T84 cell receptor binding and guanyl cyclase
`activation by Escherichia coli heat-stable toxin
`
`ALFREDO GUARINO, MITCHELL COHEN, MICHAEL THOMPSON,
`KIERTISIN DHARMSATHAPHORN, AND RALPH GIANNELLA
`Division of Digestive Diseases, Department of Internal Medicine, Veterans Administration Hospital,
`Cincinnati 45220; University of Cincinnati College of Medicine, Cincinnati, Ohio 45267; and Division of
`Gastroenterology, Department of Medicine, Medical Center, University of California,
`San Diego, California 92103
`
`GUARINO, ALFREDO, MITCHELL COHEN, MICHAEL THOMP-
`SON, KIERTISIN DHARMSATHAPHORN, AND RALPH GIAN-
`NELLA. T84 cell receptor binding and guanyl cyclase activation
`by Escherichia coli heat-stable toxin. Am. J. Physiol. 253 (Gas-
`trointest. Liver Physiol. 16): G775-G780, 1987.-Escherichia
`coli heat-stable enterotoxin (STa) induces intestinal secretion
`by binding to enterocyte receptors and activating the guanylate
`cyclase-guanosine 3',5'-cyclic monophosphate (cGMP) system.
`The intermediate steps between binding of STa and secretion
`are poorly understood, due in part to the lack of a convenient
`system to study the effects of STa at the cellular level. To
`establish such a model, we investigated the binding of ' 25I-STa,
`STa activation of guanylate cyclase, and STa-induced increase
`in cGMP production in a well-characterized human colonic cell
`line, Ts,. Binding was specific, linear with cell number, and
`time, temperature and pH dependent, and reversible. ST may
`also be internalized by these cells. Addition of unlabeled STa
`competitively inhibited binding of 125I-STa. These parameters
`closely resemble those described in intact rat enterocytes and
`cell-free membrane preparations. STa stimulated guanylate
`cyclase and cGMP production in a dose-related manner. The
`similar dose-response relationships for binding, guanylate cy-
`clase stimulation by STa, and cGMP production suggest that
`the guanylate cyclase-cGMP system is coupled to ST occupancy
`of specific receptors. These data, together with the fact that
`STa induces chloride secretion from T84 cells suggest that Ta4
`cells are a suitable and convenient system to study the cellular
`mechanism of action of STa.
`
`sine 3',5'-cyclic monophosphate (cGMP), through the
`activation of particulate guanylate cyclase activity (5),
`and impaired sodium chloride absorption and net chlo-
`ride secretion (15). The binding of STa to rat small
`intestinal cells and to brush-border membranes has been
`previously described by us and others (7, 9, 11). However,
`the biological processes that follow the binding of STa
`are poorly understood, due in part to the lack of a
`convenient and suitable model to study the effects of
`STa at the cellular level.
`We have used a human colonic cell line, the TM cell
`line (4), to establish a model suitable to study the specific
`mechanisms of STa receptor-effector interactions. We
`report here the characterization of the binding of 'I-
`STa to TM cells, as well as STa-induced stimulation of
`guanylate cyclase and cGMP production. We chose the
`T84 cell line because these cells maintain the morpholog-
`ical characteristics of well-differentiated colonic epithe-
`lial cells. In addition, they retain vectorial electrolyte
`transport properties in response to various hormones (4).
`Furthermore, these cells have been recently shown to
`secrete chloride in response to STa (13). The TM cells
`could therefore provide an experimental model to study
`STa-cell interactions from the initial binding to the final
`secretion.
`
`intestinal secretion; diarrhea; high-performance liquid chro-
`matography
`
`METHODS
`Cell culture. T84 cells were maintained and grown as
`previously described (4). Briefly, cells were maintained
`in a 1:1 mixture of Dulbecco's modified Eagle's medium
`and Ham's F-12 medium with 15 mM N-2-hydroxyethyl-
`piperazine-N'-2-ethanesulfonic acid (HEPES) buffer,
`pH 7.4, 1.2 g of NaHCO3, 40 mg penicillin, 8 mg ampi-
`cillin, and 90 mg streptomycin per liter plus 5% newborn
`calf serum. After 1 wk, confluent monolayers were split
`from one 100-mm tissue culture plate to two 24-well
`plates, using 0.1% trypsin and 0.9 mM EDTA in Ca',
`Mg2±-free phosphate-buffered saline (PBS).
`Purification and labeling of STa. STa enterotoxin was
`purified from E. coli strain 18D as previously described
`(20). Pure STa, quantitated by amino acid analysis and
`by a specific enzyme-linked immunosorbent assay (19),
`was radioiodinated by a lactoperoxidase method and
`G775
`Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 15, 2019.
`
`ESCHERICHIA COLI can cause diarrhea in humans by
`producing one or more enterotoxins. These are a large
`molecular weight heat-labile enterotoxin (LT) and small
`molecular weight heat-stable enterotoxins (ST) (16). The
`latter have been divided into two groups, designated STa
`and STb, based on different biochemical characteristics
`and on differing host susceptibility (2). STa is elaborated
`in two forms, an 18- or 19-amino acid peptide, both of
`which have been sequenced (1, 3, 21). STa induces diar-
`rhea through a mechanism that has been investigated in
`several animal models. The toxin binds to specific recep-
`tors located on the brush border of small intestinal
`enterocytes (9). This induces a prompt increase in guano-
`
`MYLAN EXHIBIT - 1037
`Mylan Pharmaceuticals, Inc. v. Bausch Health Ireland, Ltd. - IPR2022-00722
`
`

`

`G776
`
`EFFECTS OF E. COLI ST IN A HUMAN CELL LINE
`
`purified using a C-18 Sep-Pak cartridge (Millipore) and
`high-performance liquid chromatography as previously
`described (20). 'I-STa monoiodinated in the 4-tyrosine
`position was used for binding studies, since it has been
`shown to be stable, homogeneous, and retain its biolog-
`ical activity (20)
`Binding of 1251-STa to T84 cells. Binding assays were
`performed in tissue culture plates containing 24 x 16-
`mm wells. Each well contained approximately 6 x 105
`cells or 200 µg protein. The variation in number of cells
`from well to well did not exceed 15%. Prior to the start
`of each experiment, cells were washed three times with
`PBS and placed for 30 min at 37°C in a humidified CO2
`incubator. Each well received 1 ml of F-12 modified
`Eagle's medium buffered with 15 mM 2-(N-morphol-
`ino)ethanesulfonic acid (MES) and containing 100,000-
`150,000 cpm (48-73 pM) 125I-STa. Incubation was per-
`formed at 37°C for 1 h or other times as stated in the
`RESULTS. Particular experiments were also performed at
`4°C as well. The pH of the medium used for the binding
`studies was 5.8 and did not change during the course of
`the experiment. After incubation, cells were rinsed three
`times with PBS and solubilized by incubation with 0.5
`M NaOH for 1 h at room temperature. The remaining
`cell-associated radioactivity was then quantitated by
`counting the cell extracts in a Packard scintillation spec-
`trometer. Specific binding was determined by measuring
`binding of 1251-STa to Ts4 cells in the presence of excess
`unlabeled STa (1 µg/ml) and subtracting this nonspecific
`binding from total binding (binding in the presence of
`l'I-STa alone). Protein was measured by the method of
`Lowry et al. (14). Cells were counted using a hemocytom-
`eter.
`Binding of 1251-STa to T&1 membranes. T84 cell mem-
`branes were prepared as follows. T84 cells were twice
`washed with (in mM) 5 EDTA, 1 HEPES, 0.1 phenyl-
`tris(hydroxymeth-
`methylsulfonyl fluoride, and 8.2
`yl)aminomethane (Tris), pH 7.5, and harvested by
`scraping with a glass pestle. The scrapings were homog-
`enized in EDTA buffer for 1.5 min in an Omni-Sorvall
`mixer at maximum speed and centrifuged at 11,500 g for
`30 min. The pellet was resuspended in F-12 modified
`Eagle's medium and used immediately. Binding assays
`were performed in 12 x 75 glass test tubes containing a
`total volume of 1 ml of modified F-12 medium, pH 5.8,
`containing 15 µg/ml bovine serum albumin (BSA),
`-190,000 cpm (92 pM) ' 5I-STa, and membranes. Incu-
`bations were performed at 37°C. The reaction was ter-
`minated by suction filtration through Whatman GF/B
`filters, each filter was washed three times with ice-cold
`F-12 medium, and the radioactivity was counted. Specific
`and nonspecific binding were calculated as previously
`described (9).
`Guanylate cyclase assay. Confluent cells from two 100-
`mm flasks were washed twice with PBS, removed by
`scraping, and hand homogenized with a Wheaton A
`pestle for 60 s (18 strokes) in 10 ml of sucrose-EDTA-
`dithiothreitol (DTT) buffer (0.25 M sucrose, 50 mM
`Tris • HC1, 1 mM EDTA, 1 mM DTT, pH 7.9). The
`homogenate was diluted twofold in the same buffer and
`centrifuged at 32,000 g for 20 min. The pellet was washed
`
`in 5 mM Tris, pH 7.6, and resuspended to yield a final
`protein concentration of 15-30 µg/10 µl. Guanylate cy-
`clase was determined as reported by Waldman et al. (22).
`The assay was performed by incubating the samples for
`10 min at 37°C in the presence of GTP and a GTP-
`regenerating mixture (22). (Preliminary experiments
`demonstrated that 10 min, 37°C, pH 7.6, and 10-40
`of protein are optimal conditions for the assay.) cGMP
`formed was determined using an radioimmunoassay
`(RIA) assay as previously described (8).
`Coupling between 1251-STa binding to Tg4 cells and STa-
`induced increase in cGMP production. To determine
`whether binding of STa to T84 cells was coupled to cGMP
`production, cells were incubated at 37°C in 24-well plates
`with 125I-STa in the presence of increasing concentra-
`tions of unlabeled STa in modified F-12 medium, pH 5.8.
`After 15 min the reaction was terminated, and binding
`was measured as described above. In parallel wells, the
`same increasing concentrations of STa in modified F-12
`medium, pH 5.8, were added and production of cGMP
`was determined as follows. After 15 mM at 37°C, each
`well received 1 ml ice-cold 10% trichloroacetic acid
`(TCA), and after 30 min, the supernatant from each well
`was collected and centrifuged. cGMP in the supernatant
`was purified by column chromatography on Dowex
`AG50W-X8 resin and measured by RIA as previously
`described (8). [3H]cGMP added to TCA was used as a
`recovery marker.
`Data presentation and analysis. All experimental
`points of binding studies were performed in triplicate
`and each experiment was repeated at least three times.
`Results are expressed as means ± SE of the counts per
`minute of 1251-STa specifically bound per 100 µg of cell
`protein.
`ST-stimulated guanylate cyclase activity, measured as
`picomoles of cGMP formed per minute per milligram of
`protein, is expressed as fold increase over basal level.
`Incubations with STa were performed in triplicate and
`each cGMP determination was assayed in duplicate.
`Production of cGMP after addition of STa was assayed
`in triplicate and results are expressed as picomoles of
`cGMP formed per milligram of protein. Results of the
`dose-response curves were calculated using the computer
`program "Allfit" (17). Each experiment was performed
`at least three times, and the data are presented as means
`± SE.
`
`RESULTS
`
`Effect of time and protein concentration. The time
`courses of total, specific, and nonspecific binding of 1251-
`STa to T84 cells are shown in Fig. 1. The rate of binding
`was highest in the initial 15 min, and binding reached a
`plateau at —3 h. After 3 h of incubation no further
`binding was observed. In other experiments, incubation
`was continued for up to 5 h, and the radioactivity that
`was bound remained constant (data not shown). Non-
`specific binding did not exceed 15% of total binding at
`any point in time. Binding was linear with protein con-
`centration, in the range of 150-350 µg/ml (data not
`shown).
`Effect of temperature. The effect of temperature on
`
`

`

`EFFECTS OF E. COLI ST IN A HUMAN CELL LINE
`
`G777
`
`TOTAL
`-
`-
`
`SPECIFIC
`
`FIG. 1. Time course of binding of 12I-heat-stable
`enterotoxin (STa) to T84 cells. Cells were incubated in
`1 ml of F-12 Dulbecco's modified Eagle's medium buff-
`ered with 2- (N-morpholino)ethanesulfonic acid, con-
`taining '"I-STa with and without -I µg (500 nM) of
`unlabeled STa at 37°C. Total, nonspecific, and specific
`binding were determined as described in METHODS.
`Results are expressed as counts per minute bound per
`100 µg protein and represent means ± SE of 3 separate
`experiments, each carried out in triplicate.
`
`2500
`
`2000
`
`1500
`
`1000
`
`500
`
`1251-STa Binding (cpm/100pg)
`
`......
`15
`
`30
`
`45
`
`60
`
`90
`minutes
`
`120
`
`NON-SPECIFIC
`
`a
`150
`
`180
`
`2000
`
`1500
`
`1000
`
`500
`
`a
`
`a,
`C
`
`a)
`W
`Co
`
`Co
`
`0
`
`15
`
`30
`
`60
`minutes
`binding of 'I-STa to T8,4 cells is shown in Fig. 2. Binding
`was temperature dependent. At 37°C, the initial rate of
`binding was greater than at 4°C, such that after 15 min
`binding was twofold greater at 37 than at 4°C. If the
`incubation was continued, the rate of binding became
`parallel at both temperatures, and after 2 h, the binding
`at 4°C was —75% of that at 37°C.
`Effect of pH. The effect of pH is shown in Fig. 3. The
`pH of the medium was adjusted immediately prior to the
`experiment by titration with HC1 or NaOH as appropri-
`ate. Binding was dependent on the pH of the medium.
`At pH 4.5 (the lowest pH tested) binding was maximal
`and then fell rapidly as the pH was increased. The pH
`of the media did not change during the course of the
`experiment.
`Dissociation of ' 25I-STa bound to T84 membranes. As
`shown in Fig. 4, the addition of excess unlabeled STa (1
`µg/ml) resulted in the prompt dissociation of ' 25I-STa
`bound to T84 membranes. When native toxin was added
`15 min after the initiation of binding, —92.6% of 1251-
`STa dissociated by 6 h; when native toxin was added 30
`min after binding, —83.7% of 125I-STa dissociated by 6
`
`FIG. 2. Effect of temperature on specific binding of
`19 -heat-stable enterotoxin (STa) to T8,4 cells. Results
`are means ± SE of counts per minute specifically
`bound per 100 µg protein of 3 separate experiments.
`
`37°C -
`
`4° C
`
`120
`
`h; and when native STa was added 60 min after binding,
`—64.4% of 125I-STa dissociated by 6 h. However, when
`the three dissociation curves were linearized by logarith-
`mic transformation (plotted as logarithm of counts per
`minute vs. time), all three curves intersected the X-axis,
`indicating complete dissociation (data not shown).
`Competitive inhibition of binding. Native STa com-
`peted with 'I-STa for binding to Te4 cells. As shown in
`Fig. 5, when T84 cells were incubated with 1-25I-STa at
`either 37 or 4°C, together with increasing concentrations
`of unlabeled ST, a dose-dependent inhibition of 125I-STa
`binding was observed. Although the 37 and 4°C binding
`curves are parallel (suggesting similar Kas), maximal
`binding at 4°C was only approximately 63.8 ± 13.5% of
`that seen at 37°C (P < 0.05).
`Effect of ST on guanylate cyclase activity. Addition of
`STa to homogenates of T84 cells resulted in a dose-related
`increase in guanylate cyclase activity (Fig. 6), reaching a
`plateau at an STa concentration of —10-7 M. The maxi-
`mal stimulation of guanylate cyclase activity was nine-
`fold over basal level. The effect of STa on guanylate
`cyclase was extremely rapid. A significant stimulatory
`
`

`

`EFFECTS OF E. COLI ST IN A HUMAN CELL LINE
`
`FIG. o Effect of p•U on binding of 125I-heat-
`stable enterotoxin (STa) to TM cells. Incubation
`was performed at 37°C for 1 h in parallel wells
`containing 1 ml of medium, adjusted to appropriate
`pH. Results are means ± SE of 3 different experi-
`ments.
`
`4.5
`
`5.5
`
`6.5
`
`7.5
`
`pH
`
`1500
`
`a
`0
`0
`
`E a 1000
`
`500
`
`0 a
`
`CI)
`
`o
`
`10-10
`
`10-9
`
`10-8
`ST (molar)
`FIG. 5. Competitive inhibition of 'I -heat-stable enterotoxin (STa)
`binding by unlabeled STa. Cells were incubated with '"I-STa and with
`increasing concentrations of STa at 37°C (closed circles) or 4°C (open
`circles) for 1 h. Results are means ± SE of 3 separate experiments.
`
`10-7
`
`10 6
`
`G778
`
`2500
`
`° 2000
`
`1 500
`
`co
`
`— - 1000
`
`a
`
`500
`
`25
`
`20
`
`- 15
`0
`
`0 10
`
`0 E
`
` a
`
`5
`
`4
`
`5
`
`6
`
`0
`
`1
`
`2
`
`3
`HOURS
`FIG. 4. Dissociation of 19 -heat-stable enterotoxin (STa) bound to
`membranes by addition of excess nonlabeled STa. T. membranes
`were incubated with 1251-STa at 37°C, and at the times indicated by
`arrows excess cold toxin (500 nM) was added and binding was measured
`for a total of 6 h. Results are means ± SE of 3 separate experiments.
`
`effect was seen as early as 2 min (the shortest time
`tested) after the addition of STa (data not shown).
`Coupling between binding of STa and cGMP stimula-
`tion. Experiments of competitive binding and cGMP
`stimulation by STa were performed in parallel plates
`under the same pH and other conditions. Shown in Fig.
`7 are the dose-response relationships of the ability of
`unlabeled STa to inhibit binding of 125I-STa to the T84
`cells and to stimulate cGMP. Results are expressed as
`percent of maximal activity. The ability of STa to inhibit
`the binding of 125I-STa closely correspond to its ability
`to stimulate cGMP formation. The concentrations of
`STa at which a half-maximal effect was obtained in each
`
`assay were 1.5 ± 0.4 x 10' and 3.1 ± 0.3 x
`respectively.
`
`M,
`
`DISCUSSION
`The initial step in the induction of secretion by STa
`appears to involve binding of ST to a specific cell surface
`receptor (9). This binding step has been studied in iso-
`lated rat enterocytes (9), rat brush-border membranes
`(7), rat basophilic leukemia cells (18), and solubilized
`partially purified rat enterocyte receptor preparations
`(12). Although a great deal is known about STa interac-
`tion with its receptor (7, 9, 12) and the alterations in ion
`transport mechanisms induced by ST (5, 10, 15), rela-
`tively little is known about the intermediate mechanism
`of action linking receptor occupancy to secretion. In the
`present study we- wish to determine whether the human
`colonic Te4 cell line might be a suitable model to further
`investigate the mechanisms of action of STa. We have,
`therefore, examined whether these cells possess ST-re-
`
`

`

`EFFECTS OF E. COLI ST IN A HUMAN CELL LINE
`
`G779
`
`9
`
`cts 7
`•
`_• o
`E
`
`U
`a)
`-
`
`C/3 ro
`3. N
`c
`cs
`
`E
`O -0
`O
`LL
`
`3
`
`_ L I
`0 10-10
`
`10-9
`
`10-8
`ST (molar)
`FIG. 6. Stimulation of guanylate cyclase activity induced by heat-
`stable enterotoxin (STa). A homogenate of T8, cells was incubated with
`increasing concentrations of STa at 37°C for 1 h. Guanylate cyclase
`activity is expressed as fold increase over basal level. Results are means
`± SE of 3 separate experiments. Base-line level was 6.4 ± 1.7 pmol of
`cGMP formed per minute per milligram of protein.
`100 -
`
`10-7
`
`10-6
`
`-100
`
`oc I
`
`7 0
`E E
`ro
`
`o
`2
`
`"6
`
`Q,
`
`8 0 -
`
`60 -
`
`40
`
`20
`
`-80
`
`60
`
`40
`
`cn coco
`
`g
`o
`
`x j
`O
`O
`
`20
`
`H
`
`0
`
`10-10
`
`1
`10-9
`
`10-8
`
`10-7
`
`10-6
`
`ST (molar)
`FIG. 7. Dose-response relationships between ability of nonlabeled-
`heat-stable enterotoxin (STa) to inhibit binding of labeled STa and to
`increase cGMP concentration. Tg4 cells were incubated with increasing
`concentrations of STa for 15 min at 37°C, and binding and cGMP
`concentration were measured as described in METHODS. Results, ex-
`pressed as percent of maximal effect, are means ± SE of 3 separate
`experiments. Under these conditions respective ED50 were 1.5 and 3 x
`10' M.
`
`ceptors and guanylate cyclase activity, whether the gua-
`nylate cyclase-cGMP system is responsive to ST, and
`whether receptor occupancy by STa is coupled to gua-
`nylate cyclase and cGMP stimulation.
`T84 cells bound " 5I-STa and binding was highly spe-
`cific, linear with cell number, and time, temperature and
`pH dependent. These characteristics are similar to those
`reported for ST binding to isolated rat small intestinal
`cells and brush-border membranes (9, 11). The effect of
`pH on binding of ' 25I-STa to T84 cells closely resembles
`that which we have previously reported for rat intestinal
`brush-border membranes (11). Binding increased mark-
`edly as the pH of the medium was reduced. We have
`demonstrated that the increased binding seen at lower
`
`pH in the intestinal brush-border membrane system is
`explained by a greater number of receptors exposed at a
`lower pH value, while the apparent Ka of binding is
`unaltered (11).
`The fate of receptor-bound 'I-STa is uncertain. It is
`possible that a portion of 1251-STa could be internalized
`by the T84 cells and/or irreversibly bound to the surface
`receptors. When the competitive inhibition experiments
`at 37°C were compared with those done at 4°C (Fig. 5),
`maximal binding at 4°C was only —64% of that seen at
`37°C. Thus it is possible that at 37°C a portion of the
`'I-STa bound to T8.4 cells is internalized by these cells.
`To determine whether an element of irreversible re-
`ceptor binding might also be occurring, experiments were
`performed to examine the dissociability of 125I-STa from
`T8,4 membranes. Addition of excess native STa resulted
`in the dissociation of 125I-STa bound to such membranes
`(Fig. 4). The rate of dissociation, however, was quite slow
`and receptor binding did not reach zero during the du-
`ration of the experiment. Visual examination of the
`curves, however, suggests that complete dissociation
`would ultimately occur. Log transformation of the three
`dissociation curves reveals all three to be straight lines
`and to intersect the X-axis, thereby confirming the visual
`impression. Thus dissociation from TS4 membrane recep-
`tors, although slow, is ultimately complete.
`STa increases cGMP levels through the activation of
`guanylate cyclase (5, 10). We have shown that T84 cells
`possess an STa-responsive guanylate cyclase activity and
`that activation of guanylate cyclase was dose and time
`dependent, closely resembling results obtained in animal
`models (5, 10, 22). The close correlation, as reflected by
`the similar ED5os, between the dose-response relation-
`ship of STa in inhibiting binding of 'I-STa to T84 cells
`and in stimulating cGMP formation suggest that these
`processes are coupled. We have previously described the
`same correlation in rat small intestinal cells (9), and the
`ED50 for both binding of STa and cGMP stimulation
`(1.25 x 10') found in rat enterocytes are nearly identical
`to those found in T84 cells. Furthermore, we have also
`shown that the STa dose-response of activation of gua-
`nylate cyclase is also similar to the STa dose-response
`of inhibition of binding of l'I-STa (Figs. 5 and 6).
`Although these experiments were done under different
`conditions than those employed in the cGMP-'25I-STa
`coupling experiments, these similarities in stoichiometry
`are consistent with the coupling of STa binding, activa-
`tion of guanylate cyclase, and production of cGMP.
`We have, therefore, characterized the binding of STa
`to T84 cells and established a reproducible model that
`involves the use of a monoiodinated, homogeneous radi-
`oligand (20). We have also shown that the T84 cell line
`responds to STa with an increase in guanylate cyclase
`activity and cGMP formation and that the stimulation
`of the guanylate cyclase-cGMP system is likely coupled
`to the occupancy of specific receptors by STa. Liu et al.
`(13) have recently shown that STa causes an increase in
`cGMP concentration coupled to chloride secretion in
`this the same cell line. The T54 cell line, therefore, seems
`a suitable system to study the mechanism of action of
`STa at the cellular level. The T84 cell line cannot only
`
`

`

`G780
`
`EFFECTS OF E. COLI ST IN A HUMAN CELL LINE
`
`be used to further study receptor binding and the mech-
`anisms of receptor-effector coupling, but also can help
`clarify the intermediate steps between binding and se-
`cretion that are still unknown.
`
`We gratefully acknowledge the expert technical assistance of Gary
`Overmann and Marcia Osterhues and the secretarial assistance of
`Margaret Doerflein.
`This work was supported by Veterans Administration Research
`Project 539-3108-01 and by National Institutes of Health Grants AI-
`20261 and AM-28305.
`
`Received 30 June 1986; accepted in final form 28 July 1987.
`
`REFERENCES
`
`1. AIMOTO, S., T. TAKAO, Y. SHIMONISHI, S. HARA, T. TAKEDA, Y.
`TAKEDA, AND T. MIWATANI. Amino-acid sequence of a heat-stable
`enterotoxin produced by enterotoxigenic Escherichia coli. Eur. J.
`Biochem. 129: 257-263, 1982.
`2. BURGESS, M. N., R. J. BYWATER, C. M. COWLEY, N. A. MULLAN,
`AND P. M. NEWSOME. Biological evaluation of a methanol-soluble,
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