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Division of Microbiology, National Institute of Health Sciences, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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induction and NF-
B activation, was found to be
very low (almost inactive) compared with Escherichia
coli lipid A, but all of the lipid As exerted strong activity
on mouse cells and on Limulus gelation activity.
Experiments using chemically synthesized E. coli-type
hexaacylated lipid A (506) and Salmonella-type
heptaacylated lipid A (516) yielded clearer results. Both lipid A
preparations strongly induced TNF- release and activated NF-B in
mouse peritoneal macrophages and mouse macrophage-like cell line J774-1
and induced Limulus gelation activity, although the
activity of the latter was slightly weaker than that of the former.
However, 516 was completely inactive on both THP-1 and U937 cells in
terms of both induction of TNF- and NF-B activation, whereas 506
displayed strong activity on both cells, the same as natural E.
coli LPS. In contrast to the action of the lipid A
preparations, all the Salmonella LPSs also exhibited
full activity on human cells. However, the polysaccharide portion of
the LPS neither exhibited TNF- induction activity on the cells when
administered alone or together with lipid A nor inhibited the activity
of the LPS. These results suggest that the mechanism of activation by
LPS or the recognition of lipid A structure by human and mouse cells
may differ. In addition, both 516 and lipid A from
Salmonella were found to antagonize the 506 and
E. coli LPS action that induced TNF- release and
NF-B activation in THP-1 cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Escherichia coli- and Salmonella-type lipid A
have the most typical lipid A structures so far defined, and both have
been synthesized chemically (12, 13, 14) and well
characterized biologically by many investigators (15, 16, 17, 18, 19, 20).
The chemical structure of these lipid As consists of a hexa- and
heptaacylated diglucosamine bearing six and seven fatty acids,
respectively, as shown in Fig. 1
. The
only structural difference between them is the hexadecanoyl acid
attached to the hydroxyl residue of 3-hydroxy tetradecanoic acid bound
to the 2 position of reducing glucosamine.
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In view of the species-specific actions of endotoxin, it is necessary to clarify the actual action of lipid A molecules on human cells to better understand endotoxicosis, and thus the activity of endotoxin must be considered based on its actions on human cells.
In this study, we show that one of the typical lipid As from Salmonella is not an activator on human cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant TNF- standards and rabbit polyclonal antisera
against murine TNF- were obtained from Asahi Kasei Kogyo (Tokyo,
Japan). Rabbit IgG was obtained from Zymed Laboratories (South San
Francisco, CA). Anti-human and -mouse IB- antisera (1309 and 751,
respectively) were gifts from Dr. Nancy Rice (Advanced BioScience
Laboratories-Basic Research Program, National Cancer
Institute-Frederick Cancer Research and Development Center). Synthetic
lipid A analogues 506 and 516 were a gift of Daiichi Kagaku (Tokyo,
Japan). RPMI 1640 medium with glutamine and IMDM were the products of
Life Technologies (Grand Island, NY). Quantitative Limulus
assay reagent (Endospecy) was obtained from Seikagaku Kogyo (Tokyo,
Japan). Pyrogen-free water was a product of Otsuka Seiyaku (Tokyo,
Japan). LPS was extracted from E. coli 03K2a2b:H2,
Salmonella minnesota (S type), Salmonella
typhimurium LT2, and Salmonella abortus equi by the
aqueous phenol method (21). Lipid A was obtained as an
insoluble substance after 1% acetic acid treatment of LPS at 100°C
for 90 min (22). The polysaccharide portion was obtained
from the supernatant of the reaction mixture by evaporating to dryness
and washing with chloroform three times. No residual lipid A portion
was detected by either fatty acid analysis by Gas chromatography or by
Limulus gelation assay.
Gas-liquid chromatography conditions
Gas-liquid chromatography analysis was performed on a model GC-14A (Shimadzu, Kyoto, Japan) with a HiCap-CBM5 fused silica capillary column (25 m x 0.25 mm; GL Science, Toyko, Japan) and temperature programs 120°C for 3 min to 250°C at 3°C/min. Nitrogen was used as carrier gas.
Mass spectrometry
Liquid secondary-ion mass spectrometry was performed on a VG ZAB-2SEQ spectrometer (VG Analytical, Manchester, U.K.) operated at 8 kV in a negative mode. The cesium gun was operated at 30 kV. Current-controlled scans were acquired at a rate of 30 s per decade. A mixture of ethanolamine and m-nitrobenzyl alcohol (1:1) was used as the matrix.
Induction of TNF- release from mouse peritoneal macrophages,
J774-1, THP-1, and U937 cells
Mouse peritoneal macrophages were obtained by washing the
peritoneal cavity of female BALB/c mice (6–10 wk old, Japan SLC,
Hamamatsu, Japan) with 5 ml of serum-free IMDM (23). The
cell number was adjusted to 2 x 106
cells/ml. After adhesion, the cells were incubated with the stimulant
for 6 h. J774-1, THP-1, and U937 cells were grown in RPMI 1640
medium supplemented with 10% (v/v) heat-inactivated FCS, 50 µM 2-ME,
5 mM HEPES, penicillin (100 U/ml), and streptomycin (100 µg/ml) in a
5% CO2 atmosphere at 37°C. J774-1 cells were
harvested by scraping with cell scraper (Costar, Cambridge, MA) and
suspended in a fresh medium. The cells (1 x
106 cells/ml/well of 24-well dishes) were allowed
to adhere to plastic for 3 h at 37°C, washed twice with medium,
and incubated an additional 4 h for TNF- induction with the
stimulant. THP-1 cells (2 x 105
cells/ml/well of 24-well dishes) were prepared for the experiments by
adding 100 ng/ml of PMA (Sigma, St. Louis, MO) and 0.1 µM
1,25-dihydroxy vitamin D3 (Wako Pure Chemical
Industries, Tokyo, Japan) to cell suspensions in RPMI 1640 medium with
10% FCS (11, 24). The cell suspensions were allowed to
differentiate and adhere to plastic for 72 h at 37°C. After
washing, the cells were incubated an additional 24 h with the
stimulant. U937 cells (1 x 106
cells/ml/well of 24-well dishes) were prepared for the experiments by
adding 100 ng/ml of PMA to cell suspensions in RPMI 1640 medium with
10% FCS (25). The cell suspensions were allowed to
differentiate and adhere to plastic for 48 h at 37°C. After
washing, the cells were incubated an additional 8 h with the
stimulant. The supernatant of the test samples was stored at -80°C
until used to determine TNF-.
TNF- assay
The TNF- produced was measured by cytotoxicity assay against
L929 murine fibroblast cells. L929 cells were grown in tissue culture
flasks in RPMI 1640 medium (Life Technologies) supplemented with 10%
FCS, 50 µM 2-ME, 5 mM HEPES, penicillin (100 U/ml), and streptomycin
(100 µg/ml). Cells were detached with trypsin, washed, resuspended in
medium at 4 x 105 cells/ml, and 100 µl
aliquots were plated in 96-well flat-bottom plates (catalog no.
25860-96; Corning Glass, Corning, NY). After incubation for 3–5 h at
37°C in 5% CO2, 50 µl of actinomycin D (4
µg/ml) in RPMI 1640 medium was added to each well, and 50 µl of
test sample was then added to the wells (final volume; 200 µl/well).
The results are expressed as means of triplicate determinations. To
determine whether the cytotoxic activity against L929 cells was due to
TNF-, aliquots of supernatants from macrophage culture were
incubated for 12 h with polyclonal rabbit antiserum to TNF-
using nonspecific IgG as the control. The polyclonal Ab to TNF-
completely abolished the cytotoxicity of the supernatants in all the
samples tested.
Activation of NF-B
Endotoxin-mediated NF-B activation was assessed by analyzing
the degradation of IB- protein in the cytosol fraction by Western
blotting (26, 27, 28).
Crude cytosol fractions of LPS-treated and -untreated cells were prepared as follows (29). J774-1 cells (2 x 106/2.5 ml/well of 6-well dishes) or THP-1 cells (1.25 x 106/well) pretreated with 20 µg/ml of cycloheximide for 30 min were treated with the test sample for 15 and 60 min, respectively, and washed once at 4°C with 5 ml of PBS and were immediately frozen. The frozen cells were lysed on ice for 10 min with 100 µl of lysis buffer (10 mM HEPES-KOH, 5 mM EDTA, and 10 mM KCl, pH 7.9) containing 0.5% (for J774-1) or 0.1% (for THP-1) Nonidet P-40, 1 mM DTT, and a protease inhibitor mixture (Boehringer Mannheim, Mannheim, Germany). The lysate was centrifuged for 5 min at 15,000 rpm at 4°C, and the resultant supernatant was used as the crude cytosol fraction.
The crude cytosol fractions (50 µg of protein) were subjected to 10%
SDS-PAGE, and IB- was detected as follows. Protein was
transferred for 1 h onto a polyvinylidene difluoride membrane
(Millipore, Bedford, MA) with a semidry blotting apparatus. The
membrane was then probed with human (or mouse) IB- antisera
(1:1000 dilution) followed by a peroxide-labeled goat anti-rabbit
IgG Ab (Boehringer Mannheim; diluted 1:10000). Proteins were detected
by using an enhanced chemiluminescence system (Amersham, Arlington
Heights, IL).
Limulus amoebocyte gelation activity
Activation of the proclotting enzyme of the horseshoe crab was tested by a quantitative Limulus assay reagent (Endospecy). Pyrogen-free water was used to dilute the test samples. A 50-µl amount of each sample was incubated with the same volume of lysate containing chromogenic substrate in 96-well flat plates (Costar) for 30 min at 37°C. The reaction was stopped by adding 200 µl of 0.6 M acetic acid. The chromogen (p-nitroaniline) was measured at 405 nm using a microplate reader (Molecular Devices, Menlo Park, CA).
Inhibition assay of endotoxin-mediated TNF- induction and
NF-B activation in THP-1 cells
Inhibition of endotoxin-mediated TNF- induction and NF-B
activation in THP-1 cells were performed by adding the indicated
concentration of the inhibitor to the assay system followed immediately
by the addition of agonist, and TNF- production or the degradation
of IB- was compared with a control containing agonist alone.
Inhibition of TNF- induction is expressed as percent TNF-
production with production in the presence of agonist alone equal to
100%.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To examine the fatty acid composition of LPS and lipid A from the
Salmonella species used in the present study, i.e., S.
abortus equi, S. minnesota (S type), and S.
typhimurium LT2 lipid As were hydrolyzed, and the methyl-fatty
acids liberated were analyzed by gas-liquid chromatography. The results
are shown in Table I, where the molar
ratios of the fatty acids are expressed by assuming the number of
3-hydroxytetradecanoic acid molecules in each LPS to be 4. The
composition of all the lipid As was very similar, with the major
component of all these lipid A preparations being heptaacylated
diglucosamine lipid A, as also confirmed by the results of
mass-spectrometry (data not shown).
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release by mouse peritoneal macrophages and mouse macrophage-like
J774-1 cells
TNF- release into the medium was estimated by cytotoxicity
against actinomycin-D-sensitized L929 murine fibroblasts. Murine
peritoneal macrophages were found to react to stimulation by all
Salmonella LPSs from the three different species, S.
abortus equi, S. minnesota, and S.
typhimurium. As shown in Fig. 2A, the cells started to
secrete TNF- at a LPS concentration of 0.1 ng/ml, and TNF-
production by the macrophages increased dose-dependently. Although the
activity of the lipid A preparations was less than that of their
respective parent LPS, they stimulated cells at a concentration of
1–10 ng/ml.
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production was recognized
at a dose of 1 ng/ml (Fig. 2
B).
Salmonella LPS and lipid A induction of TNF-
release by THP-1 and U937 line cells
Human THP-1 and U937 line cells also reacted to stimulation with
all three Salmonella LPSs, and both lines started to secrete
TNF- at a concentration of 10 ng/ml. The LPS-induced production of
TNF- increased dose-dependently (Fig. 3). However, the cells did not produce
TNF- when exposed to any of the lipid A preparations, and
significant amounts of TNF- were first detected at a concentration
of 10 µg/ml in THP-1 cells, showing that the activity of each lipid A
was 103 times less or more than that of their
parent LPS.
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release by mouse peritoneal
macrophages and mouse macrophage-like J774-1 cells
Lipid A preparations obtained from natural LPS are usually
heterogeneous, and all of the lipid A preparations from
Salmonella LPS used in the present study partially included
a significant amount of E. coli-type lipid A and may also
have included contaminating parent LPS, both of which stimulate human
cells. Because the data obtained by using these preparations are,
therefore, not reliable, the same experiments described above were
performed with chemically synthesized Salmonella-type lipid
A (516; Fig. 1) using E. coli-type synthetic lipid A (506;
Fig. 1) as a control. The results in mouse cells are shown in Fig. 4, A and B. Murine
peritoneal macrophages reacted to stimulation with 516 in a
dose-dependent manner and started to secrete TNF- at a concentration
of 10 ng/ml (Fig. 4A). A similar response to 516 was also
observed in mouse macrophage-like J774-1 cells (Fig. 4B).
The activity of 506 in inducing TNF- was greater than that of 516,
and the activity was expressed even at 1 ng/ml in both mouse peritoneal
and J774-1 line cells, showing that its activity is about ten times
more powerful than that of 516.
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release by
THP-1 and U937 line cells
Human THP-1 and U937 line cells also reacted to stimulation with
E. coli-type hexaacylated lipid A and started to secrete
significant amounts of TNF- at a concentration of 1 ng/ml. The
LPS-induced production of TNF- by both cells increased
dose-dependently (Fig. 4, C and D). In contrast,
neither human THP-1 or U937 cells of produced TNF- at all when
stimulated with 516, even at a concentration of 10 µg/ml (Fig. 4, C and D).
Activation of NF-B in mouse macrophage-like J774-1 cells by
Salmonella LPS and lipid A, and E. coli-
and Salmonella-type synthetic lipid As
To examine endotoxin-mediated NF-B activation, J774-1 cells
were treated with LPS and lipid A from S. abortus equi, and
cell extracts were analyzed for degradation of IB- protein by
immunoblotting. The results are shown in Fig. 5A. Stimulation of the cells
with both S. abortus equi LPS and lipid A resulted in a
rapid decrease and disappearance of IB- protein at 1 ng/ml
ml and 10 ng/ml, respectively. Both synthetic lipid A preparations 506
and 516 also expressed strong IB- degrading activity in J774-1
cells (Fig. 5B). However, the activity of 506 was about ten
times stronger than that of 516, consistent with the results of TNF-
induction.
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B in THP-1 line cells by
Salmonella LPS and lipid A, and synthetic lipid As
Human THP-1 line cells also reacted to stimulation with
Salmonella LPS and E. coli-type hexaacylated
lipid A, and IB- protein degradation started at a concentration
of 1 ng/ml (Fig. 6, A and
B). In contrast, Salmonella lipid A exhibited
much less activity than the parent LPS (104 times
less), and Salmonella type synthetic lipid A did not induce
degradation of IB- at all, even at a concentration of 1 µg/ml
(Fig. 6).
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Activation of the clotting system cascade of the horseshoe crab
was used to assess differences in the activity of Salmonella
LPS and lipid A, 506 and 516. The results are shown in Fig. 7. Although the activity of 516 was
slightly lower than that of 506, all of the preparations exhibited
strong Limulus gelation activity.
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induction activity of E.
coli LPS and 506 by Salmonella lipid A and 516
Because the lipid A from Salmonella and synthetic
Salmonella-type lipid A failed to stimulate TNF-
production by human THP-1 cells, their antagonistic activity on the
response of cells to active LPS was tested by adding them to the cell
cultures together with the agonist. As shown in Fig. 8, both Salmonella lipid A and
synthetic Salmonella-type lipid A displayed inhibitory
activity on TNF- production stimulated by E. coli LPS.
Suppression by synthetic Salmonella-type lipid A was
observed in a dose-dependent manner without regard to the stimulatory
dose of the agonist (10 and 100 ng/ml). Significant inhibitory effects
were observed at an agonist to antagonist ratio of 1:10 (w/w), and
almost complete inhibition was possible at a 100-fold excess of both
antagonists to stimulant.
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B activation induced by E.
coli LPS in THP-1 cells
The antagonistic activity of synthetic Salmonella-type
lipid A on the response of human THP-1 line cells to
IB--degrading activity of 506 was tested. As shown in Fig. 9, synthetic Salmonella-type
lipid A (516) displayed inhibitory activity on the degradation of
IB- in response to 506. An almost total inhibitory effect was
observed at an agonist to antagonist ratio of 1:100 (w/w).
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and inhibition of TNF- induction by
parent LPS in THP-1 line cells
Because the activity of LPS and lipid A from Salmonella
on human cells was completely different, the action of the
polysaccharide portion from S. abortus equi was examined in
regard to TNF- induction activity and the inhibitory activity on
TNF- induction of active 506. No TNF- induction by polysaccharide
alone was recognized, even at 10 µg/ml, and no LPS activity was
recovered when the polysaccharide portion and lipid A from S.
abortus equi were added to the cells together (data not shown).
Furthermore, the polysaccharide showed no inhibitory activity on the
TNF- induction activity of its parent LPS, even when a
103 times excess of polysaccharide to lipid A
(w/w) was added to the cells (10 ng/ml of LPS plus 10 µg/ml of
polysaccharide) (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and NF-B activation than
E. coli lipid A. According to the fatty acid analysis by
gas-liquid chromatography and mass spectrometry (data not shown), the
major lipid A structure of all of the lipid A preparations from the
Salmonella strains used in the present study was
heptaacylated diglucosamine, which is referred to as
Salmonella-type lipid A, because it is the typical structure
present in Salmonella, as reported previously
(30, 31, 32). In addition to the Salmonella-type
lipid A, they also contained a significant amount of E.
coli-type hexaacylated diglucosamine lipid A structure. Because natural lipid A usually consists of a mixture of various structures originated from the biosynthesis and extraction procedure, as was also the case in the present study, and its biological activity depends on the chemical structure, experiments using natural lipid A may give rise to misunderstandings regarding structure-activity relationships.
To confirm the phenomenon observed when natural Salmonella lipid A was used, and to obtain more definitive results, experiments have been performed with synthetic E. coli- and Salmonella-type lipid A (506 and 516, respectively). The results showed that Salmonella-type lipid A is completely inactive on human cells, but is a strong agonist in mice and active in inducting Limulus gelation activity as well. In contrast, E. coli-type lipid A exhibited full activity in human cells as well.
Both of these lipid As, the Salmonella-type heptaacylated and E. coli-type hexaacylated forms, had been successfully synthesized chemically in the past (12, 13, 14), and their biological properties had been carefully tested by many investigators (15, 16, 17, 18, 19, 20). The Salmonella-type lipid A had been demonstrated to be very active in mice and rabbits and subsequently had been thought to be a typical lipid A (18, 19, 20) along with that from E. coli. However, Salmonella-type lipid A (516) was not tested for activity on human systems, and nobody at that time expected that it would be discriminated by human and mouse cells.
Although natural Salmonella lipid A contains a significant
amount of the E. coli-type hexaacylated diglucosamine
structure, as recognized by mass spectrometry, its activity on human
cells was mach lower than expected. In the present study, biologically
inactive Salmonella lipid A was found to antagonize the
agonist (E. coli LPS and 506) action of inducing TNF- and
NF-B activation in THP-1 cells. This may explain the low activity of
natural lipid A from Salmonella. The apparent activity of
Salmonella lipid A must be the result of the overall
reaction of active structures, including E. coli-type lipid
A, and the antagonistic activity of the Salmonella-type
structure.
In contrast to the inactivity of Salmonella lipid A on human
cells, all of the parent LPS preparations stimulated human cells to the
same extent as E. coli LPS and induced both TNF- release
and NF-B activation.
The results obtained in the present study are very interesting in two senses. First, the lipid A from Salmonella LPS is specifically inactive on human cells, showing that recognition of the lipid A structure by human cells is different from that of mice or rabbits. The same species-specific phenomenon had been observed with tetraacylated lipid A precursor (lipid IVA), which is also active in mice and rabbits, but inactive on human cells (9, 33). However, the meaning of these two lipid As is completely different in view of the fact that the precursor structure is an intermediate in the biosynthesis of LPS, whose structure is not found as a component of any LPS. In contract, the Salmonella-type heptaacylated lipid A actually exists as the major component of Salmonella lipid A, along with the hexaacylated lipid A, e.g., E. coli-type lipid A.
The second point is that Salmonella lipid A is inactive, but
that the LPS exhibits full activity in terms of both the induction of
TNF- release and NF-B activation in human macrophage-like cells,
while both preparations are equally active in the other assay systems
employed in the present study, including Limulus gelation
activity and the activation of mouse cells, both peritoneal macrophages
and the J774-1 line, as shown by many previous studies performed both
by us and other investigators. These facts suggest that the
polysaccharide portion plays an important role in the manifestation of
activity in human cells. However, the polysaccharide portion alone did
not activate the cells at all, and no LPS activity was recovered when
lipid A and polysaccharide of the same LPS origin were added to the
cells simultaneously. Furthermore, the activity of LPS in human cells
was not inhibited even by adding a 100 times excess amount of the
polysaccharide portion.
The reason why Salmonella lipid A, but not LPS, fails to stimulate human cells, but not mouse cells, is unclear. Until now, the active center of the endotoxic activity of LPS has been thought to reside in the lipid A portion. There have been no reports claiming that the polysaccharide portion plays a role in the activation of macrophages as well as the activation of other endotoxic activities. Although the role of polysaccharide in human cell activation is still unknown, these results suggest that the activation mechanism of LPSs may differ. Recently, Shimazu et al. have reported that MD-2, a molecule that confers LPS responsiveness on Toll-like receptor 4 (TLR4), has broader specificity in the form of the TLR4-MD2 complex than recently described for TLR2 (34), and they suggested that the polysaccharide portion selectively affects recognition by TLR2 (35). All considered, further study concerning the role of the polysaccharide portion in LPS signaling in human cells is needed and is currently under way.
The findings in the present study also suggest that the activity of Salmonella LPS introduced into the body may be altered by circumstances or by host defense mechanisms: Salmonella LPS is easily detoxified because the ketocidic linkage that links lipid A, and the polysaccharide portion is quite susceptible to acidic conditions (36), whereas inactive Salmonella lipid A may change to the active form by being split off the acyloxyacylated fatty acids by the enzyme in macrophages (37, 38). These changes may make understanding the reaction of endotoxin in sepsis complex.
Based on the results of the present and previous studies, it seems that higher animals have higher specificity for the recognition of lipid A structures as observed in the case of lipid A precursor and chemically modified lipid A analogues (39). This seems to suggest that the active endotoxin is effectively detoxified in higher animals.
Experiments concerning the structure-activity relationships of lipid As have mainly been performed in animal systems thus far, and thus an important finding in the present study had been overlooked. Accordingly, the study should be performed again from the viewpoint of human endotoxicosis by using a human system and chemically pure materials.
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Acknowledgments |
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B- antisera (1309 and 751, respectively). |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Ken-ichi Tanamoto, Division of Microbiology, National Institute of Health, Sciences, 1-18-1 Kamiyoga, Setagayaku, Tokyo 158-8501, Japan. E-mail address:
Received for publication August 9, 1999. Accepted for publication January 3, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-inducing action of LPS in both murine macrophages and a human macrophage cell line. J. Immunol. 155:5391.[Abstract]
B proteins in human monocytic THP-1 cells. J. Biol. Chem. 268:11803.B- is necessary for activation of transcription factor NF-B. Nature 365:182.[Medline]
B- ) and enhanced processing of the NF-B precursor p105 are obligatory steps in the activation of NF-B. Nucleic Acids Res. 21:5059.This article has been cited by other articles:
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L. Steeghs, J. Tommassen, J. H.W. Leusen, J. G.J. van de Winkel, and P. van der Ley Teasing apart structural determinants of `toxicity' and `adjuvanticity': implications for meningococcal vaccine development Innate Immunity, April 1, 2004; 10(2): 113 - 119. [Abstract] [PDF]
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 M. Muroi, T. Ohnishi, S. Azumi-Mayuzumi, and K.-i. Tanamoto Lipopolysaccharide-Mimetic Activities of a Toll-Like Receptor 2-Stimulatory Substance(s) in Enterobacterial Lipopolysaccharide Preparations Infect. Immun., June 1, 2003; 71(6): 3221 - 3226. [Abstract] [Full Text] [PDF]
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M. Muroi and K.-i. Tanamoto The Polysaccharide Portion Plays an Indispensable Role in Salmonella Lipopolysaccharide-Induced Activation of NF-{kappa}B through Human Toll-Like Receptor 4 Infect. Immun., November 1, 2002; 70(11): 6043 - 6047. [Abstract] [Full Text] [PDF]
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M. Muroi, T. Ohnishi, and K.-i. Tanamoto Regions of the Mouse CD14 Molecule Required for Toll-like Receptor 2- and 4-mediated Activation of NF-kappa B J. Biol. Chem., October 25, 2002; 277(44): 42372 - 42379. [Abstract] [Full Text] [PDF]
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 P. M. Hwang, W.-Y. Choy, E. I. Lo, L. Chen, J. D. Forman-Kay, C. R. H. Raetz, G. G. Prive, R. E. Bishop, and L. E. Kay Solution structure and dynamics of the outer membrane enzyme PagP by NMR PNAS, October 15, 2002; 99(21): 13560 - 13565. [Abstract] [Full Text] [PDF]
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H. Janusch, L. Brecker, B. Lindner, C. Alexander, S. Gronow, H. Heine, A. J. Ulmer, E. Th. Rietschel, and U. Zahringer Structural and biological characterization of highly purified hepta-acyl lipid A present in the lipopolysaccharide of the Salmonella enterica sv. Minnesota Re deep rough mutant strain R595 Innate Immunity, October 1, 2002; 8(5): 343 - 356. [Abstract] [PDF]
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M. Muroi, T. Ohnishi, and K.-i. Tanamoto MD-2, a Novel Accessory Molecule, Is Involved in Species-Specific Actions of Salmonella Lipid A Infect. Immun., July 1, 2002; 70(7): 3546 - 3550. [Abstract] [Full Text] [PDF]
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 I. C. Almeida and R. T. Gazzinelli Proinflammatory activity of glycosylphosphatidylinositol anchors derived from Trypanosoma cruzi: structural and functional analyses J. Leukoc. Biol., October 1, 2001; 70(4): 467 - 477. [Abstract] [Full Text] [PDF]
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 K.-I. Tanamoto, H. Kato, Y. Haishima, and S. Azumi Biological Properties of Lipid A Isolated from Flavobacterium meningosepticum Clin. Vaccine Immunol., May 1, 2001; 8(3): 522 - 527. [Abstract] [Full Text] [PDF]
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 K.-i. Tanamoto, T. Iida, Y. Haishima, and S. Azumi Endotoxic properties of lipid A from Comamonas testosteroni Microbiology, May 1, 2001; 147(5): 1087 - 1094. [Abstract] [Full Text]
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C. R.H. Raetz Regulated covalent modifications of lipid A Innate Immunity, February 1, 2001; 7(1): 73 - 78. [Abstract] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, Salmonella abortus
equi LPS; 
, lipid A; •, 506; and 
, 516.
, 10 ng/ml; •, 100 ng/ml.