From the Department of Molecular and Cellular
Biology, Harvard University, Cambridge, Massachusetts 02138-2020 and
the ¶ Departamento de Bioquímica, Instituto de
Química, Universidade de São
Paulo 26077 05599-970, SP, Brazil
Received for publication, February 6, 2001, and in revised form, March 21, 2001
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ABSTRACT |
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Regulation of antioxidant enzymes is critical to
control the levels of reactive oxygen species in cell compartments
highly susceptible to oxidative stress. In this work, we studied the regulation of a chloroplastic iron superoxide dismutase (Fe-SOD) from
Lingulodinium polyedrum (formerly Gonyaulax
polyedra) under different physiological conditions. A
cDNA-encoding Fe-SOD was isolated from this dinoflagellate, showing
high sequence similarity to cyanobacterial, algal, and plant Fe-SODs.
Under standard growth conditions, on a 12:12-h light-dark cycle,
Lingulodinium polyedrum Fe-SOD exhibited a daily rhythm of
activity and cellular abundance with the maximum occurring during the
middle of the light phase. Northern analyses showed that this
rhythmicity is not related to changes in Fe-SOD mRNA levels,
indicative of translational regulation. By contrast, conditions of
metal-induced oxidative stress resulted in higher levels of Fe-SOD
transcripts, suggesting that transcriptional control is responsible for
increased protein and activity levels. Daily (circadian) and
metal-induced up-regulation of Fe-SOD expression in L. polyedrum are thus mediated by different regulatory pathways,
allowing biochemically distinct changes appropriate to oxidative challenges.
Reactive oxygen species
(ROS)1 such as superoxide
(O Organisms combat toxic effects of oxygen with antioxidants, which
include detoxifying enzymes and low molecular weight compounds. The
enzyme superoxide dismutase (SOD) represents a first step in such ROS
scavenging systems. SOD isoforms, including the copper/zinc-containing (CuZn-SOD), manganese-containing (Mn-SOD), and iron-containing (Fe-SOD)
metalloenzymes, catalyze the dismutation of O Irradiation by visible light in the presence of a photosensitizer leads
to the production of ROS, which in plants and algae is linked to
photosynthesis (4). Because of the elevated oxygen concentration and
intense electron flux within chloroplasts, electrons inevitably react
with oxygen, thereby generating O The presence of metal ions due to human activities is another important
cause of oxidative stress in living systems (8). Such metals can
promote oxidative damage both by directly increasing the cellular
concentration of ROS (2, 9) and by reducing the cellular antioxidant
capacity (10).
In contrast to higher plants, the antioxidant response to oxidative and
environmental stress has not been investigated in dinoflagellates at
the molecular level. These are a diverse group of unicellular
eukaryotes containing bioluminescent, photosynthetic, heterotrophic,
and symbiotic members having important ecological roles as primary
producers and consumers in aquatic environments. Dinoflagellates are
responsible for red tides, with those that are toxic having the
potential for producing serious health and economic problems. They have
unique genomic features, including large amounts of DNA (up to 200 pg/nucleus) packed in permanently condensed chromosomes (11) and an
absence of classical histones (12), which make their mechanisms of
genetic regulation of great interest.
In previous studies, we found that oxidative stress is an important
mediator of metal toxicity in unicellular algae and that SOD is an
essential component of the antioxidant defense system of
dinoflagellates (13, 14). Here we show that the chloroplastic Fe-SOD
isoform of Lingulodinium polyedrum is under the control of
two different regulatory mechanisms: increases in enzyme activity caused by treatment with metal ions is attributable to higher Fe-SOD
transcript levels, whereas a daily rhythm of Fe-SOD expression is
clock-controlled at the translational level.
Culture Conditions--
Cells of the dinoflagellate L. polyedrum (strain GP 70) were cultured at 20 ± 1 °C on a
12:12-h light-dark (LD) cycle with cool white fluorescent light at an
irradiance of 150 µE·m Cell Extracts and Chloroplast Purification--
Cells were
harvested by filtration, suspended in 0.1 M sodium
phosphate buffer pH 7.8, and lysed in a nitrogen pressure apparatus. Following centrifugation at 12,000 × g for 10 min at
4 °C, the supernatant was used as crude extract for enzyme activity
and protein assays.
Chloroplasts were purified from 1-liter cell cultures on a 90%
Percoll, 0.25 M sucrose density gradient as previously
described (17). The band containing chloroplasts (1.15 g·ml Enzyme Activity Assays--
SOD activity was determined by SOD
inhibition of superoxide-initiated ferricytochrome c
reduction (18). Superoxide was generated by the xanthine/xanthine
oxidase system, and assays were carried out using either crude extracts
or chloroplast extracts. Ferricytochrome c reduction was
followed at 550 nm for 1 min. Fe-SOD activity in chloroplast extracts
was measured in the presence of 5 mM KCN. One unit of SOD
is defined as the amount causing 50% inhibition of ferricytochrome
c reduction at 25 °C.
Extracts of L. polyedrum were also electrophoresed on 12%
nondenaturing polyacrylamide gels and stained directly for SOD
activity, which appears as light bands against the deep blue gel
background (19). The distinct SOD isoforms present in L. polyedrum extracts have been previously identified by their
different sensitivities to CN PCR Cloning and Sequence Analysis--
Total RNA was isolated in
denaturing buffer (4 M guanidinium thiocyanate, 25 mM sodium citrate, 0.5% SDS, and 10 mM
2-mercaptoethanol) and precipitated with 2 M lithium
chloride, as previously described (20). Poly(A)+ RNA was
purified from total RNA with the QuickPrep Micro mRNA purification
kit (Amersham Pharmacia Biotech), according to the manufacturer's instructions.
cDNAs encoding Fe-SOD were isolated using reverse
transcription-polymerase chain reaction (RT-PCR). First strand
reactions contained 2 mM MgCl2, 200 µM dNTPs, 10 pmol of oligo(dT)16 primer, 500 ng of L. polyedrum mRNA, and 2 units of Superscript II
reverse transcriptase (Life Technologies, Inc.). After RT for 1 h
at 42 °C, the remaining mRNA was removed enzymatically, and the
cDNA products were amplified using different combinations of
degenerate Fe-SOD primers (FSD1, 5' CAYTAYGGHAARCAYCAY 3'; FSD2r, 5'
GTRTGRTTCCAVACYTGBGC 3'; FSD2, 5' GCVCARGTBTGGAAYCAYAC 3'; and FSD3r,
5' GCRTGYTCCCAVACRTC 3'), with Taq DNA polymerase and the
following PCR cycle: 3-min denaturation at 94 °C, 30 cycles of
30 s at 94 °C, 35 s at 55 °C, and 40 s at
72 °C, followed by a 10-min extension at 72 °C. The Fe-SOD 3' and
5' untranslated region sequences were obtained by rapid amplification
of cDNA ends technique, using the same PCR conditions.
Amplified cDNA fragments were gel-purified, cloned into
pGEM-T vector (Promega), and sequenced by the ABI dye-terminator method (PerkinElmer Applied Biosystems). Sequence analyses were performed with
the SeqLab software2
(Wisconsin Package 10.0, GCG, Madison, WI).
Quantification of Fe-SOD Proteins and Transcripts--
Proteins
(20 µg), electrophoresed on 12% polyacrylamide-SDS, were transferred
to a nitrocellulose membrane for Western blotting analysis (21) using
polyclonal antibodies against bacterial Fe-SOD (Sigma). After
radioactive probing with 125I-Protein A, membranes were
autoradiographed for detection of bound antibody.
Total RNA (1-10 µg/lane) and poly(A)+ RNA (1 µg/lane)
were electrophoresed on a 1.2% agarose formaldehyde gel, blotted to a positively charged nylon membrane and probed with the cloned Fe-SOD cDNA. Northern hybridizations and washes were carried out under stringent conditions. Fe-SOD transcripts were also quantified by RT-PCR
using Fe-SOD primers FSD2/FSD3r and PCR conditions described above. As
a control for variations in RNA loading, expression of the L. polyedrum luciferin-binding protein (LBP) was also monitored on
both Northern blots and RT-PCR analysis. Densitometry analyses were
performed with the NIH Image v1.54.
Statistical Analysis--
Statistically significant differences
among treatments were determined by analysis of variance complemented
by the Dunnet's test. All conclusions are based on at least a 5%
level of significance (p < 0.05).
Fe-SOD in L. polyedrum--
The isoforms Fe-SOD, Mn-SOD, and
CuZn-SOD have been previously identified in crude extracts of L. polyedrum by inhibition assays with CN
Different sets of degenerate PCR primers, designed on the basis of
amino acid sequences of conserved Fe-SOD domains, were used to amplify
overlapping sequences of the coding region of this enzyme, using
mRNA as starting template. Blast searches of the
GenBankTM database indicated that the predicted amino acid
sequence of the resulting 690-bp ORF has high sequence identity to
cyanobacterial (Synechocystis, 59%;
Synechococcus, 55%; P. boryanum, 53%), green algal (C. reinhardtii, 57%), and higher plant (A. thaliana, 52%; N. plumbaginifolia, 52%) Fe-SODs (Fig.
2). The presence of conserved residues
known to be responsible for iron binding (Fig. 2, bolded) further confirms the identity of this nuclear-encoded L. polyedrum gene as the Fe-SOD isoform. In addition, its N-terminal
region contains a hydrophobic stretch followed by several hydroxylated and basic residues, typical of transit peptide sequences (Fig. 2,
underlined). When the cloned cDNA was used as probe in
Northern blots, a specific ~750-bp transcript was detected (Fig.
1C), which is in agreement with the average size of Fe-SOD
transcripts. However, no hybridization signal could be detected if 1 µg or less of total RNA was blotted, indicating that the basal level
of Fe-SOD transcripts is low compared with other L. polyedrum genes (LBP, luciferase, and glyceraldehyde-3-phosphate
dehydrogenase).
Daily Expression of Fe-SOD Is Rhythmic and Controlled at the
Translational Level--
Organisms may be subject to oxygen toxicity
under normal conditions because of physiological activity. Because
production of ROS is associated with exposure to light and
photosynthetic activity, the concentration of ROS within chloroplasts
is expected to be greater during the day than at night. To determine
whether there is a corresponding rhythmicity in Fe-SOD, its expression was monitored in L. polyedrum cells throughout a 24-h time
interval, under a 12:12-h LD cycle. The activity of Fe-SOD was
determined on the basis of selective tests, as described above, and
found to be 4-fold higher in chloroplasts of L. polyedrum
cells harvested during the light phase (LD 6) than during the dark
phase (LD 18; Fig. 3A). In
agreement with previous data (22), total SOD activity in crude extracts
also displayed a diurnal rhythm, with a maximum occurring in the middle
of the light phase (LD 6). Western blot analysis of crude extracts
confirmed that the cellular levels of Fe-SOD also varied during the
12:12-h LD cycle with an amplitude similar to that of the enzyme
activity rhythm (Fig. 3B).
However, these changes were not reflected in the levels of Fe-SOD
mRNA; the abundance of Fe-SOD transcripts remained relatively constant over a daily cycle in cells harvested at different times, as
shown by Northern blot analyses of mRNA extracted every 3 h (Fig. 4). LBP was included in the study
first because this is a protein whose synthesis and cellular
concentration is controlled by a circadian clock (23, 24), yet its
activity is not directly involved in the metabolism of ROS. Second, the
message level of LBP is established as being constant over the LD
cycle, and the regulation of its translation is circadian-controlled
(23), also shown for another rhythmic protein,
glyceraldehyde-3-phosphate dehydrogenase (25). The parallel leads us to
the postulate that Fe-SOD synthesis and its cellular abundance is
regulated, not by ROS, but by the circadian system.
Fe-SOD Transcript Levels Are Higher after Metal Stress--
Metal
ions are known to cause oxidative stress and damage in aerobic
organisms, including L. polyedrum (26). In previous studies,
exposures of either 0.04 µM Hg2+, 4.8 µM Cd2+, 18 µM
Pb2+, or 1.6 µM Cu2+ were
reported to result in increases in total SOD activity, maximal after
6 h (14). In the present work, the cellular levels of the Fe-SOD
isoform were found to be 3- to 4-fold higher after similar exposures to
such metal ions (Fig. 5A), and
significant increases in Fe-SOD activity, measured in chloroplast
extracts, were also found (Fig. 5B).
Unlike the diurnal regulation of Fe-SOD, these increases appear
to be mediated by increased transcript levels, as shown by Northern
analyses (Fig. 6A). When an
alternate and more sensitive method for detecting transcripts was used
(RT-PCR), similar results were obtained (Fig. 6B). As in the
experiments of Fig. 5, cultures were exposed to each of the four metal
ions individually, and in each case there was a substantial increase in
the Fe-SOD transcript level, but not in that of the LBP transcripts.
This provides direct evidence that the regulatory pathways for daily
and metal-induced up-regulation of Fe-SOD expression do not involve a
common intermediate.
SOD isoforms are able to incorporate different metal ions in
their active sites depending on metal availability in the medium (27).
Although some SOD activity may be retained in such cambialistic (metal-exchangeable) SODs, the binding of metal ions without
appropriate redox capacity may lead to enzymatically inactive isoforms
(2, 28). One could speculate that, in our experiments, treatment with
other metal ions displaces iron from the active site and affects enzyme
activity. This could explain the lower enhancement of Fe-SOD activity
compared with the increases in transcript and protein levels found in
cells subjected to metal stress.
Whereas SOD genes have been isolated from many different species,
the Fe-SOD isoform has been reported from only a few, and this is the
first gene of any SOD isoform to be isolated from a dinoflagellate,
from which fewer than a dozen genes altogether have been cloned. This
Fe-SOD is nuclear-encoded; its deduced amino acid sequence is 52-59%
identical to Fe-SOD isoforms from plants and cyanobacteria,
respectively, and residues responsible for iron binding are fully
conserved. Preliminary results indicate that, as for other L. polyedrum genes so far studied, there are no introns.
Although enhanced transcription of plant Fe-SODs has been reported as a
consequence of environmental adversities in several cases (29, 30),
this is the first demonstration of such a response after exposure to
different metal ions. As in other species, SOD in L. polyedrum surely provides a mechanism for inactivation of ROS, but
different mechanisms of regulation of Fe-SOD are operative. Exposure to metal ions results in an increased level of the Fe-SOD mRNA, whereas the daily variation in Fe-SOD protein and activity levels involves translational regulation. There is no evidence in
either case for the direct regulation of Fe-SOD by ROS.
With cells grown on a light-dark cycle, Fe-SOD exhibits a robust daily
rhythm in both protein level and enzyme activity, peaking in the light
phase. However, this is not accompanied by changes in the cellular
level of the mRNA, so it is concluded that translational regulation
is responsible for the Fe-SOD rhythm. Antioxidants, such as SOD,
glutathione S-transferase, and Translational regulation of SOD expression has been reported in rats,
where Mn-SOD abundance is regulated post-transcriptionally by the
binding of a redox-sensitive trans-acting protein to the 3'
untranslated region of its transcript, and binding enhances protein
translation (36, 37). This is similar to the circadian control of LBP
synthesis in L. polyedrum, but in the case of LBP, binding
represses translation (24).
In contrast to the daily rhythm, induction of Fe-SOD by metal ions in
L. polyedrum was accompanied by 3-4-fold increases in the
amount of its cellular mRNA and protein, with a somewhat smaller increase in Fe-SOD activity. Whether this is caused by increased transcription or slower degradation of mRNA, or both, cannot be specified. Transcriptional control of the expression of other genes in
dinoflagellates has been reported recently (38, 39), and in other
eukaryotes transcription of SOD genes is activated by redox-sensitive
transcription factors such as AP-1 and NF- Although little is known about the mechanisms of SOD regulation in
plants and algae, it seems likely that metal-induced expression of
Fe-SOD in L. polyedrum is under the control of protein that binds to a metal-responsive element, as in the case of the rat CuZn-SOD
isoform (46). This is supported by the observations that Fe-SOD
transcript levels in L. polyedrum are increased by exposure
to either a redox active metal ion such as the micronutrient Cu2+ or to nonessential metal ions without redox capacity
such as Hg2+, Cd2+, and Pb2+.
In summary, our findings show clearly that regulation of L. polyedrum Fe-SOD can take place at different steps of gene
expression. Translation of Fe-SOD varies over the course of a 24-h
light-dark cycle, whereas an increase in the Fe-SOD transcript pool
occurs after exposure to toxic metal ions, a novel finding. In both
cases, a prompt induction of Fe-SOD expression is critical for
controlling the steady-state levels of O
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2·s
1.
Typically, 1 liter of f/2 medium (15) was inoculated with 150 ml of a
dense culture (104 cells·ml
1) and used
after 4-5 weeks of growth. For the metal stress experiments, cells
were exposed for 6 h to either 0.04 µM
Hg2+, 4.8 µM Cd2+, 18 µM Pb2+, or 1.6 µM
Cu2+. Metal salts were added directly to the medium at the
beginning of the light period (LD 0). Metal ion concentrations and
exposure time used in the experimental model were chosen on the basis
of previous toxicity bioassays and are known to induce oxidative stress
in L. polyedrum (14, 16).
1 density, monitored by absorbance at 670 nm) was
suspended in 1 ml of ice-cold extraction buffer (150 mM
Tris, pH 8.0, 2 mM EDTA, 0.3 M sucrose, 20 mM 2-mercaptoethanol) and lysed by high nitrogen pressure
(150 atm, 5 min, at 4 °C). Excess debris was removed by
centrifugation at 12,000 × g for 1 min, at 4 °C,
and the supernatant was used immediately for the biochemical assays.
and
H2O2 (14). Thus, identification of the
chloroplast SOD was inferred by comparison of the electrophoretic
profile of SOD isoforms from chloroplast and crude extracts.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
H2O2. Fe-SOD is CN
-resistant and
H2O2-sensitive, Mn-SOD is CN
- and
H2O2-resistant, whereas CuZn-SOD is sensitive
to both substances. From the SOD banding pattern of crude extracts on
native gels (14), several protein bands corresponding to Fe-SOD
activity could be detected in extracts of isolated chloroplasts (Fig.
1A). Although the
chloroplastic SOD bands are not resolved in the native gels, resistance
to CN
and inactivation after H2O2
treatment confirmed their Fe-SOD nature (not shown). Other evidence for
the presence of the Fe-SOD isoform in chloroplast extracts was provided
by immunodetection with heterologous Fe-SOD antibodies, which
cross-reacted with an ~32-kDa chloroplastic protein (Fig.
1B).
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Fig. 1.
Identification of Fe-SOD in L. polyedrum. A, banding patterns of three SOD
isoforms in activity gels. Equal amounts of proteins (20 µg/lane)
from crude extracts (CE) and chloroplast (Chl)
extracts were electrophoresed on native polyacrylamide gels and stained
for SOD activity. Chloroplast Fe-SOD is broadly distributed in the
light area, confirmed by other tests (see text).
B, immunodetection of Fe-SOD in chloroplast extracts by
Western blot (20 µg/lane). C, Northern analysis of Fe-SOD
transcripts using different amounts of total RNA and L. polyedrum Fe-SOD cDNA as a probe.
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Fig. 2.
Alignment of the deduced amino acid sequence
of L. polyedrum Fe-SOD (Lp; AF
289824) with those of Synechocystis sp
(Sy; BAA 18027), Synechococcus sp
(Sc; CAB 57855), Plectonema boryanum
(Pb; AAA 69954.1), Chlamydomonas
reinhardtii (Cr; AAB 04944.1),
Nicotiana plumbaginifolia (Np; AAA
34074.1), and Arabidopsis thaliana
(At; AAA 33960.1). The N-terminal region
rich in hydrophobic residues is underlined. Conserved amino
acid domains used for primer design are indicated by arrows.
Identical amino acid residues are marked with asterisks, and
those related to iron binding are in boldface.
Dots within sequences indicate gaps introduced for best
alignment.
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Fig. 3.
Daily rhythm of Fe-SOD from cells grown under
12:12-h LD cycle. A, total SOD (line) and
Fe-SOD (vertical bars) activities measured in crude extracts
and chloroplast extracts, respectively. Each data point is
the mean ± S.E. of 3 experiments. B, Western blots.
Total proteins (20 µg/lane) were extracted every 3 h, subjected
to sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), and probed with Fe-SOD antibodies. Densitometry data are
plotted as arbitrary units (au) versus time.
White and black horizontal bars represent light
and dark phases, respectively.
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Fig. 4.
Daily levels of Fe-SOD transcripts are
constant. A, Northern analyses were performed with
poly(A)+RNA (1 µg/lane), extracted every 3 h from
cells grown under 12:12 h LD cycle, and gene-specific probes.
Expression of LBP was also monitored as a control for RNA loading.
B, quantification of Fe-SOD (solid circles) and
LBP (open circles) transcripts in the respective blots.
Densitometry data are plotted as arbitrary units (au)
versus time.
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Fig. 5.
Increase of Fe-SOD following exposure to
toxic metal ions. A, cellular levels of Fe-SOD in
extracts prepared from cells exposed to either 0.04 µM
Hg2+, 4.8 µM Cd2+, 18 µM Pb2+, or 1.6 µM
Cu2+ for 6 h. Western blots were performed with total
proteins (20 µg/lane) probed with Fe-SOD antibodies. Densitometry
analysis in arbitrary units (au) is given by bar
graphs. B, Fe-SOD activity in chloroplasts prepared
from cells after treatment with metal ions as above. Control cells were
grown in the absence of toxic metals. Each data point
represents the mean ± S.E. of 3 experiments. *, significantly
different from control group (p < 0.05).
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Fig. 6.
Increased Fe-SOD transcript levels after
treatment with toxic metal ions. Densitometry analysis in
arbitrary units (au) is given by bar graphs
(Fe-SOD, solid bars; LBP, open bars).
A, Northern analyses of Fe-SOD and LBP transcript abundance
in total RNA (10 µg/lane) extracted from cells exposed to either 0.04 µM Hg2+, 4.8 µM
Cd2+, 18 µM Pb2+ or 1.6 µM Cu2+ for 6 h. B,
measurement of Fe-SOD and LBP transcript levels by RT-PCR in cells
treated as above. Poly(A)+RNA (500 ng/reaction) was
reverse-transcribed in the presence of gene-specific primers. PCR
products were electrophoresed on 1.5% agarose gel and visualized by
ethidium bromide staining.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-carotene have been reported to be controlled by the circadian clock (22, 31, 32), as are
many L. polyedrum proteins (33, 34), several of which have
been shown to be regulated at the level of translation (23, 25, 35).
The results presented here show that even with cells exposed to a
light-dark cycle, and thus expected to generate a rhythm in the amount
of O
B (40, 41). Indeed, the
promoter regions of many eukaryotic SOD genes contain multiple
regulatory elements responsible for transcriptional control in response
to a variety of stimuli (42-44). In bacteria, the expression of SOD
occurring after shifts in the cellular oxidative balance is regulated
by the transcription factors Sox R and Sox S (45).
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ACKNOWLEDGEMENT |
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We thank Dr. T. Wilson for helpful comments.
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FOOTNOTES |
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* This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo, Companha de Aperfeiçoamento de Pessoal de Nível Superior, Stiftelson för Internationalisering av högre utbildning och forskning, and National Institute of Mental Health Grant 46660.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: 16 Divinity Ave., Cambridge, MA 02138-2020. Tel.: 617-495-3714; Fax: 617-496-8726; E-mail: hastings@fas.harvard.edu
Published, JBC Papers in Press, March 22, 2001, DOI 10.1074/jbc.M101169200
2 The full-length cDNA sequence of Fe-SOD from L. polyedrum is available in the GenBankTM sequence data base under GenBankTM Accession Number AF 289824.
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ABBREVIATIONS |
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The abbreviations used are: ROS, reactive oxygen species; LD, light-dark cycle; RT-PCR, reverse transcription/polymerase chain reaction; LBP, luciferin-binding protein; SOD, superoxide dismutase..
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