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Departments of
* Biochemical Pharmacology and
Molecular Toxicology, University of Konstanz, Konstanz, Germany;
Medical School Hannover, Institute of Biochemistry, Hannover, Germany; and
Neurobiology, H. Lundbeck A/S, Valby, Denmark
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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,
IL-6, and IFN-
, whereas TNF production was strongly attenuated. In
line, MK2-deficient bone marrow-derived macrophages showed impaired
release of TNF, but not of IL-1, in response to various bacterial
stimuli in addition to decreased phagocytosis of fluorescence-labeled
bacteria. Furthermore, spleen cells from MK2-/- mice
displayed diminished IFN- synthesis after stimulation with L.
monocytogenes. In contrast, MK2 deficiency had no effect on
macrophage generation of NO or on oxidative burst activity in response
to L. moocytogenes. These results
indicate an essential role of MK2 in host defense against intracellular
bacteria probably via regulation of TNF and IFN- production required
for activation of antibacterial effector
mechanisms. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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expression (19). Targeted disruption of the MK2 gene in
mice results in strongly decreased TNF production associated with
enhanced resistance to liver damage induced by concomitant injection of
D-galactosamine plus LPS in vivo (19),
suggesting that MK2 represents an interesting pharmacological target
for specific anti-inflammatory therapy. However, an intact cytokine response is essential for efficient host defense against invading pathogens (20, 21, 22, 23, 24, 25) and global suppression of cytokine production via, for example, anti-inflammatory glucocorticoid therapy strongly increases susceptibility to microbial infection (26, 27, 28, 29). These considerations prompted us to study the effect of MK2 deficiency on host defense in a murine model of bacterialinfection in vivo and on leukocyte effector functions in vitro.
To this aim, we infected MK2-/- and control mice with a virulent strain of the facultative intracellular bacterium Listeria monocytogenes and determined the survival, bacterial load, and mediator production. Our results indicate that MK2 plays a critical role in resistance against intracellular bacteria probably via regulation of selected cytokines and phagocytosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice carrying a mutation in the catalytic domain of the MK2 gene (19) were kept at 24°C, 55% humidity, at a 12-h day-night rhythm on a diet of Altromin C 1310 (Altromin, Lage, Germany). Age- and sex-matched male and female MK2-/- and MK2+/+ littermates on a mixed background (S129 x C57BL/6) bred in the internal animal facility of the University of Konstanz were used for the experiments. All animals received humane care in accordance with the National Institutes of Health guidelines and the legal requirements in Germany.
Bacteria
The hemolytic L. monocytogenes EGD strain was generously provided by S. H. E. Kaufmann (Department of Immunology, Max Planck Institute for Infection Biology, Berlin, Germany). After passaging in vivo, bacteria were grown on tryptic soy broth (Difco, Detroit, MI) at 37°C and gentle rotation. Aliquots of 3 x 109 viable bacteria/ml in 25% glycerin were stored at -80°C. Just before use, stock aliquots were thawed and diluted in pyrogen-free saline. For in vitro experiments, aliquots were incubated for 1 h at 70°C to inactivate bacteria. These heat-killed L. monocytogenes (HKLM) were opsonized by incubation with 10% normal mouse serum at 37°C for 30 min.
Experimental infection
L. monocytogenes infection was initiated by i.v. inoculation with 104 or 1.5 x 105 bacteria per kilogram of body weight, and survival was monitored for 14 days. Bacterial load and cytokine levels were analyzed 48 and 72 h after challenge in parallel experiments. In addition, leukocyte counts were determined at 72 h.
Determination of CFU
CFU were determined from serial dilutions of organ homogenates or blood plated on Columbia blood agar plates (Heipha, Heidelberg, Germany) and incubated at 37°C for 30 h.
Leukocyte counts
Blood was obtained by cardiac puncture under terminal pentobarbital anesthesia (Narcoren; Merial, Hallbergmoos, Germany). White blood cell counts were determined microscopically in a Neubauer chamber after erythrocyte lysis with Türk’s solution (Merck, Darmstadt, Germany). Leukocyte differential counts were done on May-Grünwald/Giemsa-stained smears.
Cytokine ELISA
Cytokine levels in organ homogenates, plasma, and culture supernatants were determined by ELISA, using specific Abs purchased from PharMingen (Hamburg, Germany). For the detection of TNF in in vivo samples, the OptEia kit from PharMingen was used. The detection limits were <25 pg/ml.
Generation of BMDM
Mice were killed by cervical dislocation, and bone marrow cells were isolated from the femurs. After erythrocyte lysis with 0.17 M ammonium chloride, bone marrow cells were cultured at an initial density of 105 cells/ml on 94/16-mm microbiology plates (Greiner, Nurtingen, Germany) in 20 ml RPMI 1640 with glutamine supplemented with 10% heat-inactivated FCS (Biochrom, Berlin, Germany), antibiotics (100 U/ml penicillin, 10 mg/ml streptomycin; Life Technologies, Eggenstein, Germany), and 20% M-CSF-containing L929 supernatant for 6–9 days. Differentiated bone marrow-derived macrophages (BMDM) were harvested with cold PBS without Ca2+ and Mg2+, resuspended in medium without L929 supernatant, and used at a density of 6.5 x 104 cells/well.
Determination of cytokine production by BMDM
BMDM (6.5 x 104 cells/well) from MK2+/+ and MK2-/- mice were added to 96-well microtiter plates (Cellstar; Greiner) and allowed to adhere for at least 3 h. Then, cells were stimulated by addition of LPS (Salmonella abortus equi; Bioclot, Wustenhofen, Germany), lipoteichoic acid (LTA) from Staphylococcus aureus (in-house preparation, as described previously (30)), tetramethylrhodamine-conjugated fluorescent Escherichia coli particles (Molecular Probes, Leiden, The Netherlands), or serum-opsonized HKLM. After incubation for 6 h at 37°C, 5% CO2 in a humidified atmosphere, cytokine levels in supernatants were determined.
IFN- production by spleen cells
Spleens were removed aseptically from male
MK2+/+ and MK2-/- mice,
and single cell suspensions were generated in 1 ml saline by pressing
spleens through 50-µm nylon meshes (BD Biosciences, Heidelberg,
Germany). After lysis of erythrocytes with 0.17 M ammonium chloride,
cells were washed and resuspended in RPMI 1640 (BioWhittaker, Verviers,
Belgium) containing 10% FCS (Boehringer Mannheim, Mannheim,
Germany) and 100 IU/ml penicillin/streptomycin (Biochrom). A
total of 1 x 106 cells/well was added to
96-well culture plates (Greiner) and stimulated with serum-opsonized
HKLM for 24 h at 37°C. IFN- was determined in supernatants by
ELISA.
Determination of macrophage phagocytosis
Determination of macrophage phagocytosis was performed essentially as described (31), with the following modifications. BMDM (6.5 x 104/well) were plated in 96-well microtiter plates and allowed to adhere for at least 3 h. At different time points, tetramethylrhodamine-conjugated fluorescent E. coli particles were added to a final concentration of 5 µg/ml. In some experiments, the E. coli particles had been opsonized with 10% normal mouse serum at 37°C for 30 min before use (serum-opsonized E. coli). Phagocytosis was stopped by washing the cells twice with PBS to remove nonphagocytosed bacteria. Then, cells were lysed by addition of 100 µl/well PBS plus 0.1% Triton X-100. Fluorescence was determined at 530 nm excitation and 590 nm emission wavelengths using a fluorescence microplate reader (microplate fluorescence reader FL 600; Deelux Labortechnik, Goedenstorf, Germany). Cells without bacteria were used to determine the background fluorescence. Inhibition by low temperature or cytochalasin D was used to distinguish between binding and phagocytosis of bacteria in preliminary experiments.
Determination of NO production
BMDM (6.5 x 104 cells/well)
were pretreated with saline or 10 ng/ml recombinant murine (rmu)IFN-
(a generous gift from G. R. Adolf, Bender, Vienna, Austria) for 45
min. Then, cells were stimulated by addition of LPS, LTA, or HKLM for
24 h. NO production was determined indirectly by measuring nitrite
levels in supernatant by the Griess reaction (32).
Determination of oxidative burst
For determination of luminol-ECL (33),
105 peritoneal lavage cells from naive mice per
well were cultured in white 96-well cell culture plates (Cellstar;
Greiner) for 24 h in the presence of either 0.9% NaCl/0.1% human
serum albumin (HSA) or a combination of 10 ng/ml rmuIFN- plus 10
ng/ml LPS. Then, cells were washed twice with PBS and resuspended in
HBSS without Phenol Red (Life Technologies) containing 10% FCS
(Boehringer Mannheim). Background luminescence was recorded after
addition of luminol (5-amino-2,3-dihydro-1,4-phtalazinedione; Sigma,
Deisenhofen, Germany) dissolved in 0.04% triethylamin (Sigma) at a
final concentration of 1 mg/ml. Then, cells were stimulated by addition
of either 108 serum-opsonized HKLM or 1 mg
zymosan (Sigma) per milliliter, and luminescence was determined for
2 s every 3.2 min for a period of 70 min at 37°C in a
luminometer (VICTOR2 multilabel counter; Wallac
Instruments, Turku, Finland).
Statistics
Data are presented as SEM. Statistical differences were determined by the two-sided, unpaired Student t test after testing for normality using the Kolmogorov-Smirnov test. In case of multiple comparisons, the Bonferroni’s multiple comparison test of selected data pairs was used. In case of unequal variances (Bartlett’s test, p < 0.05), data were log transformed before analysis. Survival curves were generated according to the method of Kaplan and Meier and compared using the log-rank test. A value of p < 0.05 was considered significant. All tests were performed with GraphPad Prism, version 3.0 for Windows (GraphPad, San Diego, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been shown previously that MK2-deficient mice, probably as
a result of decreased TNF synthesis, are more resistant to inflammatory
liver damage induced by the concomitant injection of LPS plus
D-galactosamine, suggesting a decisive role of MK2 in the
inflammatory response to endotoxin (19). To study the
involvement of MK2 in inflammation and host defense during
Gram-positive bacterial infection, we determined the susceptibility of
MK2-/- mice to infection with L.
monocytogenes. MK2-/- and
MK2+/+ mice were infected i.v. with 1 x
LD10 (1.5 x 105
CFU/kg), and the survival was monitored for 14 days.
MK2-/- mice displayed significantly increased
mortality as compared with wild-type (wt) mice (survival 1 of 7
(MK2-/-) vs 9 of 10 (wt), p <
0.001; Fig. 1
). However, a residual
capacity to clear bacteria was retained in
MK2-/- mice, because all survived infection
with 104 bacteria per kilogram of body weight
(data not shown).
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To test whether the increased susceptibility of
MK2-/- mice to L. monocytogenes
challenge was due to impaired bacterial elimination, we determined the
bacterial load in blood and organ homogenates following infection with
L. monocytogenes. No significant differences in CFU numbers
were found at 48 h postinfection (data not shown). However, at
72 h postinfection, MK2-/- mice displayed
higher bacterial numbers in all organs tested (liver, spleen, lung,
blood), although only the differences in CFU counts in spleen and lung
reached statistical significance (Fig. 2). These results suggest that despite
comparable initial host defense, MK2-/- mice
failed to control bacterial replication at the later stage of L.
monocytogenes infection. Notably, the numbers of total blood
leukocytes did not differ significantly between the two genotypes at
72 h postinfection (2.7 ± 0.3 x
106/ml (MK2+/+) vs 2
± 0.3 x 106/ml
(MK2-/-), n = 5;
p = 0.12).
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Efficient host defense against the intracellular pathogen L.
monocytogenes is dependent on an intact cytokine response
(20, 21, 22, 34, 35). Because previous data demonstrated a
regulatory role of MK2 in TNF and IFN- induction after exposure to
the model inflammatory stimulus LPS (19), we hypothesized
that decreased production of these cytokines could be responsible for
deficient antibacterial defense in MK2-/-
mice.
We determined the production of several proinflammatory cytokines in
blood and homogenates of liver, spleen, and lung 48 and 72 h after
infection with 1.5 x 105
Listeria/kg. The tissue/blood levels of IL-1, IL-6, and
IFN- at either time point of analysis were not significantly
affected by the genotype (data not shown). However, TNF concentrations
were strongly reduced in L. monocytogenes-infected
MK2-/- animals at late stage of infection (Fig. 3), despite similar or even increased CFU
numbers (see above).
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To characterize the effect of MK2 on TNF production in more detail, we performed in vitro studies on TNF release by isolated macrophages from wt and MK2-/- mice. As an extension of previous studies using the Gram-negative model stimulus LPS (19), we used also the Gram-positive analog, LTA, and entire Gram-positive or Gram-negative bacteria.
MK2-/- BMDM displayed significantly reduced TNF
release in response to all stimuli tested (LPS, LTA, E.
coli, HKLM) (Fig. 4). This indicates
a key role of MK2 in TNF production elicited by upstream signal
transduction pathways involving different Toll-like receptors
(TLR). In contrast to TNF, reduction of IL-6 production was confined to
LPS challenge, and IL-1 release was even enhanced in
MK2-/- macrophages stimulated with HKLM (Fig. 4). These results support the in vivo finding of a critical involvement
of MK2 in TNF regulation, whereas production of IL-6 and especially of
IL-1 appears to be largely independent of MK2.
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production by spleen cells from
MK2-/- mice
In addition to the posttranscriptional control of TNF synthesis,
previous studies indicated a regulatory role of MK2 in the synthesis of
IFN- after LPS stimulation of spleen cells in vitro
(19). In contrast, IFN- levels in
MK2-/- mice 48 and 72 h after infection
with L. monocytogenes were not significantly different from
those of wt animals (data not shown). However, because samples were
taken at later phases of infection with significantly higher bacterial
load in MK2-/- mice (72 h), we chose a
different approach to study early IFN- production in response to
defined numbers of pathogens. To this aim, we determined in vitro
IFN- production by isolated spleen cells from
MK2+/+ and MK2-/- mice
upon stimulation with increasing concentrations of HKLM. Indeed, in
this in vitro model, IFN- levels were significantly lower in
supernatants from MK2-/- spleen cells (Fig. 5), confirming and extending previous
findings of a major regulatory role of MK2 in IFN- production.
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Engulfment of blood-borne bacteria and subsequent intracellular
killing by cytokine-activated macrophages is a key defense mechanism
during the early phase of listeriosis, restricting bacterial
replication rate before the establishment of a specific and sterilizing
T cell response (36). To test whether phagocytic activity
depended on functional MK2, we determined the uptake of
fluorescence-labeled E. coli particles by BMDM in an
optimized quantitative phagocytosis assay. Uptake of bacteria, as
measured by specific fluorescence increase, was approximately linear
over several hours and could be blocked by addition of cytochalasin D
or low temperature (Fig. 6A).
As shown in Fig. 6B, MK2-/- BMDM
displayed functional, albeit reduced, phagocytic activity compared with
wt cells. Prior opsonization of E. coli with 10% normal
mouse serum enhanced bacterial phagocytosis by cells from either
genotype by 
60% of the respective value for nonopsonized bacteria
(Fig. 6C), suggesting that complement receptors were still
fully functional in MK2-/- cells and that a
general mechanism of phagocytosis was impaired in the absence of
MK2.
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Generation of reactive oxygen intermediates (ROI) by the
NADPH-dependent oxidase has been shown to be critical for resistance
against L. monocytogenes (37, 38, 39). We
determined ROI production by adherent peritoneal cells from wt and
MK2-/- mice in response to HKLM. No difference
in peak activity or overall oxidative burst (area under the curve) was
observed between cells from either genotype (Fig. 7A). When cells were
pretreated for 24 h with a combination of LPS and rmuIFN-,
oxidative burst activity in response to HKLM (Fig. 6
B), but
not to zymosan (data not shown), was strongly increased. Although peak
levels and area under the curve were slightly reduced in macrophages
from MK2-/- mice (Fig. 7B),
functional MK2 was not essential for oxidative burst activity, arguing
against a direct regulation of macrophage NADPH oxidase by
MK2.
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In addition to activation of the NADPH oxidase, production of NO
has been suggested as an important antimicrobial killing mechanism of
activated macrophages (39, 40, 41). We determined the ability
of BMDM from MK2-/- and wt mice to generate NO
(as measured by nitrite accumulation in the medium over 24 h) in
response to stimulation with LPS, LTA, and HKLM. Nitrite production in
response to LPS stimulation was significantly decreased in
MK-2-deficient cells. LTA or HKLM alone was not sufficient to trigger
NO production in vitro (Table I). IFN-
has been shown to be a potent costimulus for expression of the
inducible NO synthase (iNOS) (42, 43). Therefore, we
examined NO production by BMDM also in the presence of exogenously
added rmuIFN-. Indeed, besides increasing NO production by LPS
stimulus, addition of this cytokine boosted NO production in response
to LTA as well as HKLM to levels comparable with those induced by LPS +
IFN-. However, NO release in response to IFN- plus LTA/HKLM was
not affected by MK2-/- deficiency, indicating
that TLR2-specific stimuli can induce iNOS independent of MK2, provided
that enough IFN- is present.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, especially, have been shown to be essential for the activation
of listericidal activity in infected macrophages (22, 39, 44, 45, 46). In line with previous findings for LPS challenge, TNF
levels were decreased in MK2-deficient mice infected with L.
monocytogenes, indicating that TNF induction by Gram-positive
infection is regulated by the MK2. Although disruption of the MK2 gene
significantly increased the susceptibility to Listeria
infection, MK2-/- mice were still able to
control infection with a lower inoculum (104/kg),
suggesting a moderate effect of MK2 on resistance compared with the
drastically increased susceptibility of TNFR or IFN- receptor
knockout mice (20, 21, 22). This can be explained by the
finding that MK2-/- mice were still able to
produce lower, but still substantial amounts of TNF and normal levels
of IFN-. Our results of an increased bacterial load in
MK2-/- mice confirm and extend recent data from
van den Blink et al. (17), who demonstrated deficient
control of bacterial replication after administration of the p38
inhibitor SB203580 in murine models of pneumonia and
tuberculosis. However, our data demonstrate reduced TNF release in
MK2-/- mice despite an increase in bacterial
numbers, which is in contrast to the enhanced TNF production observed
by van den Blink et al. (17). This difference could be due
to inefficient inhibition of p38 in macrophages in vivo when SB203580
was used. Alternatively, because Listeria infection has been
shown to induce signaling via both the p38 and the p42/p44 MAPK pathway
(47, 48, 49), impairment of additional macrophage activation
via p42/44 MAPK in MK2-/- mice could be
responsible for the diverging effects of p38 inhibition and MK2
deficiency. During the first 48 h of listeriosis,
polymorphonuclear granulocytes (PMN) play a pivotal role by engulfing
free bacteria, e.g., in the sinusoids of the liver (50).
Thus, impaired ability of MK2-/- mice to
recruit PMN from the bone marrow could be responsible for the higher
bacterial load observed in the MK2 knockout animals. Our data do not
support a major defect in leukocyte recruitment, because no differences
in total number of circulating leukocytes or leukocyte composition were
found at 72 h postinfection. However, further studies using
quantitative determination of PMN organ infiltration at earlier time
points postinfection will be necessary to definitely settle this point.
Besides infiltration of neutrophils, phagocytosis and breakdown of
bacteria by resident macrophages of the reticuloendothelial system is a
key defense mechanism to restrict bacterial dissemination and infection
of more permissive cells such as hepatocytes during the early course of
L. monocytogenes infection (51). In addition,
infiltrating monocytes and macrophages contribute to granuloma
formation and digestion of bacteria at the site of infection (52, 53). Due to this pivotal role of macrophages in early control of
bacterial replication, we were interested in determining the effect of
MK2 disruption on macrophage functions in vitro. When we studied the
uptake of fluorescence-labeled E. coli, BMDM from
MK2-/- mice showed a moderate decrease in the
rate of phagocytosis as compared with wt cells. In addition,
measurement of TNF levels in supernatants from the same experiments
indicated a drastic reduction in the capacity of
MK2-/- BMDM to produce TNF in response to
E. coli. The early onset of phagocytosis suggested that the
observed differences in bacterial uptake were independent of the defect
in TNF production. Attenuation of TNF release in
MK2-/- macrophages was neither restricted to a
certain class of stimuli, nor TLR specific, because similar results
were obtained with particular (E. coli and L.
monocytogenes) as well as soluble stimuli (LPS and LTA) signaling
either via TLR2 (L. monocytogenes (54), LTA
(55)), or TLR4 (E. coli, LPS
(56)). This observation indicates that intracellular
signaling pathways of the different receptors converge upstream of MK2.
Following uptake by cytokine-activated macrophages, bacteria in the
phagolysosome are confronted with a battery of microbicidal substances
such as reactive oxygen and nitrogen species (37, 39, 40).
Evidence has been provided that generation of NO is an essential host
mechanism in antilisterial defense because inhibition of iNOS increases
bacterial load and susceptibility of mice to L.
monocytogenes infection (40). Because previous data
indicated diminished NO production in MK2-/-
mice in response to LPS stimulation (19), we hypothesized
that defective generation of reactive nitrogen intermediates by
MK2-/- macrophages could contribute to the
impaired control of bacterial replication in vivo. However, no NO
production at all was detectable upon stimulation of BMDM from either
genotype with LTA or HKLM unless very high concentrations
(108 HKLM/ml) were used or cells were
costimulated with IFN-. The finding that HKLM plus
IFN--costimulated BMDM from MK2-/- mice
produced comparable amounts of NO as wt cells argues against a direct
involvement of MK2 in the expression of the iNOS during listeriosis. In
addition to NO production, the generation of ROI, which is considered a
key listericidal mechanism of activated macrophages, was not
significantly impaired in macrophages from
MK2-/- mice. This finding is in contrast to
previous results showing MK2-dependent oxidative burst by human
neutrophils in response to fMLP or phorbol ester stimulation
(57). This discrepancy could be due to the different cell
types (human PMN vs murine macrophages) or stimuli (fMLP or PMA vs
HKLM) used in the different studies.
Our results derived from MK2-deficient macrophages indicate that the
increased susceptibility of MK2-/- mice to
L. monocytogenes infection did not result from an inherent
deficiency of macrophage-killing mechanisms. However, NO production as
well as oxidative burst induced by HKLM strongly depended on
costimulation with IFN-. Thus, an attenuation of early IFN-
production could result in insufficient macrophage activation. Our
results on IFN- production by isolated spleen cells upon stimulation
with HKLM (Fig. 4) support this hypothesis. In contrast to the strong
reduction of IFN- release in vitro, IFN- levels in vivo in
MK2-/- mice infected with viable L.
monocytogenes were not significantly different from those of wt
mice. Nevertheless, because IFN- production by spleen cells
increased with higher concentrations of HKLM, a potentially more
drastic defect in IFN- production in vivo could have been
compensated by the increase in bacterial load in
MK2-/- mice at the time of IFN-
determination (72 h). Determination of early IFN- levels immediately
after infection will be necessary to clarify this question.
Experiments in knockout mice suggested additional macrophage
listericidal mechanisms independent of NO/ROI production that required
macrophage activation via the p55 TNFR (44). MK2
deficiency most markedly impaired TNF production, whereas the effect on
phagocytosis as well as on the production of other cytokines (IL-1,
IL-6) or NO was much less pronounced. From these results, it can be
speculated that the defect of MK2-/- mice to
restrict early growth of L. monocytogenes could be due to a
combination of inefficient activation of these yet unknown
TNF-dependent listericidal activity and the TNF/IFN--dependent
stimulation of NO generation and oxidative burst. Taken together, our
results showing a decisive role of MK2 in host defense against virulent
Gram-positive bacteria emphasize that caution must be taken when
addressing MK2 inhibition as an anti-inflammatory therapy during
acute or chronic infection.
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Acknowledgments |
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, to Siegfried Morath
(University of Konstanz) for providing LTA, and to Sonja von Aulock
(University of Konstanz) for critically reading this manuscript. |
Footnotes |
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2 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; BMDM, bone marrow-derived macrophage; HKLM, heat-killed L. monocytogenes; iNOS, inducible NO synthase; LTA, lipoteichoic acid; MK2, MAPK-activated protein kinase 2; PMN, polymorphonuclear granulocyte; rmu, recombinant murine; ROI, reactive oxygen intermediate; TLR, Toll-like receptor; wt, wild type; HSA, human serum albumin.
Received for publication August 23, 2001. Accepted for publication March 5, 2002.
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Y. Shi, A. Kotlyarov, K. Laass, A. D. Gruber, E. Butt, K. Marcus, H. E. Meyer, A. Friedrich, H.-D. Volk, and M. Gaestel Elimination of Protein Kinase MK5/PRAK Activity by Targeted Homologous Recombination Mol. Cell. Biol., November 1, 2003; 23(21): 7732 - 7741. [Abstract] [Full Text] [PDF]
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J. L. Smith, I. Collins, G. V. R. Chandramouli, W. G. Butscher, E. Zaitseva, W. J. Freebern, C. M. Haggerty, V. Doseeva, and K. Gardner Targeting Combinatorial Transcriptional Complex Assembly at Specific Modules within the Interleukin-2 Promoter by the Immunosuppressant SB203580 J. Biol. Chem., October 17, 2003; 278(42): 41034 - 41046. [Abstract] [Full Text] [PDF]
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C.-C. Wu, S.-C. Hsu, H.-m. Shih, and M.-Z. Lai Nuclear Factor of Activated T Cells c Is a Target of p38 Mitogen-Activated Protein Kinase in T Cells Mol. Cell. Biol., September 15, 2003; 23(18): 6442 - 6454. [Abstract] [Full Text] [PDF]
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