1 Research Institute and
Pulmonary Center, The patch-clamp technique was used to investigate the effects of
the isoflavone genistein on disease-causing mutations (G551D and
cystic fibrosis transmembrane conductance regulator mutations; patch clamp; nasal potential difference; flavonoids
CYSTIC FIBROSIS (CF) is a common genetic disease in the
Caucasian population and is caused by mutations in the CF transmembrane conductance regulator (CFTR), the epithelial cAMP-regulated Cl channel.
CF is characterized by the absence of a cAMP-stimulated Cl conductance
across a variety of epithelia, including the respiratory tree,
pancreatic ducts, sweat glands, and the intestine. The commonest mutation is a deletion of phenylalanine at position 508 ( The increasing knowledge about the different cell biological fates of
different CFTR mutants revealed selective cellular pharmacological targets. For example, 4-phenylbutyrate (4-PB) was shown to increase expression of Generation of recombinant adenovirus carrying the
G551D gene. Recombinant adenovirus carrying the CFTR
G551D mutation was generated by a modified ligation procedure. With the
use of PCR-mediated point mutation, G551D was generated by converting
the codon for glycine (GGT) at position 551 to an aspartic acid codon
(GAT). A plasmid containing wtCFTR cDNA, pBQ4.7, was used
as the template for the PCR reaction (6). The G551D cDNA was inserted
into a shuttle vector, pLadC, which contains the left-hand terminal of
adenovirus sequence and a cytomegalovirus promoter. The
recombinant adenovirus, AdcG551D, was generated by ligation of the
linearized pLadC-G551D to the right-hand portion of the adenovirus
sub360, which contains the deletion in the E1a and E3 regions. The
ligated products were transfected into 293 cells with Lipofectamine
(GIBCO, Life Technologies, Grand Island, NY). Correct recombinants were isolated using a procedure described in Ref. 10. AdcG551D isolated from
individual plaques were propagated in 293 cells. The inserted G551D
cDNA was confirmed using PCR protocols. This viral suspension was
divided into aliquots and stored at Cells. The cervical adenocarcinoma
cell line HeLa was cultured in H21 DMEM supplemented with 10% FCS. For
patch clamping, cells were seeded at low density on cover glasses.
Twenty-four hours before use, cells were infected with viral vectors
containing G551D-CFTR, wtCFTR, or LacZ as control at a multiplicity of
infection of ~100. A virus-based infection of cells was
used to achieve the transduction of all cells for patch clamping. The
JME cell line is a continuous cell line derived from the nasal
epithelium of a homozygous Patch-clamp recordings. Cells were
patch clamped on the stage of an inverted microscope in a constantly
perfused chamber at 37°C as described in Refs. 8 and 15. In the
whole cell mode, a 29:150 mM Cl gradient from pipette to bath was used
as a driving force while the cell membrane potential
(Vm) was
clamped to zero. Because there were no other significant gradients or
driving forces, the resulting membrane current
(Im) is a Cl
current. The bath solution had the following composition (in mM) 145 N-methyl-D-glucamine chloride (NMDG-Cl), 1.7 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, and
10 sucrose, pH = 7.4. The pipette solution contained (in mM) 27 NMDG-Cl, 2 EGTA, 1 MgCl2, 10 HEPES, 5 glucose, 110 NMDG-gluconate, 5 MgATP, and 0.1 NaGTP, pH = 7.4. Current-voltage (I-V) relations were
fitted with the Goldman equation (15), which resulted in fit estimates
for the apparent intracellular Cl concentration (Cli) and the reversal potential
(Erev).
Membrane conductance (Gm) was
calculated at 0 mV with Ohm's law of the form
Gm = Im/Erev.
The average single channel open probability
(Po) of the
population of channels in whole cell recordings was calculated from current variance ( Recordings in the cell-attached patch-clamp mode were performed with
the 145 mM NMDG-Cl solution in both bath and pipette. Po and
i were determined with pCLAMP version
7 (Axon Instruments, Foster City, CA) using standard methods, and
N was defined as the maximum number of
open levels observed in the total length of the recording.
Nasal potential difference
measurements. Patients with at least one G551D-CFTR
allele were recruited from Children's Hospital Oakland and from the
Stanford CF Center at Lucile Salter Packard Children's Hospital.
Measurements were approved by the Internal Review Board of Children's
Hospital Oakland and done exactly as described in Ref. 15. The NaCl
solution contained (in mM) 145 NaCl, 4 KCl, 1 CaCl2, 1 MgCl2, and 10 HEPES, pH = 7.4. In
Cl-free solutions all Cl salts were replaced by the respective
gluconate salts.
Chemicals. Unless otherwise mentioned,
chemicals were from Sigma (St. Louis, MO). Forskolin (Calbiochem, La
Jolla CA), genistein (4',5,7-trihydroxy-isoflavone), and
quercetin (3',3,4',5,7-pentahydroxy-flavone) were made as
100 mM stock solutions in DMSO. 4-PB was made as a 5 M stock in DMSO
and used at 5 mM. Amiloride was made as a 10 mM stock and isoproterenol
as a 100 mM stock in water.
Effects of genistein on G551D-CFTR in patch-clamp
recordings. HeLa cells infected with the
G551D-CFTR-containing adenovirus were patch clamped in the whole cell
and the cell-attached mode. Figure
1 shows a whole cell
recording from a G551D-CFTR-expressing cell. Stimulation with forskolin
(10 µM) caused only a very small increase in current (Fig.
1A). On average
forskolin-stimulated conductance was 0.19 ± 0.10 nS
(n = 4). Addition of genistein (30 µM) stimulated large Cl currents, which were time and voltage independent (Fig. 1, B and
C). Data were well fitted with the Goldman equation (Fig. 1D), which
are typical biophysical characteristics of CFTR-mediated currents.
Average forskolin-plus-genistein-activated conductance was 2.1 ± 0.61 nS (n = 4). Current
variance to mean current plots was used to calculate the average
Po of
G551D-CFTR-mediated whole cell current. Figure
1E shows an example of this analysis applied to the current trace shown in Fig.
1A. Note that current noise
significantly increased during stimulation of current.
Po remained low
in the presence of forskolin, reaching a maximum of 0.04 (Fig.
1E, open circles). Stimulation with
genistein increased Po to a maximum
of 0.46 (Fig. 1E, closed circles).
Figure 1F shows average
Po values of
G551D-CFTR compared with wtCFTR. Note the low
Po of G551D-CFTR
during forskolin stimulation (0.047 ± 0.19, n = 4), which was increased by
genistein to 0.49 ± 0.12, a value not different from wtCFTR.
LacZ-infected control HeLa cells (n = 4) or uninfected control HeLa cells (n = 4) showed no responses to treatment with forskolin and genistein
(data not shown).
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
F508) of the cystic fibrosis transmembrane conductance regulator (CFTR). In HeLa cells recombinantly expressing the
trafficking-competent G551D-CFTR, the forskolin-stimulated Cl currents
were small, and average open probability of G551D-CFTR was
Po = 0.047 ± 0.019. Addition of genistein activated Cl currents
~10-fold, and the Po of G551D-CFTR
increased to 0.49 ± 0.12, which is a
Po similar to
wild-type CFTR. In cystic fibrosis (CF) epithelial cells homozygous for
the trafficking-impaired
F508 mutation, forskolin and genistein activated Cl currents only after 4-phenylbutyrate treatment. These data
suggested that genistein activated CFTR mutants that were present in
the cell membrane. Therefore, we tested the effects of genistein in CF
patients with the G551D mutation in nasal potential difference (PD)
measurements in vivo. The perfusion of the nasal mucosa of G551D CF
patients with isoproterenol had no effect; however, genistein
stimulated Cl-dependent nasal PD by, on average,
2.4 ± 0.6 mV, which corresponds to 16.9% of the responses (to
-adrenergic
stimulation) found in healthy subjects.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
F508), which occurs on 66% of CF chromosomes world wide. The
glycine-to-aspartic acid missense mutation at codon 551 (G551D) is the
third commonest CF mutation, with a worldwide frequency of ~2% on CF
chromosomes (25, 27). Higher frequencies of G551D are found in
populations of Celtic descent, with incidences of 6.9% and 5.5% in
the Irish and Australian CF populations, respectively (3, 12, 25). A
low incidence of 0-0.9% was reported in CF populations of central European origin (9, 11, 26).
F508-CFTR is a trafficking-impaired mutation (class II mutation),
and the synthesized protein is largely degraded intracellularly. The
G551D-CFTR protein traffics to the apical membrane, but Cl channel
function is markedly reduced (class III mutation) (27). Compared with
CF patients homozygous for the
F508 mutation, patients carrying the
G551D mutation show a threefold reduction in the incidence of meconium
ileus (neonatal intestinal blockage), a trend toward later age at
diagnosis of pancreatic insufficiency (12), and milder disease in
homozygotes (22). Interestingly, both the
F508 mutation and the
G551D mutation are in NBD1 of CFTR; however, ATP binding of NBD1 is not
affected by the
F508 mutation but is significantly impaired in
G551D-CFTR (21).
F508-CFTR in the plasma membrane (24). We found that
the isoflavone genistein and related flavonoids were potent activators
of wild-type (wt) CFTR in vitro and in vivo in human subjects (15) and
suggested that genistein activates CFTR when it is available in the
cell membrane. We therefore tested its effects on G551D-CFTR, which is
present in the cell membrane, and on
F508-CFTR, once it was
translocated in sufficient quantity to the membrane after 4-PB treatment.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
85°C until use.
F508 CF patient and was cultured as
described in Ref. 16.
2) to mean
current plots (7). Data were fitted to Sigworth's parabola of the form
2 = Im · i
Im2/N,
which estimated the single channel current
(i) and the number of channels in
the recording (N).
Po was then
calculated from Po = Im/(N · i).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (22K):
[in a new window]
Fig. 1.
G551D-CFTR-mediated current is stimulated by genistein.
A: whole cell recording from a HeLa
cell expressing G551D-CFTR. Forskolin (10 µM) resulted in a very
small stimulation, and perfusion with genistein (30 µM) activated
large Cl current. During breaks in current trace current-voltage
(I-V) relations were recorded.
B and
C:
I-V step protocols. Plotted currents
are difference currents ( I)
calculated by subtracting control currents from stimulated currents.
Membrane voltage
(Vm) was
stepped from
80 to +80 mV in 20-mV increments. Initial
transients are uncompensated capacitative transients.
B was recorded with forskolin in the
bath, C with forskolin and genistein
in the bath. Note time independence of currents.
Im are
baseline subtracted
Im.
D:
I-V plots. Line is best fit of data to
Goldman equation. During forskolin (
), intracellular Cl
concentration (Cli) = 35.9 mM
and Cl conductance
(GCl) = 0.36 nS; and during forskolin + genistein (
),
Cli = 51.8 mM,
GCl =3.09 nS.
E: current variance to mean current
plot. Variance (
2), mean
current (Im),
and corresponding open probabilities
(Po,
top axis) were calculated from 40-s
intervals of current trace filtered at 10 Hz. Line is best fit of data
to Sigworth's parabola, which yielded single channel current
(i) = 0.39 pA and number of channels
(N) = 482. Data points are during
treatment with forskolin (
), forskolin + genistein (
), and
washout (
). Maximal
Po after
treatment with forskolin was 0.04, after forskolin + genistein
Po = 0.46. F: average calculated
Po values during
forskolin treatment (fsk, open bars) and forskolin + genistein
treatment (geni, solid bars) in HeLa cells expressing G551D or
wild-type (wt) CFTR. Genistein significantly increased
Po of G551D-CFTR
(P = 0.01, n = 4) and wtCFTR
(P = 0.02, n = 4, paired
t-tests).
Figure 2 shows the effects of genistein on
the G551D-CFTR channel in a cell-attached patch-clamp recording. In
this recording Po
values were calculated, assuming that two channels were present. Under
unstimulated conditions little channel activity was observed (Fig.
2A, control,
Po = 0.017).
Perfusion of the bath with forskolin stimulated few channel openings
(Fig. 2A, forskolin,
Po = 0.036). Addition of genistein increased
Po to 0.385 (Fig.
2A, forskolin + genistein). Figure
2B shows the average single channel
I-V relation for G551D-CFTR recorded
in the cell-attached mode. Single channel I-V relations were typically outwardly
rectifying in the cell-attached mode (7), with a conductance in the
positive voltage range of g = 8.7 ± 1.0 pS (n = 4, forskolin) and
9.6 ± 0.8 pS (forskolin + genistein), which were similar to wtCFTR
(g = 8.6 ± 0.91 pS, n = 3) recorded under the same
conditions.
|
Next we tested the effects of genistein in unstimulated cells using the
whole cell mode (Fig. 3). Genistein showed
no effects on Cl current, indicating that
1) HeLa cells had no endogenous genistein-activated Cl current, and
2) unstimulated G551D-CFTR could not
be activated by genistein alone. Subsequent addition of forskolin in
the presence of genistein stimulated Cl currents. This indicated that
genistein was not a direct channel opener of G551D-CFTR but relied on
regulation by the cAMP/protein kinase A (PKA) pathway and that
G551D-CFTR was only active when both genistein was present and PKA was
active. In summary, these patch-clamp data show that G551D-CFTR is a
channel with markedly reduced open probability. Treatment of G551D-CFTR
with genistein effectively restored regulation of
Po by the cAMP
pathway similar to wtCFTR.
|
Effects of genistein on F508-CFTR in patch-clamp
recordings.
F508-CFTR was investigated in a nasal
epithelial cell line derived from a CF patient homozygous for the
F508 mutation. We selected JME cells because these cells have been
shown to express several phenotypical features of CF cells, including
very low
F508-CFTR expression in the membrane (16). In whole cell
experiments, no currents were activated by forskolin or genistein (Fig.
4A, control), indicating that no measurable CFTR activity was present in
the plasma membrane of these CF cells. After pretreatment of JME cells
with 4-PB (5 mM for 3 days), Cl currents were stimulated by forskolin
and further increased by genistein (Fig.
4A, 4-PB). Both forskolin- and
genistein-activated currents showed rectification according to the
Goldman equation (Fig. 4B). Figure
4C shows the average responses of JME
cells to forskolin and genistein treatment. On average, genistein
increased the forskolin-activated Cl conductance in 4-PB-treated cells
by an additional 177% (Fig. 4C).
These data indicate that genistein stimulates
F508-CFTR when it is
available in the cell membrane, such as after treatment with 4-PB.
|
Stimulation of Cl transport by genistein in CF
patients. We performed nasal potential difference (PD)
measurements in CF patients bearing the G551D mutation on at least one
allele and in healthy human subjects. To isolate and amplify the
Cl-selective PD, the nasal mucosa was perfused with amiloride (to block
Na transport) and with Cl-free solution (to establish a driving force
for Cl) according to the protocol of Knowles et al. (20). The nasal epithelium was then stimulated with isoproterenol and with genistein. Figure 5 shows measurements
of nasal PD from a healthy volunteer (Fig.
5A) and a G551D CF patient (Fig.
5B). Average responses of nasal PD
are summarized in Fig. 5C. Perfusion
with Cl-free solution and subsequent addition of isoproterenol
hyperpolarized nasal PD in healthy subjects, whereas nasal PD was
depolarized in G551D CF patients, indicating a lack of a
-adrenergic-stimulated Cl conductance in CF patients. However,
perfusion of the nasal mucosa with genistein significantly
hyperpolarized nasal PD both in G551D CF patients and in healthy
subjects, indicative of a genistein-induced Cl conductance in both
groups. In normal subjects and CF patients genistein caused a slow
activation of nasal PD (as in Fig. 5), typical for the activation of
CFTR. In G551D CF patients, genistein caused a hyperpolarization of, on
average,
2.4 ± 0.6 mV (n = 5), which corresponds to 16.9% of the average response of normal
subjects to treatment with Cl-free solution and
isoproterenol (
14.0 ± 2.1 mV,
n = 8). Another tested flavonoid, quercetin, was also active but less potent than genistein in
stimulating nasal PD in G551D CF patients (by
0.9 ± 0.3 mV,
n = 4, significantly different from 0, one sample t-test).
|
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DISCUSSION |
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Genistein restores cAMP-dependent G551D-CFTR activity. For this study we selected the G551D mutation because the G551D-CFTR protein was described as a trafficking-competent mutant (23). We found that acute treatment of cells with genistein in the presence of forskolin resulted in G551D-CFTR activity that was functionally similar to wtCFTR during stimulation with forskolin. The genistein-stimulated G551D-CFTR had a single channel conductance of 9.6 ± 0.8 pS, showed time- and voltage-independent currents, and Po was 0.49 ± 0.12, which are typical biophysical characteristics of wtCFTR. In addition, stimulation of G551D-CFTR by genistein required intrinsic regulation of the channel by the cAMP/PKA system. The recovery of a cAMP-regulated Cl channel appears fundamental for a drug-based treatment of the Cl channel defect in CF, since CF epithelial cells express significant differently regulated Cl conductances [e.g., by intracellular Ca (28)], which do not functionally substitute for the cAMP-regulated CFTR in human CF patients.
Effects of genistein in G551D CF
patients. Our nasal PD measurements in G551D CF
patients showed the typical abnormalities present in other CF patients,
which are: a hyperpolarized basal nasal PD, an increased
amiloride-sensitive PD, a depolarization of nasal PD with Cl-free
solutions, and a lack of response to the -adrenergic agonist
isoproterenol (20). In contrast, perfusion of the nasal epithelium of
G551D CF patients with genistein resulted in a hyperpolarization of
nasal PD in all patients, which under the experimental conditions
corresponds to an activation of a Cl conductance. To our knowledge, no
other CFTR-activating drug has been shown before to hyperpolarize nasal
PDs in CF patients in vivo.
Several studies suggested that only a fraction of the normal function
of CFTR is required to ameliorate CF symptoms. Highsmith et al. (13)
have shown that the expression of only ~4% of normal CFTR mRNA leads
to an unusually mild CF phenotype. Johnson et al. (18) have shown that
6-10% of CFTR-expressing cells in an epithelial monolayer are
sufficient to correct the electrophysiological parameters in CF
epithelia. A study by Dorin et al. (5) suggested that 5% of CFTR
function per cell may be sufficient to prevent intestinal pathology in
CF mice. To relate the measured nasal PD responses to stimulation of Cl
current, the transepithelial resistance
(Rt) of the
nasal epithelium is required.
Rt cannot be
determined from nasal PD measurements directly; however,
Rt was previously
estimated in human primary airway cultures. Willumsen et al. (29) found
Rt values of 340 · cm2 in normal
subjects and slightly higher values (430
· cm2) in CF patients.
Assuming that 1)
Rt in vivo is
similar to Rt in
cultures, and 2) there are no
significant differences in unstirred layers (and thus driving forces
for Cl) between normal and CF, we can estimate the equivalent Cl
current from the measured nasal PD responses (normal,
14 mV;
CF,
2.4 mV) with Ohm's law (
41.2 and
5.6 µA/cm2 for normal and
CF, respectively). Thus the responses to genistein of nasal PD in G551D
CF patients correspond to 13.6% of normal in terms of Cl current,
which may have therapeutic significance.
CF drug development. Currently,
treatment of CF is directed toward minimizing the effects of secondary
complications caused by the defective CFTR. No treatment is in use that
targets the defect of the channel. We suggest that pharmacological
treatment strategies for CF, which are aimed at correcting the defect
in CFTR-dependent Cl secretion, will depend on the kind of mutation present in CFTR. Welsh and Smith (27) distinguished four classes of
CFTR mutations causing defective 1)
protein production, 2) processing,
3) regulation, and
4) conduction. Because these defects create vastly different pharmacological targets in the cell, it appears
likely that the different classes of mutations will need selective drug
treatment. In several studies small molecules were tested that target
the defect in protein production (14), protein processing (2, 17, 24),
and CFTR regulation (1, 4, 19, and the present study). Some of these
drugs may complement one another. For example, we show that
F508-CFTR can be used in combination with 4-PB to maximize Cl currents.
It has been shown that genistein has a variety of biological targets (see Ref. 15) and it will be important to study its toxicological properties. We suggest that genistein can serve as a lead compound for mutant-specific drug development projects that are aimed at 1) improving its selectivity as an activator of CFTR in the membrane and 2) exploiting synergistic pharmacological effects in combination with drugs that correct protein production or processing mutations.
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ACKNOWLEDGEMENTS |
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We thank all the CF patients that participated in this study.
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FOOTNOTES |
---|
This study was funded by the Commercial Endowment of Children's Hospital Oakland, by National Institutes of Health (NIH) Grant SCOR IP50HL60288-01, by a pilot project of the NIH Gene Therapy Core at the University of California San Francisco, and by NIH Grant M01RR01271-16 to the Pediatric Clinical Research Center at Children's Hospital Oakland.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: H. Fischer, Children's Hospital Oakland, Research Institute, 747 52nd St., Oakland, CA 94609 (E-mail: hfischer{at}mail.cho.org).
Received 30 April 1999; accepted in final form 15 July 1999.
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REFERENCES |
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1.
Arispe, N.,
J. Ma,
K. A. Jacobson,
and
H. B. Pollard.
Direct activation of cystic fibrosis transmembrane conductance regulator channels by 8-cyclopentyl-1,3-dipropylxanthine (CPX) and 1,3-diallyl-8-cyclohexylxanthine (DAX).
J. Biol. Chem.
273:
5727-5734,
1998
2.
Brown, C. R.,
L. Q. Hong-Brown,
J. Biwersi,
A. S. Verkman,
and
W. J. Welch.
Chemical chaperones correct the mutant phenotype of the F508 cystic fibrosis transmembrane conductance regulator protein.
Cell Stress Chaperones
1:
117-125,
1996.[Medline]
3.
Cashman, S. M.,
A. Patino,
M. G. Delgado,
L. Byrne,
B. Denham,
and
M. De Arce.
The Irish cystic fibrosis database.
J. Med. Genet.
32:
972-975,
1995[Abstract].
4.
Chappe, V.,
Y. Mettey,
J. M. Vierfond,
J. W. Hanrahan,
M. Gola,
B. Verrier,
and
F. Becq.
Structural basis for specificity and potency of xanthine derivatives as activators of the CFTR chloride channel.
Br. J. Pharmacol.
123:
683-693,
1998[Abstract].
5.
Dorin, J. R.,
R. Farley,
S. Webb,
S. N. Smith,
E. Farini,
S. J. Delaney,
B. J. Wainwright,
E. W. Alton,
and
D. J. Porteous.
A demonstration using mouse models that successful gene therapy for cystic fibrosis requires only partial gene correction.
Gene Ther.
3:
797-801,
1996[Medline].
6.
Drumm, M. L.,
H. A. Pope,
W. H. Cliff,
J. A. Rommens,
S. A. Marvin,
L.-C. Tsui,
F. A. Collins,
R. A. Frizzell,
and
J. A. Wilson.
Correction of the cystic fibrosis defectin vitro by retrovirus-mediated gene transfer.
Cell
62:
1227-1233,
1990[Medline].
7.
Fischer, H.,
and
T. E. Machen.
CFTR displays voltage dependence and two gating modes during stimulation.
J. Gen. Physiol.
104:
541-566,
1994[Abstract].
8.
Fischer, H.,
and
T. E. Machen.
The tyrosine kinase p60c-src regulates the fast gate of the cystic fibrosis transmembrane conductance regulator chloride channel.
Biophys. J.
71:
3073-3082,
1996[Abstract].
9.
Gimbovskaia, S. D.,
V. N. Kalinin,
T. E. Ivashchenko,
and
V. S. Baranov.
Molecular-genetic analysis of certain mutations of the "cystic fibrosis gene" in Moldavia. Characteristics of molecular markers and their linkage with various mutations.
Genetika
30:
1616-1620,
1994[Medline].
10.
Graham, F. L.,
and
L. Prevec.
Adenovirus-based expression vectors and recombinant vaccines.
Biotechnology
20:
363-390,
1992[Medline].
11.
Greil, I.,
K. Wagner,
E. Eber,
M. Zach,
and
W. Rosenkranz.
Molecular and clinical findings in Austrian cystic fibrosis patients with mutations in exon 11 of the CFTR gene.
Wien Klin. Wochenschr.
107:
464-469,
1995[Medline].
12.
Hamosh, A.,
T. M. King,
B. J. Rosenstein,
M. Corey,
H. Levison,
P. Durie,
L. C. Tsui,
I. McIntosh,
M. Keston,
D. J. Brock,
Cystic fibrosis patients bearing both the common missense mutation Gly-Asp at codon 551 and the
F508 mutation are clinically indistinguishable from
F508 homozygotes, except for decreased risk of meconium ileus.
Am. J. Hum. Genet.
51:
245-250,
1992[Medline].
13.
Highsmith, W. E., Jr.,
L. H. Burch,
Z. Zhou,
J. C. Olsen,
T. V. Strong,
T. Smith,
K. J. Friedman,
L. M. Silverman,
R. C. Boucher,
F. S. Collins,
and
M. R. Knowles.
Identification of a splice site mutation (2789+5 G > A) associated with small amounts of normal CFTR mRNA and mild cystic fibrosis.
Hum. Mutat.
9:
332-338,
1997[Medline].
14.
Howard, M.,
R. A. Frizzell,
and
D. M. Bedwell.
Aminoglycoside antibiotics restore CFTR function by overcoming premature stop mutations.
Nat. Med.
2:
467-469,
1996[Medline].
15.
Illek, B.,
and
H. Fischer.
Flavonoids stimulate Cl conductance of human airway epithelium in vitro and in vivo.
Am. J. Physiol.
275 (Lung Cell. Mol. Physiol. 19):
L902-L910,
1998
16.
Jefferson, D. M.,
J. D. Valentich,
F. C. Marini,
S. A. Grubman,
M. C. Iannuzzi,
H. L. Dorkin,
M. Li,
K. W. Klinger,
and
M. J. Welsh.
Expression of normal and cystic fibrosis phenotypes by continuous airway epithelial cell lines.
Am. J. Physiol.
259 (Lung Cell. Mol. Physiol. 3):
L496-L505,
1990
17.
Jiang, C.,
S. L. Fang,
Y.-F. Xiao,
S. P. O'Connor,
S. G. Nadler,
D. W. Lee,
D. M. Jefferson,
J. M. Kaplan,
A. E. Smith,
and
S. H. Cheng.
Partial restoration of cAMP-stimulated CFTR chloride channel activity in F508 cells by deoxyspergualin.
Am. J. Physiol.
275 (Cell Physiol. 44):
C171-C178,
1998
18.
Johnson, L. G.,
J. C. Olsen,
B. Sarkadi,
K. L. Moore,
R. Swanstrom,
and
R. C. Boucher.
Efficiency of gene transfer for restoration of normal airway epithelial function in cystic fibrosis.
Nat. Genet.
2:
21-25,
1992[Medline].
19.
Kelley, T. J.,
K. Thomas,
L. J. Milgram,
and
M. L. Drumm.
In vivo activation of the cystic fibrosis transmembrane conductance regulator mutant F508 in murine nasal epithelium.
Proc. Natl. Acad. Sci. USA
94:
2604-2608,
1997
20.
Knowles, M. R.,
A. M. Paradiso,
and
R. C. Boucher.
In vivo nasal potential difference: techniques and protocols for assessing efficacy of gene transfer in cystic fibrosis.
Hum. Gene Ther.
6:
445-455,
1995[Medline].
21.
Logan, J.,
D. Hiestand,
P. Daram,
Z. Huang,
D. D. Muccio,
J. Hartman,
B. Haley,
W. J. Cook,
and
E. J. Sorscher.
Cystic fibrosis transmembrane conductance regulator mutations that disrupt nucleotide binding.
J. Clin. Invest.
94:
228-236,
1994[Medline].
22.
Parad, R. B.
Heterogeneity of phenotype in two cystic fibrosis patients homozygous for the CFTR exon 11 mutation G551D.
J. Med. Genet.
33:
711-713,
1996[Abstract].
23.
Qu, B. H.,
E. H. Strickland,
and
P. J. Thomas.
Localization and suppression of a kinetic defect in cystic fibrosis transmembrane conductance regulator folding.
J. Biol. Chem.
272:
15739-15744,
1997
24.
Rubenstein, R. C.,
M. E. Egan,
and
P. L. Zeitlin.
In vitro pharmacologic restoration of CFTR-mediated chloride transport with sodium 4-phenylbutyrate in cystic fibrosis epithelial cells containing F508-CFTR.
J. Clin. Invest.
100:
2457-2465,
1997
25.
The Cystic Fibrosis Genetic Analysis Consortium. Cystic
fibrosis mutation database. Cystic fibrosis mutation distribution by
continent. [Online] The Cystic Fibrosis Genetic Analysis
Consortium. http://www.genet.sickkids.on.ca/sftr/rptTable2.html
[1998]
26.
Tummler, B.,
T. Storrs,
V. Dziadek,
T. Dork,
T. Meitinger,
A. Golla,
R. M. Bertele-Harms,
H. K. Harms,
E. Schroder,
A. Claass,
J. Rutjes,
R. Schneppenheim,
I. Bauer,
K. Breuel,
M. Stuhrmann,
J. Schmidtke,
M. Lindner,
A. Eigel,
J. Horst,
R. Kaiser,
M. J. Lentze,
K. Schmidt,
H. von der Hardt,
and
X. Estivill.
Geographic distribution and origin of CFTR mutations in Germany.
Hum. Genet.
97:
727-731,
1996[Medline].
27.
Welsh, M. J.,
and
A. E. Smith.
Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis.
Cell
73:
1251-1254,
1993[Medline].
28.
Willumsen, N. J.,
and
R. C. Boucher.
Activation of an apical Cl conductance by Ca2+ ionophores in cystic fibrosis airway epithelia.
Am. J. Physiol.
256 (Cell Physiol. 25):
C226-C233,
1989
29.
Willumsen, N. J.,
and
R. C. Boucher.
Shunt resistance and ion permeabilities in normal and cystic fibrosis airway epithelia.
Am. J. Physiol.
256 (Cell Physiol. 25):
C1054-C1063,
1989