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Vol. 53, Issue 5, 926-932, May 1998
-Inhibitory Oligodeoxynucleotides Alter the
Conformation of Interferon-
Department of Pharmaceutics, State University of New York at Buffalo, Buffalo, New York 14260-1200.
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Summary |
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Summary
Introduction
Procedures
Results
Discussion
References
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The aptamer mechanism of action involves the direct interaction of
oligonucleotide with protein and is responsible for the biological
effects of many pharmacologically active oligodeoxynucleotides. In the
work reported here, we have determined the effects of aptamers on the
secondary, tertiary, and quaternary structures of the proteins with
which they interact using interferon-
and the
interferon--inhibitory aptamer oligonucleotide, 5'-GGG GTT GGT TGT
GTT GGG TGT TGT GT, as a model system. CD, fluorescence spectroscopy
studies, and antibody binding studies in this system demonstrate that
the interferon--inhibitory aptamer oligonucleotide causes
significant changes in secondary and tertiary structures of
interferon-. These structural changes do not result in, or resemble,
protein denaturation or aggregation, and the results suggest that
aptamer oligodeoxynucleotides can significantly alter the structure of
the proteins they interact with.
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Introduction |
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Top
Summary
Introduction
Procedures
Results
Discussion
References
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Single-stranded
oligonucleotides that exert pharmacological effects by directly
interacting with proteins have been termed "aptamers" (Bock
et al., 1992
). Considerable recent evidence suggests that
the vast majority of pharmacologically active oligonucleotides exert
their effects via the aptamer mechanism and not through the antisense
or antigene mechanisms, which involve interactions with nucleic acids
(Stein, 1995).
We have previously demonstrated that certain oligonucleotides inhibit
the biological effects of the proinflammatory cytokine, IFN-
(Fedoseyeva et al., 1994; Ramanathan et al.,
1994; Tam et al., 1994). These IFN- inhibitors may have
therapeutic uses because the biological effects of this cytokine are
undesirable in inflammatory and autoimmune diseases such as multiple
sclerosis, insulin-dependent diabetes and septic shock. The
IFN--inhibitory oligonucleotides act by an aptamer mechanism because
they prevent binding of IFN- to the IFN--binding subunit of the
IFN- receptor (Ramanathan et al., 1994; Lee et
al., 1996).
With the exception of the thrombin aptamer, the mechanisms of
aptamer action, particularly the effects of aptamers on the proteins
with which they interact, are poorly understood. The thrombin aptamer
inhibits clotting by binding to the anion binding exosite of thrombin,
and NMR (Macaya et al., 1993; Schultze et al.,
1994) and X-ray crystallographic (Padmanabhan et al., 1993) evidence suggests that the aptamer forms an intramolecular G-quartet. Much of the research on aptamers has focused on the structural and
sequence characteristics of aptamer oligonucleotides (Bock et
al., 1992; Ojwang et al., 1994; Rando et
al., 1995), and relatively little data on the effects of aptamers
on the target protein is available.
In this article, using IFN--inhibitory oligonucleotides as a
model system, we highlight the effects of aptamers on the structures of
the proteins with which they interact. We address four specific questions. Do IFN--inhibitory oligonucleotides alter (i) the secondary structure, (ii) the tertiary structure, (iii) the quaternary structure, and (iv) a functional epitope of IFN-?
Our results demonstrate that oligonucleotides cause large structural
changes in the secondary structure of IFN- and that the magnitude of
the structural changes is surprising given the relatively weak
(micromolar to submicromolar) dissociation constant for the interaction
between oligonucleotides and IFN-. To our knowledge, aptamer-induced
structural changes have not been previously reported and these findings
therefore represent a novel mechanism for aptamer action. Because
aptamer-induced protein structural changes have not usually been
considered a potential mechanism for aptamer oligonucleotide activity,
its prevalence may be more widespread than currently suspected.
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Experimental Procedures |
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Summary
Introduction
Procedures
Results
Discussion
References
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Oligonucleotides and interferon-.
The phosphodiester
oligonucleotide 5'-GGG GTT GGT TGT GTT GGG TGT TGT GT (Oligo I) and its
reverse complement, 5'-ACA CAA CAC CCA ACA CAA CCA ACC CC (Oligo II)
were synthesized using standard phosphoramidite protocols by Oli-to-Go
(Dulles, VA) and Genosys (Woodlands, TX). Oligodeoxynucleotide
concentrations were determined from spectrophotometric absorbance
measurements at 260 nm by using a conversion factor of 33 µg of
oligonucleotide per unit of absorbance.
, free of macromolecular additives such as human serum
albumin, was a gift from Genentech (South San Francisco, CA). The
IFN- was dialyzed against several volumes of phosphate-buffered saline in small volume Slide-a-lyzer dialysis cassettes (Pierce, Rockford, IL) according to the manufacturer's instructions. After dialysis, protein concentrations were measured using the BioRad protein
assay reagent (BioRad, Richmond, CA). Bovine serum albumin (Fisher
Scientific, Springfield, NJ) solutions were used as calibration standards.
Thermally aggregated IFN- for fluorescence experiments was obtained
by heating IFN- to 70° for 45 min. The aggregation was independently confirmed by monitoring the absorbance at 360 nm, which
increases because of increased scattering by aggregates.
CD spectroscopy. The CD studies were carried out in 1 mm path length quartz cuvets using a Jasco J500 spectropolarimeter (Jasco, Easton, MD) calibrated with d 10-camphorsulfonic acid (Sigma Chemical, St. Louis, MO). The instrument time constant and sensitivity were set at 4 sec and 1 millidegree, respectively, and samples were scanned over the wavelength range of 260-195 nm for secondary structure analysis.
For titration, two solutions were prepared in phosphate-buffered saline, pH 7.4: solution A contained 50 µg/ml IFN- and solution B
contained 25 µM oligonucleotide plus 50 µg/ml IFN-.
The spectra of solution A were recorded and solution B was added in
aliquots to solution A. After each addition, the solutions were mixed
by inversion and the spectra were recorded.
The ellipticity data over the wavelength range of 195-240 nm were
analyzed for secondary structure with the use of the convex constraint
analysis program (Perczel et al., 1991, 1992) with a
reference set consisting of 30 proteins.
The percent reduction in ellipticity was plotted against total
oligonucleotide concentration, [Oligo], and the data were fitted to
the quadratic binding equation:
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![=<UP>Maximum </UP>% <UP>Ellipticity Reduction</UP>×<FR><NU>S−<RAD><RCD>S<SUP>2</SUP>−4[<UP>Oligo</UP>][<UP>IFN</UP>]</RCD></RAD></NU><DE>2[<UP>IFN</UP>]</DE></FR>](/content/vol53/issue5/fulltext/926/img002.gif)
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concentration,
Kd is the dissociation constant, and
S = [Oligo] + [IFN] + Kd.
The quadratic binding equation was used instead of the Michaelis-Menten
type simple binding hyperbola because the free oligonucleotide concentration cannot be made equal to the total oligonucleotide concentration. The least squares curve fitting routine in Kaleidagraph 3.08 (Synergy Software, Reading PA) was used to determine the Maximum
% Ellipticity reduction and the dissociation constant terms in the
equation.
Fluorescence spectroscopy. All fluorescence studies were done at room temperature on an SLM Aminco 8000 fluorometer (Spectronics Instruments, Rochester, NY) with 4-mm excitation and emission slits.
Tryptophan fluorescence emission spectra over the 300-400 nm wavelength range were recorded with the excitation wavelength set at 280 nm. A 295-nm long pass filter was used during the measurements to minimize the effect of Raman bands on the emission maxima. Spectra were recorded in I-shaped, 2 mm/10 mm dual path length cuvets so that corrections for the inner filter effect could be made. Oligonucleotide titrations were carried out as described for the CD studies. For the urea denaturation experiments, a phosphate-buffered saline solution containing 16 M urea plus 50 µg/ml IFN- was
substituted for solution B.
The percent reduction in fluorescence was plotted against total
oligonucleotide concentration, and the data were fitted to the
quadratic binding equation using the method used for the CD studies.
The fluorescence of ANS (Sigma Chemical, St. Louis, MO) is sensitive to
quaternary structure and was used to determine the effects of
oligonucleotides on the homodimeric quaternary structure of IFN-.
The final IFN- and the ANS concentrations were 10 µg/ml and 10 µM, respectively. Emission spectra for ANS between 400 and 600 nm were obtained with excitation set at 380 nm.
Antibody binding assay.
Antibody 202 (Seelig et
al., 1994) and antibody 3125 (Alfa and Jay, 1988) were generous
gifts from Dr. Gail F. Seelig (Schering Plow Research Institute,
Kenilworth, NJ) and Dr. Francis T. Jay (University of Manitoba,
Winnipeg, Canada). IgG control antibody, recombinant-human IFN-, and
125I-labeled IFN- were purchased from
Biodesign International (Kennebunk, ME), PeproTech (Rocky Hill, NJ),
and Amersham (Arlington Heights, IL), respectively.
antibodies for different
epitopes were dissolved in 0.2 M carbonate buffer and immobilized on enzyme-linked immunosorbent assay plates overnight at
4°. The plates were treated overnight with 200 µl of
D-phosphate-buffered saline containing 1% bovine serum
albumin at 4° to block nonspecific binding.
125I-Labeled IFN- containing 0.1, 1, or 10 µM of either experimental or control oligonucleotide
(Oligo I or II) was added followed by incubation for 1 hr at 4°. An
aliquot of supernatant was removed for measuring the unbound
125I-IFN- and the plates were washed with
0.05% Tween in phosphate-buffered saline. The radioactivity in the
supernatant and that bound to the wells was quantified in a Minaxi Auto-gamma 5000 series -counter (Packard, Downers Grove, IL).
The ratio of the bound to free radioactivity was calculated and
expressed as percentage of the ratio obtained in the absence of
oligonucleotide. The results were fitted to a Hill equation:
![<UP>Bound/Free Ratio </UP>(<UP>as % of control</UP>)=100−<FR><NU>100×[<UP>Oligo</UP>]<SUP>n<SUB>H</SUB></SUP></NU><DE><UP>IC</UP><SUB><UP>50</UP></SUB><SUP>n<SUB>H</SUB></SUP>+[<UP>Oligo</UP>]<SUP>n<SUB>H</SUB></SUP></DE></FR>](/content/vol53/issue5/fulltext/926/img003.gif)
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Results |
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Summary
Introduction
Procedures
Results
Discussion
References
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Evidence that oligonucleotides alter the secondary structure of
IFN-.
To determine the effect of oligonucleotide on the
secondary structure of IFN-, far ultraviolet-CD spectra of the
protein in the presence and absence of oligonucleotides were acquired. The CD spectrum of IFN- (Fig. 1)
exhibited a negative band around 220 nm, a shoulder at 208 nm, and a
positive band at 190 nm, all of which are characteristic of an
-helix rich protein (Fasman, 1993). Convex constraint analysis
(Perczel et al., 1991, 1992) showed 58% -helical content
and some random coil and aromatic contributions. These are consistent
with the reported crystal structure data on IFN- (Ealick et
al., 1991) and with previously reported far ultraviolet-CD data
(Hogrefe et al., 1989).
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CD spectra in the absence and presence
of varying concentrations of oligonucleotide and are corrected for
ellipticity of the added oligonucleotide. Fig. 1 shows that the IFN-
CD spectrum changes significantly from the native structure upon
addition of Oligo I. The ellipticity value at 220 nm decreased with the
addition of oligonucleotide, indicating secondary structure changes in
the protein, and quantitative estimation using convex constraint
analysis indicated a reduction in -helix content to 40% and an
increase in random coil content. CD spectra of oligonucleotide alone
were also acquired at each concentration, but because the spectra were
corrected for ellipticity of oligonucleotide alone, the data are not
shown. At the highest concentration of Oligo I, 10 µM,
the ellipticity of Oligo I alone at 220 nm was less than 15% of the
ellipticity of IFN- alone. The ellipticity of IFN- at 220 nm
decreased rapidly with increasing Oligo I concentration but became
independent of the oligonucleotide concentration at the higher Oligo I
concentrations (Fig. 1, inset). A similar decrease in the
ellipticity values was observed with Oligo II (data not shown).
Evidence that oligonucleotides alter the tertiary structure of
IFN-.
Tryptophan fluorescence emission spectra were monitored
to determine the effects of oligonucleotide treatment on the tertiary structure of IFN-. Because IFN- has only one tryptophan, W31, per
monomeric subunit, fluorescence spectroscopy yields information about
the site at which oligonucleotide interacts. The tryptophan is located
at the end of the B helix and takes part in the formation of a cleft
that accommodates the carboxyl-terminal F helix of the other monomer
(Ealick et al., 1991). Changes in tryptophan fluorescence
therefore provide information about the effects of oligonucleotide on
dimeric interface of IFN-.
and for
IFN- in the presence of various concentrations of Oligo I are shown
in Fig. 2. The fluorescence spectra of
the oligonucleotide alone were also acquired at each concentration but
are not shown because the analysis included correcting for the
fluorescence of oligonucleotide. Over the 320-370 nm wavelength range,
the fluorescence contribution of Oligo I alone at its highest
concentration, 10 µM, was less than 15% of the
fluorescence of IFN- alone.
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spectrum shows a peak maximum at 343 nm, suggesting that
the tryptophan is partially shielded from the surrounding aqueous
environment, which is consistent with the crystal structure data. The
fluorescence of the protein is reduced by the addition of Oligo I in a
dose dependent manner. A similar reduction in fluorescence was observed
with Oligo II (data not shown). Fig. 2, inset, is a plot of
percent of fluorescence quenched versus Oligo I concentration. The
quenching of IFN- fluorescence in the presence of oligonucleotide
shows saturation at higher oligonucleotide concentrations.
The quadratic binding curves for the CD data (Fig. 1, inset)
and the fluorescence quenching data (Fig. 2, inset) were
very similar and the fitted parameters were not statistically
different. The data were therefore pooled and the dissociation constant
was estimated from fitting to be 0.15 ± 0.10 µM.
These results show that Oligo I interacts with W31 and suggest that the
interactions either cause or are associated with changes in the
tertiary structure of IFN-.
Evidence that the conformational changes do not involve
denaturation of IFN-.
Changes in secondary structure and
quenching of fluorescence intensity can also occur when a protein
denatures in the presence of agents such as urea. To determine whether
the conformational changes induced by Oligo I were distinct from
denaturation, we compared the fluorescence emission spectra of IFN-
obtained in the presence of various concentrations of either urea or
Oligo I (Fig. 3).
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shows a peak maximum of 343 nm.
However, with increasing urea concentration, the fluorescence intensity
is quenched (Fig. 3, inset) and the peak maxima are shifted
to longer wavelengths (Fig. 3). Thus, the denaturation of IFN- is
associated with both fluorescence quenching and shift in the peak
maximum. In contrast, peak position does not shift in the Oligo I
treated IFN- samples, suggesting that the molecular events
associated with oligonucleotide addition are different from protein
denaturation and that the solvent exposure of the tryptophan residue,
W31, is not altered during the interaction with Oligo I.
Evidence that Oligo I does not detectably alter the quaternary
structure of IFN-.
The conformational changes observed using CD
and fluorescence could potentially also be associated with the loss of
quaternary structure of the IFN- and this could result in the
formation of larger aggregates or dissociation to individual subunits.
To investigate the aggregation or dissociation of the dimeric protein, we probed the quaternary structure by ANS fluorescence.
; Merz,
1988). Thus, ANS fluorescence in the presence of IFN- can be
expected to increase if aggregation occurs, and decrease if dissociation to subunits occurs.
However, the ANS fluorescence cannot be used to draw conclusions
regarding quaternary structure unless these structural changes are
accompanied by hydrophobicity changes. The accuracy of this underlying
premise was confirmed by measuring the fluorescence of ANS in presence
of thermally aggregated IFN-. The aggregation was independently
confirmed by measuring the absorbance at 360 nm, which was almost
10-fold higher than the absorbance of native IFN-. This positive
control sample also had a 4-fold higher ANS fluorescence intensity
(Fig. 4) than native IFN-, showing
that ANS fluorescence is sensitive to IFN- quaternary structure.
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is
higher than the fluorescence of ANS in buffer, indicating possible
binding of the probe with the dimeric IFN-. As shown in Fig. 4, the
ANS fluorescence intensities for IFN--oligonucleotide mixtures were
independent of the Oligo I concentration, indicating that the extent of
IFN- aggregation or dissociation upon interaction with Oligo I is
either small or not accompanied by detectable changes in surface
hydrophobicity.
Evidence that Oligo I alters the recognition of epitopes on IFN
-.
Antibody 202 recognizes an epitope that is spread across
amino acids (1-29, 75-96, 104-111, 118-125, 131-139) (Seelig
et al., 1994) and antibody 3125 recognizes epitope E2' (Alfa
and Jay, 1988). The sequence corresponding to E2' is not known, but it mediates antiviral activity (Alfa and Jay, 1988).
to antibody 202 was inhibited in a dose-dependent manner by Oligo I and
II. Oligo I had a greater inhibitory effect compared with Oligo II and
the IC50 value for Oligo I (1.3 ± 0.24 µM) was 10-fold lower than that for Oligo II (13 ± 8.3 µM). However, both Oligo I and II show similar
inhibition of IFN- binding to antibody 3125 (Fig. 5 B) and the
IC50 values were not statistically different.
This suggests that Oligo I and II modify epitope E2' to a similar
extent.
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by
biological macromolecules.
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Discussion |
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Top
Summary
Introduction
Procedures
Results
Discussion
References
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In this work, we have investigated the effects of
oligodeoxynucleotides on the structure of IFN- and our results
demonstrate that the interaction with oligonucleotides causes a loss of
-helical content and alterations in the tertiary structure of
IFN-.
In our previous studies, we have shown that Oligo I is more active than
Oligo II in inhibiting the biological effects of IFN- and in
inhibiting the binding of IFN- to cells (Ramanathan et al., 1994) and to the IFN--binding subunit of the receptor (Lee et al., 1996). However, in the spectroscopic assays used in
this work, both oligonucleotides altered the structure of IFN-,
suggesting that the structural changes are necessary but not sufficient
for activity. In the results displayed in Fig. 5, however, Oligos I and
II show different effects on binding of IFN- to antibody 202 but
similar effects on IFN- binding to antibody 3125. These findings are
consistent with the notion that contributions from both sequence and
backbone elements are necessary for IFN- inhibitory activity of
oligonucleotides and supports the hypothesis that the two
oligonucleotides have certain similarities and certain dissimilarities
in their actions that may contribute to the differences in biological
activity. The mechanisms by which bases of the oligonucleotide inhibit
the interaction between IFN- and its receptor are currently unknown
but several possibilities can be envisioned. For example, the bases may
cause steric hindrance to interaction or alternatively, the IFN- may
not fully recover its native structure after perturbation caused by
aptamer.
At first sight, the sensitivity of the secondary structure of IFN-
to oligonucleotides is somewhat surprising, considering that the
biological roles of IFN- do not involve DNA or RNA binding. In
contrast, the inhibitory effects of phosphorothioate oligonucleotides on the activities of the DNA polymerases, RNases H (Gao et
al., 1992), and retroviral reverse transcriptases (Ojwang et
al., 1994) are, in retrospect, not surprising because the
physiological roles of these enzymes require interactions with nucleic
acids (Gao et al., 1992). Basic fibroblast growth factor
(Jellinek et al., 1993) and vascular endothelial growth
factor (Jellinek et al., 1994) are among the non-DNA binding
proteins that are inhibited by RNA aptamers. The inhibition of these
growth factors can be rationalized, in part, by their heparin binding
ability (Stein, 1995). We speculate that the interaction of
oligonucleotides with IFN- is also perhaps an unexpected consequence
of a biologically relevant interaction between IFN- and some
negatively charged component of the extracellular matrix (Rider, 1993;
Tanaka et al., 1993). Heparin sulfate and heparin bind
IFN- with nanomolar dissociation constants (Lortat-Jakob and
Grimaud, 1991, 1992; Lortat-Jacob et al., 1991), and
depending on experimental conditions, these interactions can either
enhance (Sylvester et al., 1990; Lortat-Jakob and Grimaud,
1991) or inhibit (Daubener et al., 1995; Douglas et
al., 1997) the activity of IFN-. In in vivo
experiments, these extracellular matrix interactions of IFN- appear
to enhance the stability and activity of IFN- (Sylvester et
al., 1990; Lortat-Jacob et al., 1996).
Are protein structural changes a common feature in aptamer-protein
interactions or are these changes unique to the IFN- system? Few
protein structural data are available for most aptamers, but in the
thrombin-thrombin aptamer system, the aptamer adopts a G-quartet
structure. Significant changes to the secondary structure of thrombin
were not reported, however, in the X-ray crystal structure (Padmanabhan
et al., 1993) and using molecular modeling (Macaya et
al., 1993).
In this work, we have used spectroscopic methods that allow the structural changes associated with the interaction to be monitored and do not require separation of the bound and unbound species. Other techniques such as mobility shift assays and gel filtration are also frequently used to demonstrate binding, but in our hands, these assays have not proven useful possibly because the binding is relatively weak.
The spectroscopic changes reported here may be useful in the design of
high throughput screening strategies for identifying more potent,
specific, therapeutically useful inhibitors for IFN- from
combinatorial chemistry libraries. Effective inhibitors of IFN- are
likely to be clinically useful in diseases such as multiple sclerosis,
graft rejection, and diabetes where the proinflammatory effects of
IFN- are disease-promoting.
In conclusion, our results may potentially provide a novel mechanistic basis for the activity of aptamers because they show that oligonucleotides can cause structural changes in proteins.
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Acknowledgments |
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We thank Dr. Gail F. Seelig (Schering Plough, Kenilworth, NJ)
for providing antibody 202; Dr. Francis T. Jay (University of Manitoba,
Winnipeg, Canada) for supplying antibody 3125; and Dr. Robert
Straubinger (State University of New York, Buffalo, NY) for use of the
spectropolarimeter. We thank Dr. Gerald D. Fasman (Brandeis University,
Waltham, MA) for providing us with the CDANAL software used for the
analysis of circular dichroism spectra and we gratefully
acknowledge Genentech Inc. for generously providing us with the
interferon- used in these studies.
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Footnotes |
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Received November 25, 1997; Accepted February 5, 1998
This work was supported by Grant 1R29-GM54087-01 from the National Institute of General Medical Sciences. Funding from the National Multiple Sclerosis Society is also gratefully acknowledged. M.R. is a member of the University-to-University Cooperative Research Project between Kanazawa University, Japan, and the State University of New York at Buffalo.
Send reprint requests to: Dr. Murali Ramanathan, Dept. of Pharmaceutics, 543 Cooke Hall, SUNY at Buffalo, Buffalo, NY 14260-1200. E-mail: murali{at}acsu.buffalo.edu
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Abbreviations |
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IFN-, interferon-;
ANS, 1,8-aminonaphthalene sulfonate.
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References |
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Top
Summary
Introduction
Procedures
Results
Discussion
References
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responsible for anti-viral effector function.
J Immunol
141:
2474-2479[Abstract]..
Eur J Immunol
25:
688-692[Medline]. (IFN-gamma).
Clin Exp Immunol
107:
578-584[Medline]..
Science (Washington DC)
252:
698-702-mediated immune functions by oligonucleotides. Suppression of human T cell proliferation by downregulation of IFN-gamma-induced ICAM-1 and Fc-receptor on accessory cells.
Transplantation
57:
606-612[Medline]..
J Biol Chem
264:
12179-12186 signal transduction.
Transplantation
62:
1297-1301[Medline]. and increases its activity by limiting the processing of its carboxyl-terminal sequence.
J Biol Chem
271:
16139-16143 to a basement membrane complex (matrigel).
J Clin Invest
87:
878-883.-induced major histocompatibility complex class I and intercellular adhesion molecule-1.
J Biol Chem
269:
24564-24574-induced major histocompatibility complex class I expression by certain oligodeoxynucleotides.
Transplantation
57:
612-615[Medline]. interferon block ligand binding to human interferon receptor.
J Biol Chem
269:
358-363 induction of MHC class II and ICAM-1 by a 26-base oligonucleotide composed of deoxyguanosine and deoxythymidine.
Transplant Immunology
2:
285-292[Medline].This article has been cited by other articles:
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  E. B. Khomyakova, H. Gousset, J. Liquier, T. Huynh-Dinh, C. Gouyette, M. Takahashi, V. L. Florentiev, and E. Taillandier Parallel intramolecular DNA triple helix with G and T bases in the third strand stabilized by Zn2+ ions Nucleic Acids Res., September 15, 2000; 28(18): 3511 - 3516. [Abstract] [Full Text] [PDF]
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) or Oligo I (
). The IFN-
