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* Department of Biochemistry, Kyoritsu College of Pharmacy, Tokyo, Japan; and
Yokohama City University School of Medicine, Fukuura, and
Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-initiated responses. For this
purpose we investigated the effects of alprazolam on the
IL-1-induced production of inflammatory cytokines (IL-8 and monocyte
chemoattractant protein 1 (MCP-1)) in a human glioblastoma cell line,
T98G, and explored the signaling pathways involved. We found that
alprazolam inhibited IL-1-elicited MCP-1 production within a range
of 0.1–3 µM. In contrast, it did not inhibit IL-1-induced IL-8
production. Although NF-
B is involved in regulating the
IL-1-induced expression of MCP-1 and IL-8, the degradation of
IB- stimulated by IL-1 was not inhibited by alprazolam.
Alprazolam prevented NF-B from binding to the MCP-1 promoter region
(the A2 and A1 oligonucleotide probes), but binding of NF-B to
IL-8/NF-B was not inhibited. Moreover, alprazolam inhibited
c-Rel/p50 binding to the A2 oligonucleotide probe, but not p50/p65 from
binding to the IL-8/NF-B site. While AP-1 is involved in regulating
the IL-1-induced expression of IL-8, but not MCP-1, alprazolam
potentiated the binding of c-Jun/c-Fos to the AP-1 oligonucleotide
probe. These results show that the inhibition of IL-1-mediated MCP-1
production by alprazolam is mainly due to inhibition of c-Rel/p65 and
c-Rel/p50 binding to the MCP-1 promoter region, since alprazolam did
not affect the IL-1-mediated activation of NF-B (p50/p65) or AP-1
(c-Jun/c-Fos) binding to the IL-8 promoter region. In conclusion, a new
action of alprazolam was elucidated, as shown in the inhibition of
c-Rel/p65- and c-Rel/p50-regulated
transcription. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Rel family is essential to immunity and inflammatory responses. It
is of interest whether alprazolam is involved in the expression
regulation of cytokine and chemokine genes by these transcription
factors. Although cytokines and chemokines play important roles in
inflammation, such as the activation and mobilization of inflammatory
cells, the excess production of cytokines and chemokines may aggravate
some symptoms of chronic diseases. Therefore, the appropriate
regulation of cytokine and chemokine production is important for
biological homeostasis in host defense through their ability to induce
Ags and to produce various chemokines and cytokines. Monocyte
chemoattractant protein 1 (MCP-1) is a member of the CC subfamily of
chemokines and attracts monocytes both in vitro and in vivo
(6, 7, 8). A wide variety of cells, including monocytes,
fibroblasts, vascular endothelial cells, and smooth muscle cells,
produce MCP-1 in vitro in response to stimuli such as LPS, IL-1, or
TNF-; the expression influences the transcription factor NF-B,
which plays an important part in controlling the expression of
inflammatory cytokines. Two NF-B sites, A1 and A2, are found in the
MCP-1 promoter region, and it was elucidated that the A2 site was the
most important in MCP-1 expression (9). Both
(p65)2 and c-Rel/p65 have been shown to be
important NF-B components for MCP-1 expression, and the binding of
these proteins to the A1 and A2 sites of this gene is regulated by
tissue- and stimulus-specific transcription (9). We thus
investigated the inhibitory effect of alprazolam on the expression of
MCP-1 in the glioblastoma cell line T98G.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Alprazolam was obtained from Takeda Pharmaceutical (Osaka,
Japan). 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO). Human
rIL-1 was provided by Dainippon Pharmaceutical (Osaka, Japan).
Anti-IB-, anti-IB-
, anti-c-Jun, anti-c-Fos,
anti-c-Rel, anti-p65, and anti-p50 were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). All other reagents and
chemicals used were of the highest grade available commercially. DNase
I and T4 ligase were purchased from Takara Biomedicals (Otsu,
Japan).
Cell lines and culture conditions
The human glioblastoma cell line T98G was obtained from Japanese Cancer Research Resources Bank (Tokyo, Japan). This cell line was maintained in tissue culture dishes in RPMI 1640 medium (Nissui Seiyaku, Tokyo, Japan) supplemented with 5% heat-inactivated FCS, 2 mM glutamine, 100 U/ml penicillin G, and 100 µg/ml streptomycin. The murine macrophage cell line RAW 264.7 was also obtained from Japanese Cancer Research Resources Bank and was maintained in tissue culture dishes in serum-free medium (Cosmedium; Cosmo Bio, Koto-ku, Japan).
Nitrite assay
Nitrite concentrations in the cell culture supernatants were determined fluorometrically using the 2,3-diaminonapthalene reagent as described previously (10).
Detection of mouse (m)TNF-, mJE/MCP-1, mIL-1, mIL-12,
and human IL-8 by ELISA
The mTNF- and mJE/MCP-1 contents of the culture supernatants
under control and various test conditions were measured by ELISA using
a combination of monoclonal and biotinylated mAbs, and polyclonal and
biotinylated polyclonal Abs, respectively. The mIL-1 and mIL-12
contents of the culture supernatants under control and various test
conditions were measured using an ELISA kit (BioSource International,
Camarillo, CA). IL-8 in the cultured supernatant was measured as
described previously (11). All samples were assayed at
least in duplicate. Data are presented as the mean ± SE of three
independent experiments.
Determination of cytotoxicity
Lethal cell injury was assessed by measuring lactate
dehydrogenase (LDH) release (12), while proliferation was
evaluated by enumerating viable cells using the MTT formazan production
method (13). RAW cells (1 x
106 cells/ml) were treated with LPS (with or
without alprazolam) and then transferred to 96-well microtiter plates.
After 24-h incubation, LDH activity in the supernatants was determined,
and 20 µl MTT reagent (5 mg/ml in PBS) was added to each well. After
incubation for 3 h, formazan production was assessed by measuring
OD (OD570 nm). T98G cells
(1 x 106 cells/ml) were treated with
IL-1 (with or without alprazolam), LDH release was evaluated, and
the MTT assay was performed as described.
Northern blot analysis
Northern blot analysis was performed as reported previously
(14). Total RNA (30 µg) was subjected to agarose gel
electrophoresis, blotted onto a membrane, and then hybridized with
32P-labeled IL-8, MCP-1, TNF-, and JE/MCP-1
cDNA probes.
Plasmid construction, cell transfection, and luciferase assay
Plasmid construction, cell transfection, and the luciferase assay were conducted as described previously (14). Luciferase reporter plasmids containing the luciferase gene were constructed under control of the MCP-1 promoter, the IL-8 promoter, and the AP-1 site as described elsewhere (9, 14), and cell transfection and luciferase assay were conducted using a modification of the method described by Ueda et al. (9) and Mukaida et al. (14).
The pGLM-PRM was the 167-bp human MCP-1 promoter region between -107
and +60, and the pGLM-ENH was the 230-bp human MCP-1 enhancer region
between -2742 and -2513. The mutated constructs, pGLM-MA1, pGLM-MA2,
and pGLM-MA1A2 were chemically synthesized (14). However,
-133Luc was contained with AP-1, C/EBP/NF-IL-6, and NF-B sites.
IL-8-BLuc was included in three (IL-8-B, sites, and AP-1Luc was
included in two (AP-1) sites. Transfection was performed by the
electroporation method. T98G cells (1.0 x
107) were washed with RPMI 1640 and resuspended
in 700 µl RPMI 1640 containing 20 µg reporter plasmid DNA.
Electroporation was performed at 960 µF and 270 V using a Gene Pulser
and a Gene Pulser cuvette with a 0.4-cm electrode gap (Bio-Rad,
Hercules, CA). The cells were suspended in 40 ml RPMI 1640 containing
10% FCS, divided among six 6-cm dishes, and incubated for 24 h
before treatment with the stimulants and/or test compounds. Cell
extracts were prepared 12 h after treatment with IL-1, and
luciferase activity was measured with a Pica Gene detection kit (Tokyo,
Inc., Tokyo, Japan) and MiniLumat LB 9506 (Berthold Japan K.K., Tokyo,
Japan).
Preparation of nuclear extracts and EMSA
Nuclear extracts were prepared from T98G cells as described
previously (14), and aliquots were frozen at -80°C.
EMSA was conducted on 5% polyacrylamide gels in 1x Tris-borate/EDTA
electrophoresis buffer. A1, A2, IL-8/NF-B, AP-1, and mutant
A2 probes were synthesized by Rikaken (Aichi, Japan). The
sequences of these oligonucleotides were as follows:
5'-GATCTGGGA-ACTTCCAAAGC-3' (A-1 probe),
5'-AGAGTGGGAATTTCCACTC-A-3' (A-2 probe),
5'-AAATCGTGGAATTTCCTCG-3' (IL-8/NF-B probe),
5'-AGTGTGATGACTCAGGTT-TG-3' (AP-1 probe), and
5'-GAGTGGGAATTCGG-ACTCACT-3' (mutant A-2 probe).
Supershift analysis
The nuclear extract was preincubated with 3 µg of anti-c-Rel, anti-p65, and anti-p50 Abs for 30 min at 20°C before adding the labeled probe. Supershift analysis was then completed as described above.
Western blot analysis
T98G cells were treated with the test agents and lysed in a buffer. The amount of protein in each sample was determined by Bradford assay. Equal amounts of lysates were subjected to SDS-PAGE, and the proteins were transferred onto nitrocellulose filters. After blocking with 5% nonfat milk in TBS at room temperature for 1 h, the membrane was probed with Abs. They were incubated for 30 min with biotinylated anti-rabbit Ig Ab (Amersham Pharmacia Biotech, Arlington Heights, IL). They were then incubated for 30 min with peroxidase-streptavidin conjugate. Blots were developed using an ECL detection system (Amersham Pharmacia Biotech).
Nicking and joining of circular DNA
The experimental procedure was that described by Hayashi et al. (15) with a minor modification. Briefly, pUC19 form 1 DNA was treated with DNase I to produce an average of one nick per molecule. This open circular DNA was treated with T4 ligase at 16°C for 2 h in 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 10 mM DTT, 1 mM ATP, 10% DMSO, and ethidium bromide or alprazolam. After ligation, the mixture was analyzed by agarose gel electrophoresis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-mediated MCP-1 induction, but not IL-8
induction
As in our previous study stimulation of the human glioblastoma
cell line T98G with 10 ng/ml IL-1 for 24 h resulted in the
production of MCP-1 and IL-8 (11). The expression levels
of MCP-1 and IL-8 mRNAs in cells stimulated with IL-1 in the
presence of alprazolam were quantified. As shown in Fig. 1
, A and B, the
MCP-1 mRNA level decreased depending on the alprazolam concentration,
while the IL-8 mRNA level slightly increased. When T98G cells
pretreated with alprazolam for 30 min were washed thoroughly with PBS
to remove alprazolam, then stimulated with IL-1, mRNA expression was
not significantly different from that without pretreatment (Fig. 1C). This finding indicates that the persistent presence of
alprazolam is necessary for the inhibition of MCP-1 expression. To
clarify that the inhibitory action of alprazolam was not due to cell
toxicity, we examined cell viability. We found that alprazolam at 3
µg/ml had no significant effect on cell viability during 24-h
incubation, as evidenced from MTT and LDH assays (data not shown).
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As described above, pretreatment of T98G cells with alprazolam did not affect the expression of MCP-1 when the drug was removed after pretreatment, suggesting that the inhibitory effect was exerted in the cytoplasm.
Although new information regarding the signal after IL-1 binding to the
receptors has recently been reported, activation of the transcription
factor NF-B has the greatest importance. To determine whether the
decrease in the MCP-1 level was due to NF-B inhibition, we performed
a luciferase assay using a reporter plasmid in which the MCP-1 promoter
region had been inserted. There are two areas involving NF-B, A1 and
A2, in the MCP-1 promoter region, and their importance and specificity
in LPS-stimulated THP-1 cells have been shown by reporter assay using
A1 and A2 mutants (9). Thus, we performed a reporter assay
of the MCP-1 promoter region in IL-1-stimulated T98G cells. The
results are shown in Fig. 2A.
This assay showed that the A2 region is also important for the
expression of MCP-1 in T98G cells. We then investigated whether
alprazolam has inhibitory activity on the transcription to the MCP-1
promoter region, using a reporter plasmid (pGLM-ENH) containing the A1
and A2 regions. The results are shown in Fig. 2B. Alprazolam
inhibited transcription activity of the MCP-1 enhancer promoter region
in a concentration-dependent manner. The above findings suggested that
alprazolam inhibits NF-B binding to the MCP-1 promoter region.
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B and AP-1 binding regions) as in MCP-1. The
results are shown in Fig. 2
, C and D. Fig. 2C shows the results obtained using the reporter plasmid
containing the IL-8 promoter region. The activity increased in the
presence of 3 µM alprazolam, but was not affected at lower
concentrations. When a similar experiment was performed using a
reporter plasmid containing only the NF-B or AP-1 binding region of
the IL-8 promoter region, the activity increased only when the plasmid
contained the AP-1 binding region alone in the presence of 3 µM
alprazolam, as in the plasmid containing the IL-8 promoter region, but
the activity was not affected under other conditions (Fig. 2D). When the relative luciferase activity in the IL-8
promoter region was 33,000, the luciferase activity in the AP-1 binding
region alone corresponded to 
1180, suggesting that the AP-1
binding region only slightly affects the activation of IL-8
transcription. The above findings showed that alprazolam does not
affect IL-8 production at the transcriptional level in
IL-1-stimulated T98G cells, and AP-1 activity is induced by a
high concentration of alprazolam.
Alprazolam does not inhibit NF-B activation
The above experiments suggested that alprazolam inhibits the
binding of activated NF-B to the MCP-1 promoter region. Thus, we
first investigated whether alprazolam affects the activation of
NF-B. Inactive NF-B complexes are associated with inhibitory
proteins of the IB family that sequester NF-B in the cytoplasm.
It is well known that following various stimuli, IB proteins are
first phosphorylated, then ubiquitinated, and finally rapidly degraded
by the proteasome, allowing for the nuclear translocation of NF-B
and transcriptional initiation of NF-B-dependent genes. The IB
family includes the widely distributed IB- and IB- proteins
together with IB-
, IB-
, and Bcl-3 (16). IB
proteins are characterized by a series of ankyrin repeats that interact
with the DNA binding domain and nuclear localization signal of NF-B
(17). Therefore, the effect of alprazolam on the
activation of NF-B was determined by Western blotting with anti
IB- or -. The results show that alprazolam did not affect the
degradation of IB- (Fig. 3, A and B). Next, we investigated the distribution
of activated NF-B in cytosol and nucleus by Western blotting. It has
been shown that IL-1 activates p50 and p65 and induces IL-8 production
in T98G cells (14). The involvement of p65/c-Rel in the
induction of MCP-1 expression has also been reported (9).
Thus, we analyzed protein extracts from cytosol and nucleus by Western
blotting. The results are shown in Fig. 3C; p50, p65, and
c-Rel were activated and transferred from the cytosol to the nucleus.
Alprazolam partially inhibited the activation at 3 µM, but not at 1
µM. Alprazolam did not inhibit the transfer to the nucleus.
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-stimulated binding of NF-B to
DNA
The Western blot experiment demonstrated that alprazolam does not
inhibit the activation of NF-B or its transfer to the nucleus,
suggesting that the inhibition by alprazolam is exerted in the nucleus.
Thus, we performed EMSA using the nuclear extract. First, EMSA was
performed using A2, which is most closely associated with the
expression of MCP-1, as the probe to confirm the potential of the
binding activity (Fig. 4, A
and B). When the experiment was performed in the presence of
1–3 µM alprazolam under the same conditions, binding to the A2
region was inhibited in proportion to the concentration (Fig. 4A). We predicted that if the inhibition point of alprazolam
is not related to the activation or nuclear transfer of NF-B, i.e.,
if alprazolam targets the binding to DNA in the nucleus, inhibition may
be observed during EMSA after adding alprazolam to the nuclear extract
preparation. The inhibition was observed as expected (Fig. 4B). These results indicated that alprazolam specifically
inhibited the binding of NF-B to the A2 site. Next, to clarify the
NF-B-constituting protein that binds to this region, we performed a
supershift experiment using Abs against p65, p50, and c-Rel of the Rel
family, because the importance of these proteins as transcription
factors involved in the expression of MCP-1 and IL-8 has been shown.
The experiment revealed the involvement of p65, p50, c-Rel, c-Rel/p65,
c-Rel/p50, and p50/p65 (Fig. 4C). We also performed a
similar supershift experiment withnuclear extracts from
alprazolam-treated cells. Anti-c-Rel Ab did not decrease the bands
compared with anti-p65 and anti-p50 Abs (Fig. 4D).
To identify the factor among the Rel family involved in
alprazolam-induced inhibition, IL-1-stimulated T98G cells, which
were the positive control, and cells stimulated with IL-1 in the
presence of alprazolam were treated with anti-c-Rel Ab to remove
c-Rel, then subjected to the gel-shift experiment using A2 probe (Fig. 4E). Treatment with anti-c-Rel Ab decreased DNA binding
in the positive control in the gel-shift experiment. In cells treated
with alprazolam, no effect was observed. The above findings also
strongly suggested that alprazolam-induced inhibition of MCP-1 is due
to blockage of the binding site of the c-Rel/p65 and c-Rel/p50 complex
to DNA. Next, EMSA was performed using IL-8/NF-B, AP-1, and A1 as
the probes (Fig. 5). Alprazolam did not
exhibit inhibitory activity with IL-8/NF-B or AP-1 (Fig. 5, A and B), and p65 and p50 were confirmed to be
the molecules that bind to IL-8/NF-B probe (Fig. 5D). In
contrast, A1 exhibited inhibitory activity similar to that of A2 (Fig. 5C). Furthermore, the supershift experiment using Abs
against p65, p50, and c-Rel showed that p65, p50, and c-Rel were
associated with the bands detected in the gel-shift experiment using
the A1 probe, as in the experiment using A2 probe (Fig. 5E).
Therefore, c-Rel may be involved in the inhibition of the MCP-1
enhancer region. Thus, our results suggest that alprazolam specifically
inhibited the binding of c-Rel/p65 and c-Rel/p50 to the promoter region
of MCP-1.
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Alprazolam inhibited the production of other proteins regulated by c-Rel
The above findings suggest that alprazolam regulates the
transcriptional activation of proteins involving transcription factor
c-Rel. To confirm this, we investigated the effect on production of
various cytokines in LPS-stimulated mouse macrophage cells RAW 264.7
cells. It has been reported that LPS-stimulated RAW 264.7 cells
produced NO, TNF-, IL-1, and JE. RAW 264.7 cells pretreated with
IFN-, then stimulated with LPS, produced IL-12, and c-Rel was
involved at the transcriptional level in the production of these
proteins, excluding TNF- (18, 19, 20, 21, 22). In contrast, c-Rel
was not involved in the production of NO in the cells stimulated with
IFN-. Thus, to investigate whether the inhibitory activity of
alprazolam is related to c-Rel, we investigated its effect on each
product. The results are shown in Fig. 6.
Alprazolam markedly inhibited the production of IL-1 and JE, which
has been shown to involve c-Rel, while the inhibition of
IFN--induced NO production and IL-12 production requiring
pretreatment with IFN- was weak (Fig. 6, A–E). Moreover,
JE expression induced by LPS was reduced more than TNF- expression
in the presence of 0.01–1 µg/ml alprazolam (Fig. 6F). The
results suggested that alprazolam inhibited c-Rel-regulated
transcription, because c-Rel, p50, and p65 combined with the
LPS-stimulated inducible NO synthase, IL-1, and IL-12 promoter
regions, whereas p50 and p65 combined with the LPS-stimulated TNF-
promoter region (23). The inhibitory effect on IL-12
production may have decreased, because it was produced in RAW cells
after IFN- pretreatment in this experiment, and IFN- stimulation
does not involve c-Rel.
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G). It was also
confirmed by the supershift experiment that p65, p50, and c-Rel are
related to the A2 region (Fig. 6G). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and IFN-. Recently, two closely located NF-B binding
sites have been identified in the distal 5'-flanking region of the
human MCP-1 gene: A1 (5'-GGGAACTTCC-3') and A2 (5'-GGGAATTTCC-3')
(9). The A2 site was found to be important for
transcription of the human MCP-1 gene in TPA-, IL-1-, and
TNF-stimulated malignant glioma cells (A172) (24). The A2
sequence has a high affinity for c-Rel/p65. In contrast, the NF-B
site of the Ig chain gene (5'-GGGACTTTCC-3') has a high affinity for
p50, and the human IL-8/NF-B site (5'-TGGAATTTCC-3') has a high
affinity for only p65 (9). From EMSA experiments with
nuclear extracts from IL-1-stimulated T98G cells it became evident
that alprazolam inhibited NF-B binding to the A1 and A2 promoter
regions. The binding was markedly inhibited in the EMSA system in which
alprazolam was added to the nuclear extract preparation, suggesting
this mode of action. The involvement of A2 and A1 in the expression of
MCP-1 in T98G cells was investigated. Although the base sequences were
similar, based on the comparative experiment using the same amount of
nuclear extract and the finding of luciferase shown in Fig. 2A, the involvement of the A2 region was greater. Both IL-8
and MCP-1 were produced by IL-1-stimulated human T98G cells,
but alprazolam inhibited only MCP-1 production. In other words,
alprazolam inhibited the expression of MCP-1 proteins, which have the
A2 and A1 sequence in the promoter region.
Five Rel family members have been identified in mammalian cells: p50,
p65 (RelA), c-Rel, p52, and RelB. Our results suggested that alprazolam
inhibited the binding of c-Rel/p65 and c-Rel/p50 to the A2 region more
effectively than p50/p65, because alprazolam did not influence IL-8
production under the same conditions. Moreover, when nuclear extract
(positive control) from IL-1-stimulated cells was reacted with
anti-c-Rel Ab to remove c-Rel, DNA binding in the positive control
decreased in the gel-shift experiment; on the other hand, in cells
treated with alprazolam, no effect was observed.
Moreover, we showed that alprazolam inhibited other proinflammatory
cytokines regulated by Rel family members. Recently, it was reported
that alprazolam suppresses T cell proliferation by selectively
inhibiting the production of IL-2, but not the acquisition of IL-2R
(25). The role of Rel in the monocyte/macrophage lineage
was elucidated in mice with an inactivated c-rel gene
(26). It was clear that Rel is a positive or negative
regulator of transcription in macrophages and that Rel has distinct
roles in different macrophage populations. Venkataraman et al. showed
that c-Rel is a component of the Ag receptor-induced, B binding
proteins in both B and T cells (27). Moreover, there is
evidence that c-Rel contributes to the survival of nerve growth
factor-dependent sympathetic neurons (28), the human
12-lipoxygenase gene (29), and c-myc gene
transcription (30). Furthermore, the sequences in the
distal regulatory region, including the two NF-B (A1 and A2) sites,
are highly homologous between the human and mouse MCP-1 genes. Freter
et al. (31) recently reported the binding of a 90-kDa
phosphoprotein coactivator to mouse MCP-1. The participation of an
unknown coactivator and the effect of alprazolam upon it were
suggested.
This study demonstrated a new mode of action for alprazolam. In
addition to relieving the anxiety and tension that accompany
inflammation as well as stomach/duodenal ulcers and hypersensitivity
colitis, alprazolam is expected to prevent an excess of chemokines and
cytokines, including MCP-1, from infiltrating inflammatory regions,
i.e., to inhibit c-Rel-associated immunity and inflammation-related
substances. Plasma IL-1 concentrations were found to be higher in
patients suffering from panic disorders than in control subjects
(32), and alprazolam is expected to improve the symptoms
of these patients. As MCP-1 can be detected during inflammation,
including in the brain of patients with Alzheimer’s disease
(33), this novel action of alprazolam may be useful in
therapy.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: PAF, platelet-activating factor; LDH, lactate dehydrogenase; m, mouse; MCP-1, monocyte chemoattractant protein-1; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide.
Received for publication August 7, 2001. Accepted for publication July 11, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Eur. Cytokine Network 9:47.[Medline]
B is target for glucocorticoid-mediated interleukin 8 gene repression. J. Biol. Chem. 269:13289.B/IB proteins in cancer: implications for novel treatment strategies. Surg. Oncol. 8:143.[Medline]
B1 precursor protein p105. Biochimie 83:341.[Medline]
B/Rel in lipopolysaccharide-stimulated macrophages. J. Pharmacol. Exp. Ther. 294:548.B/Rel activation. Toxicol. Lett. 104:195.[Medline]
B/Rel nuclear translocation by dexamethasone: mechanism for the inhibition of iNOS gene expression. Biochem. Mol. Biol. Int. 45:435.[Medline]
gene expression in LPS-stimulated RAW 264.7 cells by blocking NF-B/Rel and AP-1 activation. Immunopharmacology 48:173.[Medline]
B activation in mouse macrophages and the RAW 264.7 cell line by a synthetic adenyl carbocyclic nucleoside. Biochem. Pharmacol. 60:717.[Medline]
B-like transcription factors is a positive and negative regulator of macrophage gene expression: distinct roles for Rel in different macrophage populations. EMBO J. 15:7099.[Medline]
B contributes to survival of sympathetic neurons. J. Neurosci. 18:10356.B/Rel. FEBS Lett. 363:105.[Medline]
Bp65 (RelA) and c-Rel, but not by NF-Bp50. Biochem. J. 313:39.
B and a 90-kDa phosphoprotein coactivator. J. Biol. Chem. 271:17417. concentrations in panic disorder. Psychiatry Res. 54:135.[Medline]
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, enhancer region; 
, promoter
region; X, introduced mutations; A1 and A2, NF-
