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Antimicrobial Agents and Chemotherapy, October 1999, p. 2350-2355, Vol. 43, No. 10
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Inhibition of Human Immunodeficiency Virus Type 1 Replication in
Acutely and Chronically Infected Cells by EM2487, a Novel Substance
Produced by a Streptomyces Species
Masanori
Baba,1,*
Mika
Okamoto,1 and
Hitoshi
Takeuchi2
Division of Human Retroviruses, Center for
Chronic Viral Diseases, Faculty of Medicine, Kagoshima University,
Kagoshima 890-8520,1 and Department of
Exploratory Drug Research, Eisai Company, Tsukuba, Ibaraki
300-2635,2 Japan
Received 11 January 1999/Returned for modification 16 April
1999/Accepted 15 July 1999
 |
ABSTRACT |
In a search for effective HIV-1 transcription inhibitors, we have
evaluated more than 75,000 compounds for their inhibitory effects on
Tat-induced human immunodeficiency virus type 1 (HIV-1) long terminal
repeat (LTR)-driven reporter gene expression and found that EM2487, a
novel small-molecule substance produced by a Streptomyces
species, is a potent and selective inhibitor of HIV-1 replication in
both acutely and chronically infected cells. Its 50% effective
concentration for acute HIV-1 infection was 0.27 µM in peripheral
blood mononuclear cells (PBMCs), while the 50% cytotoxic
concentration for mock-infected PBMCs was 13.3 µM. EM2487 proved
inhibitory to a variety of HIV-1 strains and HIV-2 in acutely infected
T-cell lines (MOLT-4 and MT-4). The compound could suppress tumor
necrosis factor alpha (TNF-
)-induced HIV-1 production in
latently infected cells (OM-10.1 and ACH-2) as well as
constitutive viral production in chronically infected cells (MOLT-4/IIIB and U937/IIIB) without showing any
cytotoxicity. EM2487 did not affect early events of the HIV-1
replication cycle, as determined by proviral DNA synthesis in
acutely infected MOLT-4 cells. In contrast, the compound selectively
prevented viral mRNA synthesis in OM-10.1 cells, suggesting that HIV-1
inhibition occurs at the transcriptional level. Furthermore, EM2487 did
not inhibit TNF--induced HIV-1 LTR-driven reporter gene expression
but did inhibit that induced by Tat, irrespective of the presence or
absence of the nuclear factor 
B binding sites in the LTR. These
results suggest that the mechanism of action is attributable in part to the inhibition of Tat function.
| |
INTRODUCTION |
The progress of combination
chemotherapy with human immunodeficiency virus type 1 (HIV-1)
reverse transcriptase (RT) and protease inhibitors has achieved
long-sustained suppression of viral replication in HIV-1-infected
individuals (8, 17). However, considering the high cost and
low patient compliance of long-term combination chemotherapy
(12), discovery of novel anti-HIV-1 agents with different
mechanisms of action is still highly desirable. In addition, recent
studies have revealed that replication-competent virus can be recovered
from resting CD4+ T cells even in patients with prolonged
suppression of plasma viremia (more than 100 weeks) by combination
chemotherapy (13, 32). Therefore, it is clear that the
current chemotherapy cannot be terminated unless such reservoir cells
have been eradicated or viral recovery from these cells can be
completely suppressed. In this regard, inhibitors that selectively
prevent HIV-1 gene expression have the potential of inhibiting the
recovery of latent virus from resting CD4+ T cells as well
as infected macrophages, which are also considered to be a
long-surviving chronically infected cell population in HIV-1-infected
patients (26). In our extensive search program for HIV-1
transcription inhibitors, we have evaluated more than 75,000 compounds
for their inhibitory effects on Tat-induced reporter gene expression in
cell cultures and found that EM2487 (Fig.
1), a novel small-molecule substance
produced by a Streptomyces species, is a potent and
selective inhibitor of HIV-1 replication in acutely and chronically
infected cells.
| |
MATERIALS AND METHODS |
Compounds.
Preparation and purification of EM2487
(Mr, 829) carried out in collaboration with
Mercian Corporation (Tokyo, Japan) will be described elsewhere
(27a). The purity of the final preparation was more than
99.9% (data not shown). The Tat antagonist Ro24-7429 (18)
was synthesized by Eisai Co. (Tsukuba, Japan). The HIV-1 transcription
inhibitor K-12 (3) and the protease inhibitor nelfinavir
were kindly provided by Daiichi Pharmaceutical Co. (Tokyo, Japan) and
Japan Tobacco Co. (Takatsuki, Japan), respectively. The HIV-1 RT
inhibitors lamivudine and MKC-442 (4) were supplied from
Mitsubishi Chemical Corporation (Yokohama, Japan). Dextran sulfate was
purchased from Sigma Chemical Co. (St. Louis, Mo.). Except for dextran
sulfate, all compounds were dissolved in dimethyl sulfoxide at 20 mM or
higher concentration to exclude any antiviral or cytotoxic effect of
dimethyl sulfoxide. Dextran sulfate was dissolved in distilled water.
Cells and viruses.
MOLT-4 cells (20), MT-4 cells
(21), peripheral blood mononuclear cells (PBMCs), OM-10.1
cells (7), ACH-2 cells (9), MOLT-4/IIIB cells, and U937/IIIB cells were
used in the antiviral assays. OM-10.1 and ACH-2 cells are clones of
HL-60 and CEM cells latently infected with HIV-1, respectively.
MOLT-4/IIIB and U937/IIIB cells are MOLT-4 and
U937 cells chronically infected with HIV-1 (IIIB strain),
respectively. PBMCs were obtained from healthy donors and stimulated
with phytohemagglutinin. Establishment of W-3 and KM-3 cells and their
reporter gene constructs will be reported elsewhere (27a).
These cells are clones of CEM cells that stably integrate a HIV-1 long
terminal repeat (LTR)-driven secreted alkaline phosphatase gene
(14). The integrated HIV-1 LTR contains two intact
NF-B-binding sites in W-3 cells, whereas both of the sites are
mutated in KM-3 cells. Three strains of HIV-1 (IIIB, HE,
and Ba-L) and one strain of HIV-2 (EHO) were used in the antiviral
assays. HE and Ba-L are a T-cell line-tropic clinical isolate
(24) and a macrophage-tropic strain, respectively.
Antiviral assays.
The activities of the compounds against
acute HIV-1 and HIV-2 infections were based on the inhibition of
virus-induced cytopathicity in MOLT-4 and MT-4 cells and p24 antigen
production in PBMCs, as previously described (1). MOLT-4 and
MT-4 cells (105/ml) were infected with the virus at a
multiplicity of infection of 0.1 and 0.02, respectively, and cultured
in the presence of various concentrations of the test compounds. After
a 4-day incubation at 37°C, the MOLT-4 cells were subcultured at a
ratio of 1:5 with fresh culture medium containing appropriate
concentrations of the test compounds and further cultured. The number
of viable cells was measured by the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
method on day 7 (MOLT-4 cells) and day 4 (MT-4 cells) after virus
infection (25). For the assays in PBMCs, the cells (105/ml) were infected with HIV-1 at a multiplicity of
infection of 0.1. After virus adsorption for 2 h, the cells were
extensively washed to remove unadsorbed virus particles and cultured in
the presence of various concentrations of the test compounds. After a
6-day incubation at 37°C, the culture supernatants were collected and
their p24 antigen levels were determined with a sandwich enzyme-linked immunosorbent assay kit (Cellular Products, Buffalo, N.Y.). The cytotoxicities of the test compounds were evaluated in parallel with
their antiviral activities. They were based on the viability of
mock-infected cells, as determined by the MTT method.
The activities of the compounds against chronic HIV-1 infection were
based on the inhibition of p24 antigen. OM-10.1 and ACH-2 cells
(105/ml) were incubated in the absence or presence of the
test compounds for 2 h, stimulated with 1 ng of tumor necrosis
factor alpha (TNF-) (Boehringer-Mannheim, Mannheim, Germany)/ml, and
further incubated. On the other hand, MOLT-4/IIIB and
U937/IIIB cells (105/ml) were cultured in the
absence or presence of the test compounds without any stimulation.
After a 3-day incubation at 37°C, the culture supernatants were
collected and examined for their p24 antigen levels. The cytotoxicities
of the test compounds for the chronically infected cells were also
determined by the MTT method.
PCR analysis.
The effects of the compounds on HIV-1 proviral
DNA synthesis were analyzed by PCR (23). MOLT-4 cells
(2.5 × 105) were infected with HIV-1 at a
multiplicity of infection of 1.0 and cultured in the absence or
presence of the test compounds. After a 24-h incubation at 37°C,
total DNA was extracted from the cells with a DNA extraction kit (Wako,
Osaka, Japan). The extracted DNA was subjected to PCR amplification
with the HIV-1 gag-specific primer pair SK38 and SK39
(23) and the control primer pair GH20 and PC04 for
-globin (5). The amplification was carried out for 35 cycles (94°C for 30 s, 55°C for 30 s, and 72°C for 1 min). The amplified products were electrophoresed in an agarose gel and
visualized by ethidium bromide staining.
Northern blot analysis.
OM-10.1 cells (106/ml)
were incubated in the absence or presence of EM2487 for 2 h,
stimulated with 10 ng of TNF-/ml, and further incubated. After a
24-h incubation at 37°C, total RNA was extracted from the cells with
an RNA extraction kit (RNAzol B; Tel-Test, Friendswood, Tex.). The
extracted RNA (20 µg) was electrophoresed and transferred to
Hybond-N+ membrane (Amersham, Little Chalfont,
Buckinghamshire, United Kingdom). The blot was hybridized with a
32P-labeled full-length HIV-1 molecular clone, HXB2
(27).
Reporter gene assays.
W-3 and KM-3 cells (3 × 106/ml) were either treated with 10 ng of TNF-/ml or
transfected with 10 µg of a plasmid expressing HIV-1 Tat, containing
the second exon, under the control of the simian virus 40 promoter
(modification of pSV2tat72) by electroporation (300 V; 1,000 µF). The cells were cultured in the presence of various
concentrations of the test compounds. After a 2-day incubation at
37°C, the culture supernatants were collected, incubated at 65°C
for 30 min to inactivate the alkaline phosphatase activity of fetal
calf serum, and examined for secreted alkaline phosphatase levels. At
the same time, the number of viable cells was determined by the MTT method.
| |
RESULTS |
Antiviral activity in acutely infected cells.
When we
evaluated EM2487 for its inhibitory effects on HIV-1 (IIIB
strain) replication in PBMCs, it completely inhibited p24 antigen
production in culture supernatants at a concentration of 4 µM (Fig.
2A). EM2487 did not reduce the
proliferation and viability of mock-infected PBMCs at this
concentration. Its 50% effective concentration (EC50) was
0.27 µM, while the 50% cytotoxic concentration (CC50)
was 13.3 µM (Table 1). Thus, the
selectivity index (SI), based on the ratio of its CC50 to
its EC50, was 49. EM2487 was also active against the
macrophage-tropic strain Ba-L and a macrophage-tropic clinical isolate
(KK strain) in PBMCs. The EC50s for Ba-L and KK were 2.1 and 0.072 µM, respectively (Table 1 and data not shown). EM2487
proved effective against HIV-1 replication in MOLT-4 and MT-4 cells
(Table 1). Like other HIV-1 transcription inhibitors, such as Ro24-7429
and K-12, EM2487 appeared to be a less potent inhibitor of HIV-1 in
MT-4 cells than in MOLT-4 cells. EM2487 also inhibited the replication
of HE (an HIV-1 clinical isolate) and HIV-2 (EHO strain) in MOLT-4 cells (Table 1). Furthermore, the compound was equally inhibitory to
AZT (zidovudine)-sensitive and AZT-resistant strains (data not shown).
EM2487 was active against simian immunodeficiency virus but inactive
against human T-cell lymphotropic virus type I (data not shown).
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FIG. 2.
Inhibitory effects of EM2487 on HIV-1 replication in
acutely infected PBMCs (A) and TNF--stimulated OM-10.1 cells (B). In
acute infection, phytohemagglutinin-stimulated PBMCs were infected with
HIV-1 (IIIB strain) and cultured in the presence of various
concentrations of the test compound. In chronic infection, OM-10.1
cells were incubated in the absence or presence of the test compounds
for 2 h, stimulated with TNF- (1 ng/ml), and further incubated.
After a 6-day incubation for PBMCs and a 3-day incubation for OM-10.1
cells, the p24 antigen levels of the culture supernatants ( ) were
measured by antigen capture ELISA. At the same time, the number of
viable cells ( ) was determined by the MTT method. The p24 antigen
levels of the control culture supernatants (in the absence of EM2487)
were 70 ± 15 and 174 ± 11 ng/ml in PBMCs and OM-10.1 cells,
respectively. The experiments were repeated three times, and
representative results are shown.
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Antiviral activity in chronically infected cells.
In the next
experiment, we examined whether EM2487 could inhibit HIV-1
replication in chronically infected cells. OM-10.1 cells produce little
or no HIV-1 under basal conditions but do produce a significant level
of virus after stimulation with TNF- or phorbol 12-myristate
13-acetate (7). In fact, the level of HIV-1 p24 antigen in
culture supernatants was 0.2 to 0.5 ng/ml in the absence of TNF-,
yet it increased more than 200-fold after stimulation with 1 ng of
TNF-/ml (data not shown). As shown in Fig. 2B, EM2487 suppressed p24
antigen production in TNF--stimulated OM-10.1 cells in a
dose-dependent manner. The compound completely prevented antigen
production at a concentration of 0.8 µM. However, it did not reduce
the viability and proliferation of OM-10.1 cells at concentrations up
to 4 µM. The EC50 and CC50 were 0.075 and 12.5 µM, respectively (Table 2),
indicating that EM2487 is a potent and selective inhibitor of HIV-1
replication in chronically infected cells. EM2487 was also inhibitory
to HIV-1 replication in TNF--stimulated ACH-2 cells as well as
MOLT-4/IIIB and U937/IIIB cells, both of which
constitutively produce a large amount of virus without stimulation
(data not shown). When the anti-HIV-1 activity of EM2487 was compared
with those of Ro24-7429, K-12, and lamivudine, K-12 was found to be a
slightly more potent inhibitor of HIV-1 than EM2487 in OM-10.1 and
MOLT-4/IIIB cells. Ro24-7429 was less active, and the RT
inhibitor lamivudine was totally inactive in these cell lines (Table
2).
Effect on HIV-1 proviral DNA synthesis.
To gain insight into
its mechanism of action, we examined whether EM2487 could inhibit HIV-1
proviral DNA synthesis in acutely infected cells. The PCR analysis
revealed that, like the protease inhibitor nelfinavir, EM2487 did not
affect the synthesis of HIV-1 proviral DNA even at a concentration of 5 µM, which was more than 50-fold higher than its EC50 in
MOLT-4 cells (Fig. 3A). In contrast, apparent suppression of HIV-1 proviral DNA synthesis was observed in
the presence of the adsorption inhibitor dextran sulfate (5 µM) and
the nonnucleoside RT inhibitor MKC-442 (1 µM). These results indicate
that EM2487 does not interfere with early events of the viral
replication cycle.
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FIG. 3.
(A) Effect of EM2487 on HIV-1 proviral DNA synthesis in
MOLT-4 cells. The cells were mock infected (lane 1) or infected with
HIV-1 (lanes 2 to 6) and cultured in the absence (lane 2) or presence
of either 5 µM EM2487 (lane 3), 5 µM dextran sulfate (lane 4), 1 µM MKC-442 (lane 5), or 1 µM nelfinavir (lane 6). After a 24-h
incubation, total DNA was extracted and subjected to PCR amplification
with the HIV-1 gag-specific primer pair SK38 and SK39 (a)
and the control primer pair GH20 and PC04 (b) for -globin. The
amplified products were electrophoresed and visualized by ethidium
bromide staining. (B) Inhibitory effect of EM2487 on HIV-1 mRNA
synthesis in OM-10.1 cells. The cells were incubated with the compound
for 2 h, stimulated (+) with TNF- (10 ng/ml), and further
incubated. After a 24-h incubation, total RNA was extracted from the
cells, blotted, and hybridized with a 32P-labeled
full-length HIV-1 molecular clone, HXB2.
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Inhibitory effect on HIV-1 transcription.
Since EM2487 was
selected through screening in a Tat-induced reporter gene expression
system, the compound was expected to be an HIV-1 transcription
inhibitor. Therefore, Northern blot analysis was conducted to determine
whether EM2487 could prevent HIV-1 mRNA synthesis in TNF--stimulated
OM-10.1 cells. As shown in Fig. 3B, EM2487 selectively suppressed
TNF--induced HIV-1 mRNA synthesis at concentrations nontoxic to the
host cells, indicating that EM2487 inhibits HIV-1 replication at the
transcriptional level. To elucidate whether EM2487 primarily inhibits
Tat or the cellular transcriptional factor NF-B, experiments
involving transfection of the Tat expression plasmid into W-3 and KM-3
cells were conducted. Transfection with the Tat expression plasmid or
treatment with 10 ng of TNF-/ml induced approximately 450- or
5.6-fold increase of alkaline phosphatase production in W-3 cells,
respectively (Fig. 4A). Under such
conditions, EM2487 could reduce the Tat-induced alkaline phosphatase
production in a dose-dependent fashion (Fig. 4B). Interestingly, EM2487
appeared to enhance the TNF--induced alkaline phosphatase production
in W-3 cells. On the other hand, KM-3 cells, in which the HIV-1 LTR
contained two mutated NF-B binding sites, did not respond to TNF-
stimulation but did strongly respond to Tat (Fig. 4C). EM2487 also
reduced the Tat-induced alkaline phosphatase production in KM-3 cells
(Fig. 4D). In both cell systems, the compound did not affect basal
alkaline phosphatase production (data not shown). Furthermore, the
inhibitory effect of EM2487 on Tat-induced gene expression was
confirmed by a cotransfection experiment with an HIV-1 LTR-driven
chloramphenicol acetyltransferase-expression plasmid and the
Tat-expression plasmid in HeLa cells (data not shown). These results
suggest EM2487 is inhibitory to HIV-1 Tat rather than NF-B.
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FIG. 4.
Characterization of W-3 and KM-3 cells and effects of
EM2487 on Tat- or TNF--induced transactivation in these cells. W-3
(A and B) and KM-3 (C and D) cells were either transfected with the Tat
expression plasmid (10 ng) or treated with TNF- (10 ng/ml). The
cells were cultured in the presence of various concentrations of the
compound. After a 2-day incubation, the culture supernatants were
collected and examined for their alkaline phosphatase levels. At the
same time, the number of viable cells was determined by the MTT method.
(A and C) Alkaline phosphatase (AP) activities of culture supernatants
in the absence of EM2487. (B and D) Effects of EM2487 on Tat-induced
() or TNF--induced ( ) transactivation and viable cell number
().
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| |
DISCUSSION |
Control of HIV-1 gene expression is an attractive approach to
chemotherapy of AIDS. Although the cellular transcription factor NF-B is a potent activator of HIV-1 gene expression (15,
22), the viral transactivator protein Tat seems to play a more
important role in sustaining a high level of viral replication in
acutely infected cells. Since several lines of evidence suggest that
repetitive acute infection accounts for most plasma viruses in
HIV-1-infected individuals (26), a selective Tat inhibitor
may have great potential as a candidate for the treatment of HIV-1
infection. In addition, TNF--triggered activation of NF-B leads
to rapid production of Tat, which is necessary for continuous HIV-1
gene expression in latently infected cells, such as OM-10.1 and ACH-2.
Therefore, an effective Tat inhibitor suppresses TNF--induced HIV-1
gene expression in these cells, assuming that it is also able to
suppress the recovery of latent virus from resting CD4+ T
cells as well as infected macrophages in vivo.
The Tat inhibitors first described in the literature are Ro5-3335
[7-chloro-5-(2-pyrryl)-3H-1,4-benzodiazepin-2(H)-one]
and its congener Ro24-7429 (18, 19). These compounds were
shown to be active against HIV-1 in both acute and chronic infections. In fact, we also confirmed that Ro24-7429 was a selective inhibitor of
HIV-1 replication in acutely infected MOLT-4 cells and chronically infected cells (OM-10.1 and MOLT-4/IIIB) (Tables 1 and 2).
However, as previously reported, Ro24-7429 did not display selective
inhibition of HIV-1 replication in MT-4 cells (31) (Table
1). Ro24-7429 is assumed to target a host cellular factor that binds to
the transactivating response element (TAR) (6). Clinical
trials of Ro24-7429 were halted due to lack of efficacy and some side effects in patients (11). More recently, it was reported
that CGP64222, a hybrid peptoid-peptide oligomer of nine residues, inhibited HIV-1 replication in peripheral blood lymphocytes by blocking
the formation of the Tat-TAR RNA complex (16). GCP64222 was
discovered from a pool of 3.2 × 106 individual
chemical entities, suggesting the extreme difficulty in discovering
this class of compounds by random screening.
In this study, we have identified EM2487, a novel substance produced by
a Streptomyces species and a potent and selective inhibitor
of HIV-1 replication in acutely and chronically infected cell cultures.
Among 75,000 compounds examined for their inhibitory effects on
Tat-induced HIV-1-driven reporter gene expression, less than 10 compounds were found to be active (data not shown). The active
compounds were further evaluated for their inhibitory effects on HIV-1
replication in acutely infected MOLT-4 cells. EM2487 was the only
compound that displayed selective inhibition of HIV-1 replication. The
chemical structure of EM2487 is unique (Fig. 1), which hampers its
modification and structure-activity relationship studies. EM2487
totally differs in chemical structure from CGP64222 or the
fluoroquinoline derivative K-12. The latter has recently been reported
as a potent and selective inhibitor of HIV-1 transcription
(3).
K-12, a representative of the fluoroquinoline derivatives, inhibited
HIV-1 replication in both acutely and chronically infected cells, and
its anti-HIV-1 activity appeared to be similar to that of EM2487
(Tables 1 and 2). Unlike Ro24-7429, both EM2487 and K-12 displayed
selective inhibition of HIV-1 replication in acutely infected MT-4
cells, although their SIs were smaller than those in MOLT-4 cells
(Table 1). Since K-12 is able to suppress Tat-induced transactivation,
it is possible that EM2487 and K-12 share the same target molecule for
Tat inhibition. However, as shown by recent studies of its mechanism of
action, K-12 inhibits the Tat function in a TAR-independent fashion
(unpublished data). In addition, K-12 was also inhibitory to murine
retroviruses, which are devoid of accessory genes such as
tat and rev, and some herpes viruses (30). K-12 reduced the production of TNF- and
interleukin-6 in mitogen-stimulated PBMCs (2). Thus, K-12
does not seem to directly block Tat itself or the Tat-TAR interaction
but may prevent the interaction of some cellular factors with Tat.
Although we have not completed extensive studies of the EM2487
mechanism of action, a recent preliminary study revealed that neither
EM2487 nor K-12 interfered with Tat-induced transactivation in a
cell-free assay system (data not shown), suggesting that EM2487 does
not interrupt the Tat-TAR interaction. In conclusion, EM2487 appears to
be a selective inhibitor of Tat, yet we cannot exclude the possibility
that EM2487 also interacts with known and unknown cellular
transcriptional factors involved in the Tat-induced transactivation (10, 28, 29, 33-35).
| |
ACKNOWLEDGMENTS |
OM-10.1 cells and pSV2tat72 were obtained through the
AIDS Research and Reference Reagent Program, National Institute of
Allergy and Infectious Diseases, Bethesda, Md. (contributors were S. Butera [OM-10.1 cells] and A. Frankel [pSV2tat72]).
This work was supported in part by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Science, Sports, and Culture.
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FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Human Retroviruses, Center for Chronic Viral Diseases, Faculty of
Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan. Phone: (81) 99-275-5930. Fax: (81) 99-275-5932. E-mail: baba{at}med3.kufm.kagoshima-u.ac.jp.
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Antimicrobial Agents and Chemotherapy, October 1999, p. 2350-2355, Vol. 43, No. 10
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
