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Antimicrobial Agents and Chemotherapy, January 2000, p. 14-18, Vol. 44, No. 1
0066-4804/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Effects of Atovaquone and Diospyrin-Based Drugs on Ubiquinone
Biosynthesis in Pneumocystis carinii Organisms
Edna S.
Kaneshiro,1,*
Donggeun
Sul,1 and
Banasri
Hazra2
Department of Biological Sciences, University
of Cincinnati, Cincinnati, Ohio 45221,1 and
Department of Pharmacy, Jadavpur University, Calcutta 700-032, India2
Received 8 March 1999/Returned for modification 21 September
1999/Accepted 8 October 1999
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ABSTRACT |
The naphthoquinone atovaquone is effective against
Plasmodium and Pneumocystis carinii carinii. In
Plasmodium, the primary mechanism of drug action is an
irreversible binding to the mitochondrial cytochrome
bc1 complex as an analog of ubiquinone.
Blockage of the electron transport chain ultimately inhibits de novo
pyrimidine biosynthesis since dihydroorotate dehydrogenase, a key
enzyme in pyrimidine biosynthesis, is unable to transfer electrons
to ubiquinone. In the present study, the effect of atovaquone was examined on Pneumocystis carinii carinii coenzyme Q
biosynthesis (rather than electron transport and respiration) by
measuring its effect on the incorporation of radiolabeled
p-hydroxybenzoate into ubiquinone in vitro. A triphasic
dose-response was observed, with inhibition at 10 nM and then
stimulation up to 0.2 µM, followed by inhibition at 1 µM. Since
other naphthoquinone drugs may also act as analogs of ubiquinone,
diospyrin and two of its derivatives were also tested
for their effects on ubiquinone biosynthesis in P. carinii
carinii. In contrast to atovaquone, these drugs did not
inhibit the incorporation of p-hydroxybenzoate into
P. carinii carinii ubiquinone.
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INTRODUCTION |
Ubiquinone (coenzyme Q [CoQ])
(Fig. 1A) plays a pivotal role in
cellular respiration by participating in inner mitochondrial membrane
electron transport by accepting electrons from a number of
dehydrogenase enzymes and passing them to the cytochromes and eventually to molecular oxygen (6, 8, 30). Biosynthesis of
ubiquinone (1, 22, 26, 29, 33) and the reactions that occur
in different cellular compartments are probably best understood from
studies with subcellular fractions of rat liver cells (1, 22,
29). Ubiquinone is composed of a benzoquinone ring and a
lipophilic polyprenyl chain. In the rat liver, the precursor of the
benzoquinone moiety, p-hydroxybenzoic acid (PHBA), is formed
in the cytosol from the aromatic amino acids tyrosine or phenylalanine,
whereas PHBA is formed directly from chorismic acid in organisms that
have the shikimic acid pathway (i.e., plants, bacteria, and some
fungi). The polyprenyl chain of CoQ is formed by a branch pathway in
isoprenoid biosynthesis by the polymerization of five-carbon
isopentenyl units; the number of the isopentenyl units in the chain
distinguishes the CoQ homologs. The major CoQ homolog has been used as
a taxonomic criterion for the verification of the phylogenetic
relationships between organisms (32). There appears to be
two independent sites where ubiquinone is synthesized in eukaryotic
cells: one is the endoplasmic reticulum (ER)-Golgi system and the other
is the mitochondrion (13, 33). After the transfer of a
polyprenyl P-P chain to PHBA, followed by several additional reactions
(1, 22, 26, 29, 33), the completed CoQ homolog is produced.
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FIG. 1.
Structures of compounds relevant to the present study.
(A) Ubiquinone (CoQ); n, number of isopentenyl units. (B)
Atovaquone (556C80). (C) Diospyrin (R = H) and diospyrin
dimethylether (R = CH3). (D) Diospyrin dimethylether
hydroquinone (R = CH3).
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Purified Pneumocystis carinii carinii (P. carinii) isolated from methylprednisolone-immunosuppressed
rats was shown to contain CoQ10 as the major CoQ
homolog (smaller amounts of CoQ9 were also detected)
(10). Since CoQ10 was not detected in the lungs
of healthy, untreated or in the lungs of immunosuppressed, uninoculated rat controls, this suggested that the pathogen was at least capable of
synthesizing CoQ10. Recently, the incorporation in vitro of chorismate, PHBA, tyrosine, and mevalonate into P. carinii
CoQ was demonstrated (10, 24, 28); thus, this organism can
synthesize de novo both moieties of ubiquinone. The P. carinii pentafunctional gene for enzymes in the shikimic acid
pathway has been cloned and characterized (2), suggesting
that the pathway is functional in this organism. This gene is localized
in the nucleus; hence, it is likely that ubiquinone biosynthesis in
this organism occurs in the ER-Golgi system, although synthesis in the
mitochondria cannot be ruled out.
Several hydroxynaphthoquinone drugs that are effective against
protozoan infections (e.g., malaria, trypanosomiasis, and
leishmaniasis) also have activity against P. carinii.
Atovaquone (Fig. 1), first used as an antimalarial agent, was also
found to have therapeutic activity against P. carinii
pneumonia (PCP). This has been demonstrated both in animal models
(19) and in humans (7, 14, 20). The mechanism of
action of atovaquone against Plasmodium is believed to
result from the irreversible binding of the drug to a 11.5-kDa protein
of the mitochondrial cytochrome bc1 complex,
thus inhibiting electron transport (12, 14-16). Since
dihydroorotate dehydrogenase (DHOD), a key step in de novo pyrimidine
synthesis, is coupled to the electron transport chain at complex III
and because the parasite cannot salvage host pyrimidines, the mechanism
of cidal drug action is thought to be the blockage of pyrimidine
biosynthesis as a consequence of electron transport inhibition.
It was previously reported that the 50% inhibitory concentration
(IC50) for P. carinii O2 consumption
was 5 × 10
8 M atovaquone (14, 15). Thus,
the respiratory chain was also implicated as the site of action in
P. carinii. It was hypothesized, however, that unlike
Plasmodium, P. carinii could salvage host pyrimidines, and the depletion of ATP (resulting from inhibition of
respiration) was proposed as a mode by which P. carinii is killed by the drug (15). Moreover,
unlike Plasmodium DHOD activity, which is inhibited by 1 nM
atovaquone (12), the activity of this enzyme in P. carinii lysates was not inhibited by concentrations of 
10 µM
(21). Although atovaquone and other hydroxynaphthoquinone drugs are recognized as ubiquinone analogs, details on the mechanisms of their antimicrobial activities in different organisms remain unclear.
To test the hypothesis that oxidative phosphorylation is highly active
in the respiratory chain of P. carinii and that the consequence of the drug's efficacy against PCP is the disruption of
ATP synthesis, direct measurements of cellular ATP were performed. The
effect of atovaquone on the ATP content of P. carinii
organisms is described in a separate report (M. T. Cushion,
et al., submitted for publication). In the present study, the
effect of atovaquone on CoQ biosynthesis in P. carinii
was examined by the incorporation in vitro of radiolabeled PHBA into
CoQ. The results were compared with those obtained with another group
of naphthoquinoid drugs which appear to be promising as antiparasitic
agents (Fig. 1). Diospyrin, a natural product of Diospyros
montana stem bark, and two of its derivatives (17, 18)
exhibit activity in vitro against Plasmodium,
Leishmania, and Trypanosoma at micromolar concentrations (18, 34). In an attempt to better understand the mechanism of antiparasitic activities of different quinoid drugs,
these compounds were also examined for their effects on ubiquinone
biosynthesis in P. carinii.
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MATERIALS AND METHODS |
Organisms.
P. carinii was isolated from infected rat
lungs by using the corticosteroid-immunosuppressed rat model of Boylan
and Current (4). Viral antibody-negative female Lewis rats
(Harlan Sprague-Dawley, Indianapolis, Ind.) were immunosuppressed with
methylprednisolone acetate (Depo-Medrol; Upjohn Co., Kalamazoo, Mich.)
and were twice inoculated intratracheally with cryopreserved organisms
containing 106 to 107 mixed-life-cycle stages
by previously described methods (23). After 8 to 10 weeks of
immunosuppression, moribund rats were killed and their lungs were
perfused, excised, and cut into small pieces. The P. carinii
organisms were isolated by homogenization (Stomacher; Tekmar,
Cincinnati, Ohio) by the procedures described earlier; the mucolytic
agent glutathione was included to detach organisms from host cells and
other P. carinii organisms. Purification involved sieving and a series of centrifugation steps at different speeds, followed by membrane filtration. The purities of these preparations (>95 to 100%) were quantified by microscopic, biochemical, and immunochemical analyses (23). Routinely, these preparations contained 10 to 30% cystic forms and 80 to 95% viable organisms (23). Aliquots of the final organism preparations were used for total protein analysis (25).
Incorporation in vitro of PHBA into P. carinii
CoQ.
Organisms (108 to 109) were
centrifuged into a pellet at 925 × g for 10 min, and
then the pellet was resuspended in 10 ml of serum-free RPMI 1640 medium supplemented with 100 U of penicillin per ml, 100 µg of
streptomycin per ml, and the radiolabeled precursor (24,
28). The organisms were incubated with radiolabeled
[U-14C]PHBA for two days at 37°C in a 5%
CO2 atmosphere. The organisms were washed with 0.85% NaCl
and centrifuged into a packed pellet, and then the lipids were
extracted. p-Hydroxybenzoate (specific activity, 33 mCi/mmol) was obtained from Tracer Lab (Boston, Mass.) or from Sigma
(11.3 mCi/mmol); in each assay 5 or 10 µCi was added to the medium.
To test the effects of naphthoquinone drugs on
P. carinii
CoQ synthesis, atovaquone was dissolved in the primary solvent ethanol;
diospyrin and its analogs were dissolved in dimethyl sulfoxide
(DMSO). Two separate preparations of each diospyrin-based drug
were
tested in these CoQ biosynthesis studies. Various concentrations
of
each drug were added to radiolabeled PHBA before the organisms,
suspended in the RPMI 1640 medium, were introduced into the
reaction
mixture. The ethanol concentration in the final incubation
mixture
was <0.2%, and the DMSO concentration was 0.1%.
Extraction of lipids and determination of P. carinii
CoQ radioactivity.
The lipid extraction, purification, and
fractionation methods used for studies on the incorporation in vitro of
radiolabeled precursors into P. carinii ubiquinones were as
described previously (28). Briefly, total lipids were
extracted by a neutral solvent system as described by Bligh and Dyer
(3) and were purified by biphasic partitioning as described
by Folch et al. (11). The neutral lipid fraction was
obtained by adsorption column chromatography (Unisil; Clarkson Co.,
Williamsport, Pa.) by elution with chloroform (CHCl3)
and was then resolved by 1-dimensional thin-layer chromatography (TLC)
on 0.25-mm Silica Gel H glass-backed plates (Analtech, Inc., Newark,
Del.) prewashed with methanol. The TLC plates were developed with the
solvent system petroleum ether-diethyl ether-acetic acid (80:20:1;
vol/vol/vol) (9, 10). After visualization with I2 vapor, the ubiquinone band was scraped off the TLC plate
and its radioactivity was determined by liquid scintillation spectrometry.
Incorporation of PHBA into the
P. carinii total ubiquinone
TLC fraction was expressed as picomoles of PHBA per milligram of
protein from the original organism preparations. The effects of
the
drugs on CoQ biosynthesis were compared to those of vehicle
controls.
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RESULTS |
Effects of atovaquone on the incorporation in vitro of PHBA
into P. carinii ubiquinones.
Incorporation of
PHBA into P. carinii CoQ exhibited a triphasic response
to increasing atovaquone concentrations (Table
1; Fig. 2).
A dramatic inhibition of CoQ biosynthesis was detected at 10 nM, an
effect that was reproducibly observed. At this concentration, incorporation was less than 50% of that for the untreated controls. At
between 0.1 and 0.2 µM, incorporation occurred at levels comparable to or higher than those for the controls. The higher
IC50 in the triphasic dose-response curve was observed at
1.0 µM.
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TABLE 1.
Effects of naphthoquinone drugs on incorporation in
vitro of radiolabeled p-hydroxybenzoate into P. carinii ubiquinonea
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FIG. 2.
Effects of atovaquone on P. carinii CoQ
biosynthesis. Purified organisms were incubated with
[U-14C]PHBA for 2 days in the presence of different
concentrations of the drug. There was a reproducible decrease in PHBA
incorporation into CoQ at 10 nM, which then increased to control levels
or higher at increased atovaquone concentrations. A decrease to half of
the control value (IC50) was then observed with 1 µM
atovaquone. Values represent means ± standard errors of the means
for four separate experiments.
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Effects of diospyrin and its derivatives on the incorporation of
PHBA into P. carinii ubiquinones.
Diospyrin, diospyrin
dimethylether, and diospyrin dimethylether hydroquinone did not
inhibit the incorporation in vitro of PHBA into P. carinii
ubiquinones when incubation was done for 48 h at concentrations up
to 100 µM (Table 1; Fig. 3);
dose-dependent reductions in CoQ biosynthesis were not observed.
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FIG. 3.
Effects of diospyrin (circles), diospyrin dimethylether
(squares), and diospyrin dimethylether hydroquinone (triangles).
Purified organisms were incubated with [U-14C]PHBA for 2 days in the presence of different concentrations of a compound. Values
represent means ± standard errors of the means for three or six
separate experiments. Inhibition of ubiquinone biosynthesis was not
observed at concentrations of these naphthoquinone drugs up to 100 µM.
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DISCUSSION |
Effects of atovaquone on P. carinii metabolism and
respiration.
Atovaquone is effective in clearing organisms from
patients with PCP with low to moderate numbers of organisms
(7, 12, 20). It was previously reported that the drug
inhibited P. carinii respiration (measured
polarographically) at an IC50 of 50 nM (14, 15).
Since it is known that atovaquone's activity in Plasmodium is the consequence of binding to the mitochondrial cytochrome bc1 complex (13-15) and that it
apparently also binds to the P. carinii bc1
complex (14), it is not surprising that inhibition of
P. carinii respiration was observed.
The present study addresses an effect of atovaquone other than electron
transport and respiration. Atovaquone had a triphasic
dose-effect
on PHBA incorporation into
P. carinii ubiquinone.
Inhibition
was observed at 10 nM atovaquone (the lowest concentration
tested),
which was lower than the concentration reported to be
required for
detectable inhibition of respiration (
14), reduction
of
cellular ATP (Cushion et al., submitted), inhibition of DHOD
activity,
or inhibition of organism proliferation in primary cultures
(
21).
In a previous study, CoQ
7 and CoQ
8 were not
detected by high-pressure liquid chromatography (HPLC) and/or
gas-liquid chromatography-mass
spectroscopy (
10)
methods, indicating that these homologs do
not accumulate to
readily detectable levels in the organism. By
using more sensitive
metabolic radiolabeling techniques, it was
recently found that the HPLC
fractions eluting with authentic
CoQ
7, CoQ
8,
CoQ
9, and CoQ
10 were all radioactive (D. Sul et al.,
unpublished data). The high specific activity of the
shorter homologs
and the conversion of radiolabeled CoQ
8 to
CoQ
9 and CoQ
10 suggest
that the longer homologs
can be formed by elongation of the polyprenyl
chains of completed CoQ
molecules. The biosynthesis of CoQ homologs
by elongation of CoQ
polyprenyl chains would represent a novel
mechanism for
CoQ
10 biosynthesis.
Information on the regulatory mechanisms that control steps in CoQ
biosynthesis in any cell type is severely lacking in the
literature. On
the basis of the available data on
P. carinii and
what
is currently known about ubiquinone biosynthesis in general,
we
propose the following hypotheses or scheme as a working model
to
explain the observations on atovaquone's effect on
P. carinii (Fig.
4). (i) At the low (10 nM) concentration, as an analog of
CoQ, atovaquone may inhibit de novo
CoQ biosynthesis by activating
putative product feedback mechanisms
that reduce the incorporation
of PHBA into CoQ. In untreated cells, the
enzyme would be regulated
by the accumulation of free CoQ
(CoQ
10). This probably occurs
at a cellular site closely
associated with the mitochondrion or
the ER-Golgi, where PHBA (which
has also been transported into
the organelle from the cytosol)
condenses with heptaprenyl P-P
or octaprenyl P-P. Following several
reactions, including three
S-adenosylmethionine
(SAM)-dependent methyltransfer steps, the
intermediate is converted to
the completed CoQ
7 molecule (a homolog
radiolabeled with
CoQ precursors in
P. carinii). The completed
CoQ product is
translocated to the cytosolic side of the Golgi
(or ER) membrane, where
elongation of the polyprenyl chain occurs
by the sequential addition of
isopentenyl units, producing the
major homolog CoQ
10, which
accumulates in the organism (
10).
Alternatively, completed
polyprenyl chains could be formed prior
to condensation with PHBA. In
this scheme, it is proposed that
feedback control involving inhibition
by accumulation of CoQ
10 in a free pool decreases the
PHBA-hexaprenyltransferase activity
in the Golgi-ER system.
Translocation of CoQ
10 to the inner mitochondrial
membrane
may require binding to a carrier protein which can target
it to the
mitochondrion. In the mitochondrion, CoQ participates
in electron
transport and interacts with the membrane, forming
quinol and quinone
pools in the membrane (
6,
30). Thus, atovaquone
is effective
as a ubiquinone analog at triggering this feedback
control of
PHBA-polyprenyltransferase activity. (ii) At concentrations
between 20 nM and 0.2 µM, atovaquone competes for sites on a carrier
and/or binds to some (but not all) cytochrome
bc1 complexes in
the mitochondrial inner
membrane, resulting in a reduction in
the level of electron transport.
At these concentrations, atovaquone
may displace and prevent the
binding of ubiquinone from some cytochrome
bc1
complexes; i.e., the drug binds irreversibly to some of the
bc1 complexes present in the membrane. This
would result in detectable
inhibition of respiration
(decreased respiration in
P. carinii carinii was
detected with atovaquone at 50 nM [
14,
15]). The
inhibition of respiration may then trigger upregulation of the
biosyntheses of components of CoQ intracellular transport (e.g.,
carrier) and/or the electron transport chain (e.g., ubiquinone)
in response to the need to increase the cell's respiratory
capacity.
The upregulation of these biosynthetic activities might
override
the negative, end product feedback control(s) which
atovaquone,
at lower concentrations, could activate as a ubiquinone
analog.
Thus, with atovaquone at between 20 nM and 0.2 µM, CoQ
biosynthesis
(incorporation of PHBA into
P. carinii
CoQ) is increased to normal
or higher levels. However, at these drug
concentrations, ample
electron transport could be maintained by CoQ
molecules still
in place within the mitochondrial inner membrane; thus,
O
2 consumption
is only slightly affected, and the
inhibition of oxidative phosphorylation,
as measured by ATP levels in
the cell, is not detectable (ATP
pools may also be maintained by
synthesis in the glycolytic pathway).
(iii) At the higher drug
concentrations (>1 µM), we hypothesize
that sufficient amounts
of atovaquone become irreversibly bound
to most cytochrome
bc1 complexes in the mitochondrion, resulting
in
a detectable reduction in cellular ATP levels. The reduction
of
ATP would result in decreased cellular metabolism, including
those
involved in de novo CoQ biosynthesis (reduction of PHBA
incorporation
into
P. carinii CoQ). At these high atovaquone
concentrations,
the reduced level of synthesis of ATP probably also
inhibits
P. carinii folate biosynthesis. Inhibition of
p-aminobenzoate incorporation
into folates by atovaquone was
observed at the IC
50 of the drug
(1.4 µM) for
P. carinii (
5). Detectable growth inhibition of
P. carinii organisms would then become obvious at higher
drug
concentrations. It was shown that the proliferation of short-term
primary cultures of
P. carinii was inhibited with 3 µM but
not
with 0.3 µM atovaquone (
21).
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FIG. 4.
Proposed scheme for ubiquinone biosynthesis in P. carinii and the effects of atovaquone (Av). The precursors PHBA
and geranyl P-P are formed in the cytosol. Elongation of the polyprenyl
precursor by the addition of isopentenyl units may occur at the outer
surface of the ER, and then heptaprenyl P-P is translocated to the ER
and then to the Golgi apparatus lumen. At 10 nM atovaquone, the drug
acts as an analog of CoQ10 and inhibits the
PHBA-polyprenyltransferase activity ( ), reducing the
incorporation of PHBA into CoQ. In this model, it is proposed that
decreased respiration stimulates the upregulation of biosyntheses of
components involved in the intracellular translocation of CoQ (e.g.,
carrier) and/or electron transport (e.g., ubiquinone), which can
override (++) the negative, end product feedback control(s). With
atovaquone at concentrations of >1 µM, respiration is sufficiently
inhibited and the reduction in ATP production by oxidative
phosphorylation becomes measureable. The lack of ATP causes a decline
in overall cellular metabolism, resulting in a decrease () in the
rate of PHBA incorporation into CoQ. Ca, carrier.
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Experiments on the effects of atovaquone on the
PHBA-polyprenyltransferase activity in
P. carinii were not
directly tested
in the present study, but it is now feasible to perform
these
studies. Procedures by which efficient incorporation of
radiolabeled
precursors into
P. carinii molecules occurs
have been developed
with a cell-free system (
28). The
effects of the drug on PHBA
incorporation into
P. carinii
CoQ will be examined as part of
a separate study with this cell-free
system. These experiments
would represent among the few studies
conducted on the regulation
of PHBA-polyprenyltransferase in eukaryotic
cells.
The present study demonstrated that atovaquone has potent effects on
P. carinii ubiquinone biosynthesis, suggesting that there
may be several possible mechanisms of action of the drug on this
pathogen, and these mechanisms may also occur with other organisms,
such as
Toxoplasma and
Plasmodium.
Atovaquone-resistant strains
have been identified among these
organisms. Mutations in the cytochrome
b gene appear to
explain the development of some
P. carinii-resistant
strains
(
31). Since atovaquone was found to have a profound
effect on other processes besides electron transport, it is
possible
that atovaquone resistance may also involve changes in
components
that function in the biosynthesis or intracellular
translocation
of
ubiquinone.
Effects of diospyrin, diospyrin dimethylether, and diospyrin
dimethylether hydroquinone on P. carinii ubiquinone
biosynthesis.
It has been suggested that the mechanisms of
action of quinoid drugs with antiparasite activity, as well as quinoid
metabolites of some other drugs (e.g., primaquine), are by their
action as analogs of ubiquinone (8, 15). Since diospyrin and
its derivatives did not have an effect on P. carinii CoQ
biosynthetic rates, this strongly suggests that the mechanism of action
of atovaquone differed from those of the diospyrin-based quinoid drugs.
Additional studies on the effects of the diospyrin-based drugs on the
metabolic processes of the parasitic organisms shown to be sensitive to
these compounds (18, 33) would aid in understanding the
mechanism by which these drugs clear those infections. Recently,
evidence for the inhibition of type I DNA topoisomerase activity by
diospyrin was obtained in Leishmania donovani promastigotes
(27). This observation is consistent with our results
indicating that the drug reduces the cellular ATP content of P. carinii (Cushion et al., submitted), with no effect on the
incorporation of PHBA into CoQ.
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ACKNOWLEDGMENTS |
We thank W. Gutteridge for kindly providing us with atovaquone.
This study was supported by NIH/NIAID grants RO1 AI38758 (to E.S.K.)
and International Foundation for Science, Stockholm, Sweden, grant
F/1836 (to B.H.).
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biological Sciences, University of Cincinnati, Cincinnati, OH
45221-0006. Phone: (513) 556-9712. Fax: (513) 556-5280. E-mail:
Edna.Kaneshiro{at}uc.edu.
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Antimicrobial Agents and Chemotherapy, January 2000, p. 14-18, Vol. 44, No. 1
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Abstract
Introduction
