; pH 6.5) with BSA (0.1% wt/vol; Sigma) for 1 h with gentle agitation at 15-min intervals. Oocytes were then transferred to and maintained in modified Barth's solution [in mM: 88 NaCl, 2.4 NaHCO3, 1 KCl, 0.82 MgSO4, 0.41 CaCl2, 0.3 Ca(NO3)2, 10 HEPES, pH 7.5] at 1820°C until current recordings were made 1 to 5 days later.
Membrane conductance and permselectivity.
The two-electrode voltage-clamp technique was employed. Oocytes were impaled with two 3-M KCl-filled electrodes having resistances of 0.52 M. The electrodes were connected to a GeneClamp 500 current-voltage clamp amplifier (Axon Instruments, Foster City, CA) via Ag-AgCl pellet electrodes and referenced to a Ag-AgCl pellet that communicated to the bath via an agarose bridge (3% agarose in 1 M KCl). The voltage clamp was controlled by an analog-digital interface (Digidata 1200b) using a Pentium-based computer running pClamp software (version 9.0; Axon Instruments) for command potential and current and voltage recording. Two voltage-pulse protocols were employed. In the first, membrane potential (Vm) was held at 30 mV (approximately the resting Vm) and pulsed to 90 mV for 1,000 ms, returned to 30 mV for 1,000 ms, and then pulsed to 0 mV for 1,000 ms. This pulse protocol was repeated at 4,128-ms intervals throughout the experimental period to verify that stable conductance levels were achieved with each change of bath solution. Current-voltage (I-V) relationships were generated at the end of each treatment period (baseline, peptide-exposed, ion-substituted) with a repeating three-step protocol. Vm was held at 30 mV for 500 ms, pulsed to one of nine voltages (100 to +60 mV in 20-mV increments) for 1,000 ms, and returned to 30 mV for 500 ms. The average voltage and current during the final 500 ms of each voltage pulse was used to construct each I-V relationship.
Data were recorded in solutions of four ionic compositions: ND-96 (in mM: 96 NaCl, 1 KCl, 1 MgCl2, 1.8 CaCl2, 5 HEPES), reduced Na+ and Cl (in mM: 173 mannitol, 9.6 NaCl, 1 KCl, 1 MgCl2, 1.8 CaCl2, 5 HEPES), reduced Cl (in mM: 92.3 Na-gluconate, 3.7 NaCl, 1 KCl, 1 MgCl2, 1.8 CaCl2, 5 HEPES), and reduced Na+ (in mM: 86.4 NMDG·Cl, 9.6 NaCl, 1 KCl, 1 MgCl2, 1.8 CaCl2, 5 HEPES). pH was adjusted to 7.5 for all solutions. The final osmolality of all solutions was between 185 and 200 mosmol/kgH2O. I-V relationships were initially recorded in ND-96 in the absence of any synthetic peptide. Peptide (8 µl, 5 mM in H2O) was then added to the 400-µl bath and mixed to attain a final concentration of 100 µM. After stable parameters were attained (<5 min), an I-V relationship was again recorded. Subsequently, the bath composition was changed to an alternative ion composition by adding 200 µl of the new solution, mixing, and removing 200 µl of the bathing medium 15 times (>99.7% bath replacement). Peptide was again added to the bathing medium (100 µM final concentration), and the stability of membrane conductance was verified before an I-V relationship was recorded. The bathing medium was repeatedly changed with this technique. In every case, however, recordings were made in ND-96 before (and typically after) recording in an alternative ionic composition to verify reversibility of bath composition-induced changes and to allow for comparisons to temporally close controls. Visual inspection suggested that a linear I-V relationship was present, as expected. Thus linear regression (Sigmaplot v. 2000 for Windows, SPSS, Chicago, IL; and Excel, v. 9.0.38, Microsoft, Redmond, WA) was employed to determine the slope conductance and reversal potential (Vrev) in each condition. I-V relationships were mathematically adjusted for junction potentials by using the appropriate pClamp module. Permselectivity (P) for Cl vs. Na+ was estimated by using Eq. 1, which is derived from the Goldman-Hodgkin-Katz constant field equation (13)
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FITC-dextran permeability assay. Epithelial permeability to uncharged solutes of various sizes was assessed with monolayers grown on Snapwell tissue culture inserts as described in Cell culture. Confluent monolayers were washed once with Ringer solution and placed in one of three treatments containing FITC-conjugated dextran (Sigma) in the apical compartment: 1) Ringer solution in the apical and basolateral compartments; 2) Ringer solution in the apical and basolateral compartments with NC-1059 (100 µM) in the apical solution; or 3) EDTA (3 mM) in Ringer solution that was diluted 1:1 with distilled water in both the apical and basolateral compartments. Monolayers were incubated at 37°C for 60 min, and the solution in the basolateral well was sampled to quantify fluorescently labeled dextran. Monolayers were then washed with Ringer solution to remove peptide, EDTA, and dextran, placed in tissue culture medium, and returned to the incubator for 2 days before the assay was conducted again.
Confocal microscopy. Immunoreactivity to antibodies raised against tight junction-associated proteins was assessed by confocal microscopy (Zeiss, Thornwood, NY). Samples for visualization were prepared from monolayers used in electrophysiological studies. After being removed from Ussing chambers, monolayers were washed in Ringer solution and fixed overnight in 10% neutral buffered formalin. Monolayers were washed three times in PBS, permeabilized with 0.1% Triton X-100 in PBS, blocked with goat serum, and then exposed to primary antibody in a 1:500 dilution (rat anti-zonula occludens-1, catalog no. MAB120; Chemicon, Temecula, CA, or rabbit anti-occludin, catalog no. 71-1500; Zymed, San Francisco, CA) for 1 h at room temperature. After being washed three times in PBS, FITC-conjugated goat anti-rat (catalog no. AP136F; Chemicon) or goat anti-rabbit (catalog no. Fl-1000; Vector Laboratories, Burlingame, CA) secondary antibodies were employed (1:1,000 dilution) with exposure occurring for 1 h at room temperature. TRITC-labeled phalloidin (0.1 mg/ml in methanol; Sigma) used for F-actin localization was applied concurrently with the secondary antibody. A KrAr laser was used to excite the fluorophores. Filter sets used for fluorescein were band pass (BP) 485 ± 20 nm for excitation and BP 515 ± 540 nm for emission, and for rhodamine the sets used were BP 530 ± 585 nm for excitation and long pass (LP) 590 nm for emission.
Chemicals and stock solutions. 1-EBIO (Acros; Fisher Scientific, Pittsburgh, PA) was prepared as a 1 M stock solution in DMSO. Forskolin (Coleus forskohlii) was purchased from Calbiochem (La Jolla, CA) and prepared as a 10 mM stock in ethanol. All other chemicals were purchased from Sigma and were of reagent grade unless otherwise noted. Unless otherwise stated, synthetic peptide was suspended in water at 5 mM just before experimental addition.
Data analysis. All results are presented as means ± SE. Fitting of user-defined functions to data sets was conducted with Sigmaplot. The difference between treatment groups was analyzed by using Student's t-test (Microsoft Excel 2002). The probability of making a type I error <0.05 is considered statistically significant. The reported value of n is the number of independent observations.
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RESULTS |
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NC-1059-induced changes in gTE require apical exposure. Experiments were conducted to test for the relative efficacy of apical and basolateral NC-1059 on changes in ion transport and barrier function. Results presented in Fig. 2 demonstrate that apical exposure is required to observe a significant effect of the peptide on these parameters. Results from a typical experiment are presented in Fig. 2, AD. When apically exposed to NC-1059 (300 µM), Isc rapidly increases to a peak value and then declines (Fig. 2, A and B), whereas the increase in gTE is delayed (Fig. 2, C and D, consistent with Fig. 1). Exposure of the basolateral membrane to NC-1059 produces no obvious effect, regardless of the order of exposure. Results from these and five additional monolayer pairs are summarized in Fig. 2E. On a pairwise basis, effects were never observed with basolateral exposure and were always observed with apical exposure. These results might suggest that NC-1059 interacts with a cellular component that is accessible only from the apical aspect of the monolayer, although additional experiments would be required to fully test this hypothesis.
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Experiments designed to determine whether the NC-1059 activity in solution declines over time were conducted, although it was previously reported that NC-1059 does not aggregate in solution, as was seen with related peptides (3). Results from a typical experiment are presented in Fig. 4. Paired monolayers were mounted in Ussing chambers, with some being immediately exposed to NC-1059. NC-1059 (60 and 100 µM) elicited expected increases in Isc and gTE that reversed over time. Sixty percent of the apical solution was then transferred from the apical side of a treated monolayer to an untreated monolayer, as indicated by the arrows (Fig. 4, A to C and E to G). A previously untreated monolayer was exposed to 60 µM of freshly dissolved NC-1059 at the same time (Fig. 4, D and H). The results clearly demonstrate that active NC-1059 continued to be present in the apical solution even as the effects on Isc and gTE were reversing. The possibility remained that peptide activity was slowly decreasing such that the response declined incrementally as activity diminished. This possibility was excluded by experiments in which a peptide-induced response was generated and the apical solution was partially replaced with Ringer solution containing freshly dissolved peptide. No increment in either Isc or gTE was observed (n = 3). Together, these results suggest that the effect of NC-1059 on MDCK electrical parameters is transient.
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DISCUSSION |
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NC-1059 induces a concentration-dependent increase in Isc across epithelial cell monolayers with a concurrent increase in gTE. This peptide is the only sequence designed so far that has demonstrated the ability to increase gTE to this magnitude, which is in excess of conductance changes expected for apical channel formation, because related peptide sequences provide comparable increases in Isc but do not exhibit the dramatic effects on transmural conductance (3). This additional functionality suggests that the ability of the peptide to support anion secretion is separate from, but perhaps related to, the effect on gTE.
The mechanism by which NC-1059 modulates gTE is unclear. The simplest interpretation, that NC-1059 forms conductive pores in the apical membrane that fully account for the change in gTE, is inadequate. Similar peptide sequences (i.e., with the first 16 amino acid residues being identical) cause an equal increase in Isc across MDCK epithelial monolayers but do not affect transepithelial resistance to the same extent (3). Comparison of the data presented in Fig. 1D to an earlier report (3) shows that the concentration dependence for changes in gTE is right shifted compared with the concentration dependence of Isc. Additionally, permeation of 9.5-kDa dextran across the epithelium strongly suggests that a paracellular rather than a transcellular route is involved. A second simple possibility that can be discounted is that NC-1059 is cytotoxic and that a loss of cells accounts for the change in gTE (1). Visual inspection provides no indication that cells are absent from the epithelium following NC-1059 exposure as they are following EDTA exposure. Furthermore, tight junction proteins are not redistributed in response to NC-1059, and both the selectivity (Na+ > NMDG+; Cl > gluconate) and the finite size of the permeation pathway (9.5 but not 77 kDa dextran) suggest that a selective paracellular pathway is opened. Rather, the results suggest a specific interaction of NC-1059 with the cellular components involved in modulating gTE. This conclusion is bolstered by the "sidedness" of effects in that changes in electrical parameters are observed only with apical exposure. There is clearly precedence for metabotropic receptors selectively modulating the size exclusion of the paracellular pathway (21, 37), although evidence has not yet been acquired to suggest a metabotropic effect of NC-1059.
That NC-1059 is effective only from the apical aspect of the epithelium suggests that a "receptor-type" mechanism might be involved. There is at this time, however, no definitive evidence to support such a claim. Results presented in Fig. 2 might indicate that there is limited access of the peptide across the tissue culture support. However, the membrane was permeable to 2.5 MDa, and 77-kDa dextran permeated the membrane in the presence of cells following EGTA exposure. Thus size exclusion by the culture support is unlikely. An alternative to the "receptor" hypothesis is that the apical membrane exhibits a unique lipid milieu with which NC-1059 interacts. This possibility by necessity includes the supposition that a similar milieu must be present in Xenopus oocytes because NC-1059 quite effectively modulated membrane conductance in this system. A third possibility is that NC-1059 exhibits pleiotropic effects by interacting at multiple cellular sites. At this time it remains unclear whether channel formation (i.e., ion transport) is a prerequisite for modulation of gTE. The possibility exists that NC-1059 may interact with the apical membrane to form ion channels by mechanisms similar to those that have been indicated for closely related peptides and that effects on gTE require interaction with another epithelial target.
The NC-1059-induced change in gTE is transient in nature, reaching a peak value within the first 1030 min of exposure. Several events might account for the transient nature of the response. It is possible that, due to charge neutralization or shielding, the peptide may undergo some aggregation and/or precipitation in Ringer solution (31), thus reducing the effective concentration. It was previously reported (3) that NC-1059 (initially termed NK4-Ala) does not aggregate in solution. However, the analysis was conducted with H2O as the solvent instead of Ringer solution. The ionic strength of the Ringer solution may promote peptide aggregation (a competing and irreversible reaction) that would tend to decrease the effective concentration of NC-1059 in the bath and in the cell membrane. Alternatively, the response may diminish due to protease degradation of the peptide, in turn due to uptake of peptide from the apical membrane that subsequently leads to proteolysis. Data presented in Fig. 4 suggest that peptide aggregation or inactivation cannot account for the transient nature of the effects. The possibility remains that there may be some downregulation of the metabolic process that modulates junction integrity. Data presented in Fig. 3 argue against this latter possibility in that the response to a submaximal concentration remains above baseline for at least 2 h and the epithelium subsequently responds to a higher concentration of NC-1059. Nonetheless, these and other possibilities may in part account for the transient nature of the response; additional experiments must be conducted to more fully evaluate these hypotheses.
There are numerous clinical situations in which modulation of an epithelial barrier presents therapeutic benefits. Drug absorption across intestinal, airway, or dermal epithelium could be enhanced with transient decreases in barrier function, making oral, inhaler, or topical administration of what are now parenteral drugs possible (9, 11). Oral or inhalant formulations of medications such as insulin would be less expensive to produce and more easily delivered than parenteral formulations (37). The permeability of the small intestinal epithelium to both insulin and immunoglobulin G was increased in rabbits when coadministered with ZOT (11). However, ZOT is limited in its therapeutic application because it is only effective in the small intestine (10). Additionally, ZOT is a 45-kDa protein that must be recombinantly produced and purified, although it has been shown that the majority of biological activity can reside in the COOH-terminal 15-amino acid segment (6). The M2GlyR-derived peptides are <3 kDa and can be prepared synthetically or recombinantly expressed.
Gene therapy for epithelium-associated diseases such as CF provides a second therapeutic setting in which modulation of transepithelial permeability is desirable (24, 32). Stable transfection of DNA sequences into epithelial cells in culture (7, 27) provides proof that genetic epithelial diseases can be treated or cured. However, bronchiolar epithelial cell viral receptors are located primarily in the basolateral membrane (17, 25, 38, 39), leading to a low efficiency of gene transfer from apical exposure to viral vectors. Thus high viral titers and long incubation times are required to increase transfection efficiency, which can lead to a decrease in the effectiveness of repeated treatments. Increased transfection efficiency has been achieved with some chemical modulators of tight junctions (e.g., EGTA, perfluorochemicals, fatty acids), although these treatments were sometimes associated with inflammation (5, 17). Modulators of epithelial barrier function would be the ideal agents to augment gene therapy, provided that they have a rapid onset, transient duration of action, and favorable safety profile, and do not decrease viral titer (35). Initial observations with NC-1059 suggest that it may fulfill these criteria, although additional experiments will be required to determine whether viral access is limited due to size exclusion (i.e., <77 kDa). Additional experiments would also be required to test for inflammation. In this regard it is encouraging that similar effects on gTE have been observed when an all-D-amino acid form of NC-1059 was used (unpublished observations).
NC-1059 also presents the potential for developing an increased understanding of physiological and pathophysiological processes that modulate tight junctions. Various epithelia throughout the body exhibit transepithelial electrical resistances that vary over four orders of magnitude (37), some of which change depending on the hormonal state (e.g., mammary; Refs. 22, 29) or physical environment (e.g., small intestine following a meal; Ref. 23). Signaling pathways that affect the paracellular pathway are not fully defined for any epithelium, and it is not known which mechanisms are broadly applicable and which are species or tissue specific. In this regard, it is noteworthy that NC-1059 stimulates gTE across male porcine reproductive epithelia, porcine ileal epithelia (IPEC-J2), and human colonic epithelia (Caco-2; unpublished observations). Thus, unlike ZOT, which affects only the small intestine (11), NC-1059 affects a broader spectrum of epithelia and, unlike Clostridium perfringens enterotoxin (19), causes no discernible cell damage. Thus NC-1059 can be used to survey a variety of tissues to identify common regulatory mechanisms.
In summary, NC-1059 is a channel-forming peptide that reversibly modulates conductance through the epithelial paracellular pathway. The response is fully repeatable and occurs without overt indications of cytotoxicity. This functionality provides a unique opportunity to conduct research regarding the mechanisms that selectively modulate tight junction maintenance. Additionally, NC-1059 represents a novel lead compound that might be developed to augment other forms of therapy that are currently limited by the epithelial barrier.
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GRANTS |
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ACKNOWLEDGMENTS |
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This manuscript represents contribution number 03-126-J from the Kansas Agricultural Experiment Station.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Multiple peptide-treated and control monolayers were surveyed over numerous microscope fields to determine whether any evidence of cell sloughing could be identified in either of these conditions. A confluent monolayer was observed in all cases.
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