Tài liệu hạn chế xem trước, để xem đầy đủ mời bạn chọn Tải xuống
1
/ 11 trang
THÔNG TIN TÀI LIỆU
Thông tin cơ bản
Định dạng
Số trang
11
Dung lượng
434,72 KB
Nội dung
Globinsandhypoxiaadaptationinthe goldfish,
Carassius auratus
Anja Roesner
1
, Stephanie A. Mitz
1,2
, Thomas Hankeln
3
and Thorsten Burmester
2
1 Institute of Zoology, University of Mainz, Germany
2 Institute of Zoology, University of Hamburg, Germany
3 Institute of Molecular Genetics, University of Mainz, Germany
Many freshwater environments are characterized by
spatial, temporal or seasonal fluctuations in oxygen
availability. Therefore, various fish species have
evolved physiological, anatomical and behavioral
mechanisms for coping with extended periods of
hypoxia [1–8]. These strategies include O
2
saving by
reduction of the metabolic rate, improved O
2
uptake
by enhanced ventilation, aquatic surface respiration,
expansion of the gill surface andthe increased O
2
affinity of hemoglobin [9,10]. Cyprinid fishes of the
genus Carassius (the crucian carp Carassius carassius
and its domestic Asian form, the goldfish C. auratus)
routinely experience hypoxiaand even anoxic phases
in their environment of isolated ponds. Carassius dis-
plays remarkable tolerance against O
2
deprivation.
This tolerance is conveyed by high glycogen stores in
brain and liver, increased buffering capacities, meta-
bolic rate depression andthe ability to convert the
lactate produced by anaerobic glycolysis into ethanol,
which is excreted via the gills [2,3,8,11–14].
In recent years, hypoxia has received much attention
in biomedical research [8,15] and, because of global
Keywords
cytoglobin; goldfish; hemoglobin; myoglobin;
neuroglobin
Correspondence
T. Burmester, Institute of Zoology,
University of Hamburg, Biozentrum Grindel,
Martin-Luther-King-Platz 3, D-20146
Hamburg, Germany
Fax: +49 40 42838 3937
Tel: +49 40 42838 3913
E-mail: thorsten.burmester@uni-hamburg.de
Database
The nucleotide sequences have been sub-
mitted to the GenBank database ⁄ EMBL
Data Bank under accession numbers
AM933143 (Hba), AM933144 (Hbb),
AM747267 (Mb1), AM747268 (Mb2),
AM933145 (Ngb) and AM933146 (Cygb1)
(Received 6 March 2008, revised 6 May
2008, accepted 15 May 2008)
doi:10.1111/j.1742-4658.2008.06508.x
Goldfish (Carassius auratus) may survive in aquatic environments with low
oxygen partial pressures. We investigated the contribution of respiratory
proteins to hypoxia tolerance in C. auratus. We determined the complete
coding sequence of hemoglobin a and b and myoglobin, as well as partial
cDNAs from neuroglobin and cytoglobin. Like the common carp (Cypri-
nus carpio), C. auratus possesses two paralogous myoglobin genes that
duplicated within the cyprinid lineage. Myoglobin is also expressed in non-
muscle tissues. By means of quantitative real-time RT-PCR, we determined
the changes in mRNA levels of hemoglobin, myoglobin, neuroglobin and
cytoglobin in goldfish exposed to prolonged hypoxia (48 h at
Po
2
6.7 kPa, 8 h at Po
2
1.7 kPa, 16 h at Po
2
6.7 kPa) at 20 °C. We
observed small variations inthe mRNA levels of hemoglobin, neuroglobin
and cytoglobin, as well as putative hypoxia-responsive genes like lactate
dehydrogenase or superoxide dismutase. Hypoxia significantly enhanced
only the expression of myoglobin. However, we observed about fivefold
higher neuroglobin protein levels in goldfish brain compared with zebrafish,
although there was no significant difference in intrinsic myoglobin levels.
These observations suggest that both myoglobin and neuroglobin may con-
tribute to the tolerance of goldfish to low oxygen levels, but may reflect
divergent adaptive strategies of hypoxia preadaptation (neuroglobin) and
hypoxia response (myoglobin).
Abbreviations
ARP, acidic ribosomal phosphoprotein P0; Cygb, cytoglobin; GbX, globin X; Hb, hemoglobin; LDH-A, lactate dehydrogenase A; Mb,
myoglobin; Ngb, neuroglobin; SOD, superoxide dismutase.
FEBS Journal 275 (2008) 3633–3643 ª 2008 The Authors Journal compilation ª 2008 FEBS 3633
warming, has become an important environmental
concern [16,17]. Fish have become a prime model to
investigate hypoxia tolerance strategies at the organism
level. Alternative metabolic pathways, as well as other
physiological responses are associated with major
changes in gene expression patterns. For example,
genes encoding enzymes of the glycolytic pathway and
fermentation are expressed more strongly after long-
term hypoxiain some fish species [9,18,19]. By con-
trast, genes required for oxidative energy production in
the tricarboxylic acid cycle or the respiratory chain, or
for the highly energy-consuming translation process
were found to be repressed. Hypoxia may even cause
developmental arrest, which is reflected by the repres-
sion of growth or cell cycle-associated genes [19,20].
Hypoxia also affects the O
2
-binding respiratory
proteins, which are represented in vertebrates by the
globin superfamily [21–23]. Globins are small globular
proteins that bind to O
2
by virtue of a heme-bound
Fe
2+
ion. To date, five types of globins have been
identified in fish. Hemoglobin (Hb) is included in the
red blood cells and serves for the transport of O
2
within the circulatory system. Hb is a heterotetramer
that consists of two a- and two b-chains. Monomeric
myoglobin (Mb) supplies O
2
within the striated muscle
and heart of most vertebrates [24]. Whereas Mb is a
single copy gene in most species, the common carp
(Cyprinus carpio) possesses two paralogous Mb genes
(Mb1 and Mb2) [25]. Surprisingly, Mb1 is ubiquitously
expressed in various tissues, whereas Mb2 is restricted
to the brain. Neuroglobin (Ngb) is located inthe cen-
tral and peripheral nervous systems [26], the retina [27]
and some endocrine tissues [28]. The exact role of Ngb
remains uncertain; it may supply O
2
to metabolically
active neurons, although other functions such as the
detoxification of noxious reactive oxygen or nitrogen
species are conceivable [29–31]. Cytoglobin (Cygb) is
located inthe fibroblast cell lineage as well as in dis-
tinct populations of neurons [32,33]. The function of
Cygb may be related to reactive oxygen species detoxi-
fication or the supply of O
2
to particular enzymatic
reactions [30,33]. Fish possess two paralogous Cygb
genes, which show divergent expression in neurons and
non-neuronal tissues [34]. The most recently identified
vertebrate globin, which has been referred to as glo-
bin X (GbX), is restricted to fish and amphibians
[35,36]. The physiological role of GbX, which is
expressed at low levels in a broad range of tissues, is
currently unknown.
Because hypoxia reduces the availability of O
2
to
mitochondria and respiratory proteins, it can be
assumed that low O
2
levels change the expression of
globins. Previously, we investigated the effect of
hypoxia on globin mRNA and protein levels in the
zebrafish Danio rerio [23]. Zebrafish exhibit moderate
tolerance to hypoxia, surviving extended periods at
Po
2
4 kPa. It might be expected that increased
expression of respiratory proteins should be advanta-
geous at low O
2
partial pressures, however, we found
different globin responses. Whereas Hb mRNA levels
decreased under hypoxia, Mb and Ngb protein and
mRNA levels increased significantly. The data suggest
that these globins are involved in conveying hypoxia
tolerance to zebrafish. Here we investigate the response
of globin levels inthe extremely hypoxia-tolerant
goldfish.
Results
Cloning and analyses of goldfish globins
Partial and complete cDNA sequences of goldfish
Hba,Hbb, Mb1, Mb2, Ngb and Cygb1 were obtained
by RT-PCR from RNA extracted from various tissues
(Fig. 1 ). The complete coding sequences, including the
5¢- and 3¢-ends, of Hba,Hbb and Mb1 were then
obtained from a mixed tissue cDNA library (supple-
mentary Figs S1–S3). The cDNA of Mb2 was obtained
by RT-PCR (supplementary Fig. S4). The 3¢-end of
the coding sequence of Ngb was missing, and we
obtained only the middle part of Cygb1 (supplemen-
tary Figs S5 and S6). Cygb2 could not be obtained by
RT-PCR. Because our study was mainly aimed at
investigating the regulation of globin expression, which
requires only fragments of globin cDNA, we ignored
the missing parts of the coding sequences. The coding
sequence of GbX had been obtained in a previous
study [36].
We first compared goldfish globin sequences with
their zebrafish orthologs (Fig. 1). Both Hb cDNAs
represent adult chains, whereas embryonic Hbs were
not considered in this study. The Hba chain we
obtained by screening the cDNA library is 97% identi-
cal at the nucleotide level to the goldfish Hba cDNA
sequence available inthe databases (accession number
AF528157), suggesting allelic variation or the presence
of multiple isoforms inthe C. auratus genome. Gold-
fish and zebrafish Hba proteins are 87.4% identical
(99.3% similar; considering isofunctional replace-
ments). The Hbb chains of these two fish species are
92.6% identical ⁄ 98.0% similar; within the overlapping
regions, the Cygb1 proteins of the two species are
79.7% identical ⁄ 95.8% similar and Ngb is 92.4% iden-
tical ⁄ 98.1% similar. GbX proteins are highly con-
served, with scores of 98.0% identity and 99.0%
similarity.
Goldfish globins under hypoxia A. Roesner et al.
3634 FEBS Journal 275 (2008) 3633–3643 ª 2008 The Authors Journal compilation ª 2008 FEBS
Recently, Fraser et al. [25] found two distinct Mb
sequences inthe common carp (Cy. carpio). Although
Mb1 could be readily obtained by screening the cDNA
library, Mb2 was identified by RT-PCR using oligonu-
cleotide primers designed according to the C. carpio
Mb2 sequence. Goldfish and carp Mb1 proteins are
93.9% identical and 98.0% similar; the Mb2 proteins
are 88.4% identical and 96.6% similar (Fig. 2 ). The
paralogs are 78% identical and 90% similar.
When compared with zebrafish Mb, the scores for
goldfish Mb1 are 81.6% identical ⁄ 91.8% similar, and
for Mb2 78.2% identical ⁄ 88.4% similar. Using zebra-
fish anti-Mb serum, we examined the presence of Mb
protein in goldfish organs (Fig. 3 ). We detected Mb
protein in all investigated tissues (brain, gills, heart,
liver, kidney and swimbladder). As expected, the Mb
signal was strongest in heart (note the different
amounts of total proteins applied per lane), but we
also observed apparently high Mb concentration in the
goldfish gills.
Changes in gene expression in hypoxic goldfish
In previous studies with zebrafish, we employed acidic
ribosomal phosphoprotein P0 (ARP) as the nonregu-
lated reference gene [23]. Fragments of ARP were
amplified from goldfish RNA using primers that had
been designed according to known zebrafish sequences.
Fig. 1. Comparison of zebrafish (Dre) and goldfish (Cau) globins. The amino acid sequences from Hba (HbA), Hbb (HbB), Mb, Ngb, Cygb
and GbX were aligned. The secondary structure of human neuroglobin is superimposed inthe upper row, with alpha-helices designated
A–H, the globin consensus numbering is given below the sequences. Strictly conserved amino acids are shaded in gray. Invariable (B12.2
and G7.0) and variable (E10.2 and H10.0) intron positions in vertebrate globin genes are indicated by arrows inthe upper row.
Fig. 2. Comparison of zebrafish (Dre) myoglobin andthe paralogous carp (Cca) and goldfish (Cau) Mb1 and Mb2. The predicted secondary
structure of zebrafish Mb is superimposed inthe upper row, with alpha-helices designated A–H, the globin consensus numbering is given
below the sequences. Strictly conserved amino acids are shaded in gray, functionally important residues (PheCD1, HisE7 and HisF8) are
white on a black background.
A. Roesner et al. Goldfish globins under hypoxia
FEBS Journal 275 (2008) 3633–3643 ª 2008 The Authors Journal compilation ª 2008 FEBS 3635
ARP mRNA levels remained constant in most tissues;
however, in brain and eye we found significant upregu-
lation of ARP mRNA by a factor of 1.7
(P < 0.01). Therefore, all expression levels were subse-
quently normalized according to total RNA content.
However, none of the conclusions presented here was
affected if expression levels were normalized according
to ARP (data not shown).
We applied mixed chronic and acute hypoxia
regimes that lasted 3 days. First, mild chronic hypoxia
was induced by a reduction in Po
2
to 6.7 kPa for
48 h. Acute hypoxia was achieved at Po
2
1.7 kPa
for 8 h, followed by Po
2
6.7 kPa for an additional
16 h before RNA extraction. Reduction of Po
2
to
close anoxia (< 0.5 kPa) led to the death of all experi-
mental animals within 16 h. Normoxic controls were
kept at Po
2
18.4 kPa. The expression levels of lac-
tate dehydrogenase A (LDH-A), Hba,Hbb, Cygb1
and Mb1 were first analyzed in goldfish body (car-
casses without heart, brain and eyes) (Fig. 4A ). Thus
the majority of tissue represents skeletal muscle, but
also includes blood vessels. We observed a mild
( 25%) downregulation of Hba,Hbb and Cygb1
mRNA under hypoxia, which was, however, not signif-
icant. LDH-A levels were unchanged, whereas Mb1
mRNA was found to be heavily upregulated ( 18-
fold; P < 0.05). In heart, Mb1 mRNA levels were
essentially unaffected (Fig. 4B). In brain, we observed
a twofold increase in Mb2 mRNA (P < 0.01),
although Mb1, Ngb, LDH-A and superoxide dismu-
tase (SOD)-1 mRNA levels remained essentially con-
stant (Fig. 4C). No significant changes in mRNA
levels of the investigated genes were observed in total
eye (Fig. 4D).
Quantitative western blotting
To compare the protein levels of Ngb and Mb from
goldfish and zebrafish, we performed quantitative wes-
tern blotting. We used specific antibodies that had
Brain
Gill
Heart
Liver
Kidney
Swimbladder
18 kDa
Fig. 3. Myoglobin expression in goldfish tissues. Protein extracts
from selected goldfish tissues were analyzed by western blotting
employing a zebrafish anti-Mb serum. Protein extracts (100 lg)
were applied on each lane for brain, gills, liver and kidney; 50 lg
was loaded for heart and swimbladder plus associated tissues. The
position of the 18 kDa molecular mass marker is indicated on the
right side.
A
B
C
D
Fig. 4. Expression of goldfish globins at different oxygen levels.
mRNA quantities were determined by quantitative real-time RT-
PCR. The white columns represent mRNA levels from goldfish kept
at normoxia (P
O
2
18.4 kPa), gray columns are mRNA levels from
goldfish kept at hypoxia (48 h P
O
2
6.7 kPa, 8 h PO
2
1.7 kPa,
16 h at P
O
2
6.7 kPa). RNA was extracted from carcasses (A),
heart (B), brain (C) or eye (D). Bars represent SD. The significance
of the data was estimated with a Student’s t-test, with n =4.
**P < 0.01. Gene abbreviations: Cygb, cytoglobin; HbA, hemo-
globin a; HbB, hemoglobin b; LDH-A, lactate dehydrogenase A;
Mb1, myoglobin 1; Mb2, myoglobin 2; Ngb, neuroglobin; SOD-1,
superoxide dismutase-1.
Goldfish globins under hypoxia A. Roesner et al.
3636 FEBS Journal 275 (2008) 3633–3643 ª 2008 The Authors Journal compilation ª 2008 FEBS
been raised against recombinant zebrafish Ngb or Mb.
First, we evaluated Ngb and Mb protein levels in
normoxic and hypoxic goldfish. No discernable
changes of Ngb in protein extracts from brain and eye,
or Mb in extracts from brain, heart and liver were
observed (supplementary Fig. S7). For interspecific
comparisons, total proteins were extracted from brains,
total eyes and hearts of individual goldfish and zebra-
fish specimens that had been kept under normoxic con-
ditions. We applied a constant amount of total protein
extracts to SDS ⁄ PAGE (100 lg per lane for brain and
eye; 15 lg for heart) and western blotting (Fig. 5 ). To
avoid variations due to different treatments, samples
from both species were applied to the same gel and
transferred to a single membrane, which was then
incubated with the antibodies. We observed an approx-
imately fivefold higher Ngb protein level inthe gold-
fish brain compared with zebrafish brain (Fig. 5A).
The difference was highly significant, as estimated by a
Student’s t-test (P < 0.001). In protein extracts from
goldfish eyes, Ngb protein levels were 3.2-fold higher
than in zebrafish eyes (P < 0.05). We observed no dif-
ferences in Mb levels in goldfish and zebrafish, either
in brain or in heart (Fig. 5B).
Discussion
Many fish species have evolved strategies that allow
them to survive phases of acute and chronic hypoxia
[2,5–8]. Carassius species are particularly hypoxia toler-
ant, with various mechanisms that help them to better
survive hypoxiaand even anoxia [2,11,37]. We recently
analyzed the expression regulation and putative roles
of the various globinsin another, less hypoxia-tolerant
species of the Cypriniformes, the zebrafish D. rerio
[23]. Comparison of these data with those obtained
from C. auratus will help to delineate the contribution
of globin expression regulation to hypoxia tolerance.
In particular, we focused on Mb and Ngb; although a
respiratory role for Mb is well documented, our results
provide better understanding of the physiological role
of the recently discovered Ngb.
Two paralogous Mb genes in Cyprininae
Mb is an intracellular respiratory protein that mainly
facilitates the diffusion of O
2
from the capillaries to
the mitochondria and stores O
2
[37]. Until recently, it
had been commonly assumed that there is only a single
Mb gene in vertebrates and that Mb expression is con-
fined to muscle tissue. Mb protein is present at high
concentrations inthe skeletal and heart muscles of
most vertebrates [37], but has also been identified in
smooth muscle [38,39]. However, Fraser et al. [25]
demonstrated that the common carp possesses two
paralogous Mb genes, of which one (Mb1) is ubiqui-
tously present also in nonmuscle tissues; Mb2 expres-
sion is restricted to the carp’s brain. We confirmed
these results ingoldfish, which also possesses two dis-
tinct Mb genes (Fig. 2) and exhibits ubiquitous expres-
sion of Mb (Fig. 3). This suggests that the duplication
of Mb genes andthe altered expression patterns
occurred before the divergence of the genera Cyprinus
and Carassius (both belonging to the subfamily
Cyprininae), which separated 11 million years ago
[40]. As revealed by database searches, the zebrafish
AB
Fig. 5. Comparison of neuroglobin (A) and
myoglobin (B) protein levels in goldfish and
zebrafish. Protein levels were estimated by
quantitative western blotting (cf. Fig. S8).
Three individual zebrafish specimens and
four goldfishes were used for the experi-
ments. The units of the y-axis are arbitrary,
with zebrafish brain = 1. Note that the rela-
tive levels of Mb in brain and heart cannot
be compared. ***P < 0.001; *P < 0.05
(t-test), n.s., not significant.
A. Roesner et al. Goldfish globins under hypoxia
FEBS Journal 275 (2008) 3633–3643 ª 2008 The Authors Journal compilation ª 2008 FEBS 3637
D. rerio genome harbors only a single Mb gene. It
may be assumed that the emergence of an additional
Mb gene is linked to a genome duplication event,
which occurred inthe Cyprininae 12–16 million years
ago [40]. We observed Mb protein expression in
goldfish as well as in zebrafish brain, suggesting that
nonmuscle expression of Mb emerged before the diver-
gence of D. rerio and Cyprininae within the lineage of
the Cypriniformes. As proposed by Fraser et al. [25],
the occurrence of Mb in tissues other than muscle, its
hypoxia-inducible expression, as well as the occurrence
of the brain-specific isoform Mb2 may be part of the
strategy of the Cyprininae for better survival during
prolonged periods of hypoxia.
Expression-regulation of globinsand their
function in hypoxia
As in zebrafish [23], we observed a mild downregula-
tion of Hba and Hbb mRNA levels at hypoxia com-
pared with normoxic controls (Fig. 4A). The hypoxia
response of Hb has been investigated in various fish
species, with conflicting results. For example, Timmer-
man and Chapman [41] reported an increase in Hb
levels inthe sailfin molly (Poecilia latipinna), whereas
Person Le Ruyet et al. [42] observed no difference in
normoxic and hypoxic juvenile turbot (Scophthal-
mus maximus) and seabream (Sparus aurata). In zebra-
fish, the Hb mRNA levels decrease under hypoxia
[23,41]. The contribution of the transcription regula-
tion of Hb to hypoxia tolerance is species specific and
may also depend on thehypoxia regime [43]. Never-
theless, Carassius Hb is 50% saturated even at
Po
2
= 0.33 kPa, thereby contributing to hypoxia
tolerance [44]. Most likely, the O
2
-affinity of fish Hb is
largely regulated on a post-translational level, i.e. via
alteration of O
2
affinities by means of modulators such
as ATP and GTP [45]. Therefore, alterations in Hb
mRNA levels are not necessarily required. Mb1 and
Mb2 were the only globin-types in goldfish to show
hypoxia induction at the mRNA level. It may be
assumed that enhanced expression of Mb is associated
with hypoxia tolerance in goldfish. Additional Mb in
various tissues increases the availability of O
2
to the
respiratory chain of the mitochondria, thereby prom-
oting the survival of the cells.
Putative role of Ngb in preadaptation of the brain
to hypoxia
Altered expression levels of certain proteins may help to
improve the animal’s survival under unfavorable condi-
tions. For example, interspecific variations in heat
shock proteins have been found in marine gastropods
and have been attributed to the acclimatization to dif-
ferent habitats [46,47]. The high Mb content inthe mus-
cles of marine mammals such as whales and seals is
considered an adaptation to long-term dives [48]. Noth-
ing is known about the more recently discovered Ngb.
Although the localization, expression, regulation and
evolution of Ngb have been thoroughly investigated in
recent years, its exact role in vertebrate neurons is not
well understood [29,30]. Whereas some studies point to
a Mb-like role of Ngb in supplying O
2
[26,49], thereby
enhancing the survival of neurons under hypoxia [50],
other authors have proposed a function for Ngb in the
detoxification of reactive nitrogen species and NO
[51,52] or hypoxia-related signaling [53,54]. Most stu-
dies agree that in vivo hypoxia does not significantly
enhance Ngb expression inthe mammalian brain [21].
This is not surprising, because under normal condi-
tions most mammals never experience low O
2
environ-
ments during their adult life. However, as already
pointed out, many fish live in hypoxic environments.
In fact, we previously observed an increase in Ngb
levels of up to 5.7-fold in hypoxic zebrafish brain
compared with normoxia controls, which suggests the
involvement of this protein inhypoxia response in this
fish species [23].
It is well established that theCarassius brain has
evolved various strategies to survive very low oxygen
levels [37]. However, C. auratus, which is actually more
hypoxia-tolerant than zebrafish, does not show a
hypoxia response in Ngb expression. At first sight, this
is difficult to reconcile with the hypothesis that Ngb is
involved in O
2
supply for respiration or has any other
Po
2
-related function such as reactive oxygen species
detoxification. However, we did not observe an
increase inthe expression of the typically hypoxia-
responsive gene LDH-A, or of the reactive oxygen
species-defense gene SOD, although the fact that Mb1
is heavily upregulated shows that thehypoxia regime
we applied in this study actually induce changes in
gene expression. As shown in Fig. 5, there is an appro-
ximately fivefold higher level of Ngb protein in the
goldfish brain compared with the related, less hypoxia-
tolerant zebrafish. This is the first time that higher
Ngb concentrations could be correlated with hypoxia
tolerance, which may be interpreted as a preadaptation
of the goldfish brain. A similar observation has been
made inthe subterranean mole rat Spalax ehrenbergi,
a mammal that can survive extended periods of
hypoxia without neuronal damage, and which has con-
stitutively higher expression levels of Ngb compared
with rats (A. Avivi, F. Gerlach, T. Burmester, E. Nevo
& T. Hankeln, unpublished results). These data
Goldfish globins under hypoxia A. Roesner et al.
3638 FEBS Journal 275 (2008) 3633–3643 ª 2008 The Authors Journal compilation ª 2008 FEBS
provide additional support for an adaptive role of Ngb
in hypoxia tolerance of neurons, and for a possible func-
tion of Ngb in O
2
storage or facilitated O
2
diffusion.
Distinct roles of Mb and Ngb in hypoxia
adaptation
Changing environmental oxygen concentrations have a
significant impact on the expression of intracellular
respiratory proteins, which increase the availability of
O
2
to the tissues. In mammals, the results on the
impact of hypoxia on Mb expression are variable
[55,56]. Data on hypoxia-regulation of Ngb in mam-
malian systems are also not consistent. Although Ngb
was found to be more highly expressed in hypoxic cell
and tissue culture systems, no changes in Ngb mRNA
levels were found in whole-animal experiments [21].
Here we have demonstrated that both Mb and Ngb
may contribute to the extreme hypoxia tolerance of
goldfish. The increased expression of Mb under
hypoxia andthe high intrinsic Ngb levels in goldfish
neuronal tissues agree with the proposed function of
these proteins in O
2
supply. However, neuronal tissues
require a consistent supply with sufficient O
2
and any
shortage results in severe defects inthe brains of most
vertebrates. The function of the nervous system thus
requires an uninterrupted O
2
supply. The high intrinsic
concentration of Ngb may guarantee its immediate
availability upon the onset of hypoxia, and may be
part of the strategy that secures a constant flow of O
2
to the highly energy-demanding neurons. In contrast
to neurons, striated muscle cells can survive via anaer-
obic fermentation; therefore, a delayed increase in Mb
concentration (as reflected by enhanced Mb mRNA
levels) is sufficient to ensure the supply of O
2
to the
muscle cells. Together with the high-affinity Hb [44],
high levels of Mb and Ngb may contribute to the fact
that Carassius are able to maintain normal O
2
con-
sumption rates down to oxygen levels of 5–10% of air
saturation in water [57].
Experimental procedures
Experimental animals
Adult goldfish (C. auratus L.) were purchased in a local pet
shop and kept for several months in a large tank. Animals
used for thehypoxia or normoxia control experiments
(weighing around 4 g each) were directly transferred to a
100 L aquarium and kept at 14 h light ⁄ 10 h dark cycle and
a temperature of 20 °C. Water was filtered with a thermofil-
ter (Ekip 350; Hydor, Bassano del Grappa, Italy). Water
quality was checked periodically (Multi Check; Amtra,
Rodgau, Germany) and partial water changes were carried
out when necessary. Animal handling and experiments were
conducted according to a protocol that had been approved
by the county government office (Bezirksregierung Rhein-
hessen-Pfalz, AZ 1.5 177-07 ⁄ 021-30).
Hypoxia treatment
Groups of four goldfish were randomly assigned to hypoxia
treatment or control groups. The animals were not fed for
24 h before or during the experiments. Hypoxia treatment
was performed in a 40 L aquarium with loosely fitting
covers. Water was bubbled with gas mixtures (2% O
2
in N
2
or 100% N
2
; Air Liquide, Du
¨
sseldorf, Germany). A ther-
mopump (Ekip 350; Hydor) was used to ventilate the water
and to keep the temperature constant at 20 °C. O
2
par-
tial pressure and temperature were measured every 15 min
using an oxygen sensor (Oxi 340i, WTW, Weilheim,
Germany). Hypoxia treatment was started by reducing the
Po
2
to 6.7 kPa ( 50 Torr) for 48 h, Po
2
1.7 kPa
( 13 Torr) for 8 h, followed by Po
2
6.7 kPa
( 50 Torr) for additional 16 h. O
2
partial pressure
remained constant (± < 0.5 kPa) during experimental
time. Control animals were kept under the same conditions,
but the water was gassed with room air (Po
2
18.4 kPa,
138 Torr). After the experiment, specimens were cooled
on ice and killed by decapitation. Organs were removed,
shock-frozen in liquid N
2
and stored at )80 °C until use.
RNA extraction
RNA samples from total goldfish or single organs were
extracted using the RNeasy Mini Kit by Qiagen (Hilden,
Germany). Tissues were weighed and homogenized in the
required volume of RLT buffer (Qiagen). To avoid contami-
nation with genomic DNA, a DNase digestion was per-
formed on the Qiagen columns. Quality and amount of RNA
were checked photometrically and with gel electrophoresis.
cDNA amplification, cloning and sequencing
Total RNA was extracted from goldfish brain, liver, heart,
skeletal muscle, spleen, eyes and gills as described above.
Partial or complete cDNA sequences of C. auratus Hba,
Hbb, Mb1, Mb2, Ngb, Cygb1, acidic ribosomal protein
(ARP; also known as rplp0), LDH-A and Cu ⁄ Zn-SOD-
1were amplified via RT-PCR with the OneStep RT-PCR
kit (Qiagen) using degenerated or specific oligonucleotide
primers (supplementary Table S1). The cDNA fragments
were cloned into the pCR4-TOPO-TA (Invitrogen,
Karlsruhe, Germany) or the pGEMTeasy vector (Promega,
Mannheim, Germany). Poly(A)
+
RNA was purified
from total RNA using the PolyATract
TM
kit (Promega);
5 lg poly(A)
+
RNA were used for the construction of a
A. Roesner et al. Goldfish globins under hypoxia
FEBS Journal 275 (2008) 3633–3643 ª 2008 The Authors Journal compilation ª 2008 FEBS 3639
directionally cloned cDNA expression library applying the
Lambda ZAP-cDNA synthesis kit (Stratagene, Heidelberg,
Germany) according to the manufacturer’s instruction. The
library was then screened with digoxigenin-labeled cDNA
fragments of the globins. Positive phage clones were con-
verted to plasmid vectors using the material provided in the
cDNA synthesis kit. cDNAs inserted inthe pBK-CMV
vector were sequenced on both strands by a commercial
sequencing service (Genterprise, Mainz, Germany). In some
cases, the incomplete clones were extended by 5¢- and
3¢-RACE (Invitrogen) with a series of nested oligonucleo-
tide primers according to the manufacturer’s instructions.
The sequences were obtained after the cloning of the
PCR products into pCR4-TOPO-TA (Invitrogen) or
pGEM-Teasy vector (Promega).
Quantitative real-time RT-PCR
RNA extractions and cDNA synthesis were carried out
from tissues of single specimens. Quantitative real-time
RT-PCR was performed according to a two-step protocol.
First, total RNA was converted into cDNA employing
Superscript II RNase H
)
Reverse Transcriptase (Invitrogen)
and an oligo(dT)
16
primer according to the manufacturer’s
instructions. The cDNA samples were diluted with the same
volume of DNase-free water. Real-time RT-PCR experi-
ments were carried out on an ABI Prism 7000 SDS (Applied
Biosystems, Darmstadt, Germany) using the Power SYBR
Ò
Green PCR Master Mix (Applied Biosystems). Levels of
mRNA of ARP, LDH-A, SOD-1, Hba,Hbb, Mb1, Mb2,
Ngb and Cygb1 were evaluated. To avoid amplification of
genomic DNA, all primer pairs included one intron-span-
ning oligonucleotide. The oligonucleotide primers were
obtained from Sigma-Genosys (Hamburg, Germany) (sup-
plementary Table S2). Reactions were run in triplicate with
one or two repetitions, using 1 lL of diluted cDNA as tem-
plate in a reaction volume of 25 lL. Primer concentrations
were 0.13 lm for each oligonucleotide. The Taq DNA poly-
merase was activated for 15 min at 95 ° C, followed by 40
cycles of a standard PCR protocol (15 s at 95 °C, 30 s at
60 °C, 30 s at 72 °C). The efficiency of the reaction was
measured by the slope of a standard curve. First evaluation
of results was performed inthe ABI Prism 7000 sds pro-
gram; for normalization and calibration data were exported
to qBase (http://www.medgen.ugent.be/qbase/). Final data
analyses were carried out with the Microsoft
Ò
excel 2003
spreadsheet program (Microsoft, Redmond, WA, USA).
The significance of the data was evaluated by Student’s
t-test.
Recombinant protein expression and antibody
preparation
The complete coding sequences of D. rerio Ngb was cloned
into the pET3a and Mb into pET15b expression vectors
(Novagen, Darmstadt, Germany) employing PCR-generated
restriction sites. Plasmids were transformed into Escherichia
coli BL21(DE3)pLys and grown at 25 °C in TBY medium
(0.5% NaCl, 1% tryptone, 0.5% yeast extract, pH 7.4)
containing 100 lgÆmL
)1
ampicillin, 30 lgÆmL
)1
chloram-
phenicol and 1 mmolÆL
)1
d-aminolevulinic acid. The culture
was induced at D
600
= 0.8 by isopropyl-b-d-thiogalacto-
pyranoside (0.4 mmolÆL
)1
). After 16 h, bacteria were har-
vested by centrifugation and resuspended in 50 mmolÆL
)1
Tris ⁄ HCl, pH 8.0, 1 mmolÆL
)1
EDTA, 0.5 mmolÆ L
)1
di-
thiothreitol, 8 lgÆmL
)1
DNase and 4 lgÆmL
)1
RNase
supplemented with CompleteÔ proteinase inhibitor mixture
(Roche Applied Science, Mannheim, Germany) and Pefab-
loc (Roth, Karlsruhe, Germany). The cells were broken by
freeze–thaw cycles in fluid nitrogen followed by ultrasonica-
tion. DNA and RNA were digested for 2 h at 37 °C. Cell
debris was removed by centrifugation (1 h at 4 °Cat
10 000 g). Ngb was purified by ammonium sulfate precipi-
tation, followed by DEAE ion-exchange column and size
exclusion chromatography. His-tagged Mb was purified by
affinity chromatography (Protino
Ò
Ni 2000 prepacked
columns; Macherey and Nagel, Du
¨
ren, Germany). Final
globin fractions were analyzed by gel electrophoresis,
pooled, concentrated and stored frozen at )20 °C. Protein
concentrations were determined using the Bradford [58]
method. Purified recombinant D. rerio Ngb and Mb were
used to raise a polyclonal antibody in rabbits. Specific Ngb
antibodies were affinity-purified from crude rabbit serum
using recombinant D. rerio Ngb coupled to a HiTrapÔ
NHS-activated HP column (Amersham Biosciences,
Munich, Germany) according to the manufacturer’s instruc-
tions. The antibody was stored at )70 °C in 50 mmolÆL
)1
Tris, 100 mmolÆL
)1
glycine, pH 7.4 or supplemented with
0.1% NaN
3
at 4 °C.
Protein extraction and western blotting
Tissues were removed from the animal (goldfish or zebra-
fish) and immediately homogenized in 1· NaCl ⁄ P
i
(140 mmolÆL
)1
NaCl, 2.7 mmolÆL
)1
KCl, 8.1 mmolÆL
)1
Na
2
HPO
4
, 1.5 mmolÆL
)1
KH
2
PO
4
) by ultrasonication. The
debris was precipitated by centrifugation for 10 min at
13 000 g at 4 °C andthe supernatant was stored at ) 20 °C
until use. Protein concentrations inthe samples were deter-
mined according to Bradford [58]. Protein extracts (100 lg)
were diluted in sample buffer (31.25 mmolÆL
)1
Tris ⁄ HCl,
pH 6.8, 1% SDS, 2.5% b-mercaptoethanol, 5% glycerol)
and heat-denatured for 5 min at 95 °C. Samples were
applied to a 15% SDS-polyacrylamide gel and run at 100–
120 V. Proteins were transferred to a nitrocellulose mem-
brane for 2 h at 0.8 mAÆcm
)2
. Nonspecific binding sites
were blocked by incubating for 45 min with 2% BSA in
NaCl ⁄ Tris (10 mmolÆL
)1
Tris, pH 7.4, 140 mmolÆ L
)1
NaCl). Membranes were then incubated for 2 h with
anti-Ngb or anti-zebrafish-Mb serum, both diluted 1 : 500
Goldfish globins under hypoxia A. Roesner et al.
3640 FEBS Journal 275 (2008) 3633–3643 ª 2008 The Authors Journal compilation ª 2008 FEBS
in 2% BSA ⁄ NaCl ⁄ Tris, and washed four times for 5 min
with NaCl ⁄ Tris. Membranes were incubated with the goat
anti-(rabbit IgG) coupled with alkaline phosphatase
(Dianova, Hamburg, Germany) for 1 h, diluted 1 : 10 000
in 2% BSA ⁄ NaCl ⁄ Tris, and washed as described above.
Detection was carried out with nitro blue tetrazolium
chloride and 5-bromo-4-chloro-3-indolyl-phosphate salt as
substrates. The membranes were scanned at 1200 dpi and
the images were imported into the scion image program
(version Beta 4.0.3). Protein levels were estimated by analy-
ses of grey values. Mean gray values of the background of
empty gel lanes were subtracted from the measurements of
Ngb or Mb protein levels. Data were imported into Micro-
soft
Ò
excel 2003 spreadsheet program (Microsoft). Statisti-
cal analyses were performed by Student’s t-tests.
Acknowledgements
We thank F. Gerlach for his advice on the real-time
PCR experiments and critical reading of the manu-
script. This work has been supported by grants of the
Deutsche Forschungsgemeinschaft (Bu956 ⁄ 5, Bu956 ⁄ 11
and Ha2103 ⁄ 3) andthe Fonds der Chemischen
Industrie.
References
1 van den Thillart G & van Waarde A (1985) Teleosts in
hypoxia: aspects of anaerobic metabolism. Mol Physiol
8, 393–409.
2 Lutz PL & Nilsson GE (1997) Contrasting strategies for
anoxic brain survival – glycolysis up or down. J Exp
Biol 200, 411–419.
3 Lutz PL & Nilsson GE (2004) Vertebrate brains at the
pilot light. Respir Physiol Neurobiol 141, 285–296.
4 Nikinmaa M (2002) Oxygen-dependent cellular func-
tions – why fishes and their aquatic environment are a
prime choice of study. Comp Biochem Physiol A Mol
Integr Physiol 133, 1–16.
5 Nikinmaa M & Rees BB (2005) Oxygen-dependent gene
expression in fishes. Am J Physiol Regul Integr Comp
Physiol 288, R1079–R1090.
6 Nilsson GE & Renshaw GM (2004) Hypoxic survival
strategies in two fishes: extreme anoxia tolerance in the
North European crucian carp and natural hypoxic pre-
conditioning in a coral-reef shark. J Exp Biol 207,
3131–3139.
7 Cossins AR & Crawford DL (2005) Fish as models for
environmental genomics. Nat Rev Genet 6, 324–333.
8 Walsh PJ, Veauvy CM, McDonald MD, Pamenter ME,
Buck LT & Wilkie MP (2007) Piscine insights into com-
parisons of anoxia tolerance, ammonia toxicity, stroke
and hepatic encephalopathy. Comp Biochem Physiol A
Mol Integr Physiol 147, 332–343.
9 van der Meer DL, van den Thillart GE, Witte F, de
Bakker MA, Besser J, Richardson MK, Spaink HP,
Leito JT & Bagowski CP (2005) Gene expression
profiling of the long-term adaptive response to hypoxia
in the gills of adult zebrafish. Am J Physiol Regul Integr
Comp Physiol 289, R1512–R1519.
10 Sollid J & Nilsson GE (2006) Plasticity of respiratory
structures – adaptive remodeling of fish gills induced by
ambient oxygen and temperature. Respir Physiol Neuro-
biol 154, 241–251.
11 Shoubridge EA & Hochachka PW (1980) Ethanol:
novel end product of vertebrate anaerobic metabolism.
Science 209, 308–309.
12 Shoubridge EA & Hochachka PW (1983) The integra-
tion and control of metabolism inthe anoxic goldfish.
Mol Physiol 4, 165–195.
13 van Waversveld J, Addink ADF & van den Thillart G
(1989) Simultaneous direct and indirect calorimetry on
normoxic and anoxic goldfish. J Exp Biol 142, 325–335.
14 Lushchak VI, Lushchak LP, Mota AA & Hermes-Lima
M (2001) Oxidative stress and antioxidant defenses in
goldfish Carassiusauratus during anoxia and reoxygen-
ation. Am J Physiol Regul Integr Comp Physiol 280,
R100–R107.
15 Bickler PE (2004) Clinical perspectives: neuroprotection
lessons from hypoxia-tolerant organisms. J Exp Biol
207, 3243–3249.
16 Po
¨
rtner HO & Knust R (2007) Climate change affects
marine fishes through the oxygen limitation of thermal
tolerance. Science 315, 95–97.
17 Wu RS (2002) Hypoxia: from molecular responses to
ecosystem responses. Mar Pollut Bull 45, 35–45.
18 Gracey AY, Troll JV & Somero GN (2001) Hypoxia-
induced gene expression profiling inthe euryoxic fish
Gillichthys mirabilis.
Proc Natl Acad Sci USA 98, 1993–
1998.
19 Ton C, Stamatiou D & Liew CC (2003) Gene expres-
sion profile of zebrafish exposed to hypoxia during
development. Physiol Genomics 13, 97–106.
20 Padilla PA & Roth MB (2001) Oxygen deprivation
causes suspended animation inthe zebrafish embryo.
Proc Natl Acad Sci U S A 98, 7331–7335.
21 Burmester T, Gerlach F & Hankeln T (2007) Regula-
tion and role of neuroglobin and cytoglobin under
hypoxia. Adv Exp Med Biol 618, 169–180.
22 Nikinmaa M (2001) Haemoglobin function in verte-
brates: evolutionary changes in cellular regulation in
hypoxia. Respir Physiol 128, 317–329.
23 Roesner A, Hankeln T & Burmester T (2006)
Hypoxia induces a complex response of globin expres-
sion in zebrafish (Danio rerio). J Exp Biol 209, 2129–
2137.
24 Wittenberg JB & Wittenberg BA (2003) Myoglobin
function reassessed. J Exp Biol 206, 2011–2020.
A. Roesner et al. Goldfish globins under hypoxia
FEBS Journal 275 (2008) 3633–3643 ª 2008 The Authors Journal compilation ª 2008 FEBS 3641
25 Fraser J, de Mello LV, Ward D, Rees HH, Williams DR,
Fang Y, Brass A, Gracey AY & Cossins AR (2006)
Hypoxia-inducible myoglobin expression in nonmuscle
tissues. Proc Natl Acad Sci USA 103, 2977–2981.
26 Burmester T, Weich B, Reinhardt S & Hankeln T
(2000) A vertebrate globin expressed inthe brain.
Nature 407, 520–523.
27 Schmidt M, Giessl A, Laufs T, Hankeln T, Wolfrum U
& Burmester T (2003) How does the eye breathe? Evi-
dence for neuroglobin-mediated oxygen supply in the
mammalian retina J Biol Chem 278, 1932–1935.
28 Reuss S, Saaler-Reinhardt S, Weich B, Wystub S, Reuss
MH, Burmester T & Hankeln T (2002) Expression anal-
ysis of neuroglobin mRNA in rodent tissues. Neuro-
science 115, 645–656.
29 Burmester T & Hankeln T (2004) Neuroglobin: a respi-
ratory protein of the nervous system. News Physiol Sci
19, 110–113.
30 Hankeln T, Ebner B, Fuchs C, Gerlach F, Haberkamp
M, Laufs TL, Roesner A, Schmidt M, Weich B,
Wystub S et al. (2005) Neuroglobin and cytoglobin
in search of their role inthe vertebrate globin family.
J Inorg Biochem 99, 110–119.
31 Pesce A, Bolognesi M, Bocedi A, Ascenzi P, Dewilde S,
Moens L, Hankeln T & Burmester T (2002) Neuro-
globin and cytoglobin. Fresh blood for the vertebrate
globin family. EMBO Rep 3, 1146–1151.
32 Burmester T, Ebner B, Weich B & Hankeln T (2002)
Cytoglobin: a novel globin type ubiquitously expressed
in vertebrate tissues. Mol Biol Evol 19, 416–421.
33 Schmidt M, Gerlach F, Avivi A, Laufs T, Wystub S,
Simpson JC, Nevo E, Saaler-Reinhardt S, Reuss S,
Hankeln T et al. (2004) Cytoglobin is a respiratory
protein in connective tissue and neurons, which is up-
regulated by hypoxia. J Biol Chem 279 , 8063–8069.
34 Fuchs C, Luckhardt A, Gerlach F, Burmester T &
Hankeln T (2005) Duplicated cytoglobin genes in teleost
fishes. Biochem Biophys Res Commun 337, 216–223.
35 Fuchs C, Burmester T & Hankeln T (2006) The
amphibian globin gene repertoire as revealed by the
Xenopus genome. Cytogenet Genome Res 112, 296–306.
36 Roesner A, Fuchs C, Hankeln T & Burmester T (2005)
A globin gene of ancient evolutionary origin in lower
vertebrates: evidence for two distinct globin families in
animals. Mol Biol Evol 22, 12–20.
37 Nilsson GE (2001) Surviving anoxia with the brain
turned on. News Physiol Sci 16, 217–221.
38 Schuder S, Wittenberg JB, Haseltine B & Wittenberg
BA (1979) Spectrophotometric determination of myo-
globin in cardiac and skeletal muscle: separation from
hemoglobin by subunit-exchange chromatography. Anal
Biochem 92, 473–481.
39 Qiu Y, Sutton L & Riggs AF (1998) Identification of
myoglobin in human smooth muscle. J Biol Chem 273,
23426–23432.
40 David L, Blum S, Feldman MW, Lavi U & Hillel J
(2003) Recent duplication of the common carp (Cypri-
nus carpio L.) genome as revealed by analyses of micro-
satellite loci. Mol Biol Evol 20, 1425–1434.
41 Timmerman CM & Chapman LJ (2004) Behavioral and
physiological compensation for chronic hypoxiain the
sailfin molly (Poecilia latipinna ).
Physiol Biochem Zool
77, 601–610.
42 Person Le Ruyet J, Boeuf G, Zambonino Infante J,
Helgason S & Le Roux A (1998) Short-term physiologi-
cal changes in turbot and seabream juveniles exposed to
exogenous ammonia. Comp Biochem Physiol A Mol
Integr Physiol 119, 511–518.
43 Affonso EG, Polez VL, Correa CF, Mazon AF, Araujo
MR, Moraes G & Rantin FT (2002) Blood parameters
and metabolites inthe teleost fish Colossoma macropo-
mum exposed to sulfide or hypoxia. Comp Biochem
Physiol C Toxicol Pharmacol 133, 375–382.
44 Burggren WW (1982) ‘Air gulping’ improves blood oxy-
gen transport during aquatic hypoxiainthe goldfish
Carassius auratus. Physiol Zool 55, 327–334.
45 Vaccaro Torracca AM, Raschetti R, Salvioli R, Ricc-
iardi G & Winterhalter KH (1977) Modulation of
the root effect in goldfish by ATP and GTP. Biochim
Biophys Acta 496, 367–373.
46 Hofmann GE & Somero GN (1996) Interspecific varia-
tion inthe heat shock response of the congeneric mus-
sels, Mytilus trossulus and Mytilus galloprovincialis. Mar
Biol 126, 65–75.
47 Tomanek L & Somero GN (2002) Interspecific- and
acclimation-induced variation in levels of heat-shock
proteins 70 (hsp70) and 90 (hsp90) and heat-shock tran-
scription factor-1 (HSF1) in congeneric marine snails
(genus Tegula): implications for regulation of hsp gene
expression. J Exp Biol 205, 677–685.
48 Ramirez JM, Folkow LP & Blix AS (2007) Hypoxia
tolerance in mammals and birds: from the wilderness to
the clinic. Annu Rev Physiol 69, 113–143.
49 Bentmann A, Schmidt M, Reuss S, Wolfrum U, Hankeln
T & Burmester T (2005) Divergent distribution in vascu-
lar and avascular mammalian retinae links neuroglobin
to cellular respiration. J Biol Chem 280, 20660–20665.
50 Sun Y, Jin K, Mao XO, Zhu Y & Greenberg DA
(2001) Neuroglobin is up-regulated by and protects neu-
rons from hypoxic–ischemic injury. Proc Natl Acad Sci
USA 98, 15306–15311.
51 Brunori M, Giuffre A, Nienhaus K, Nienhaus GU,
Scandurra FM & Vallone B (2005) Neuroglobin, nitric
oxide, and oxygen: functional pathways and conforma-
tional changes. Proc Natl Acad Sci USA 102, 8483–
8488.
52 Herold S, Fago A, Weber RE, Dewilde S & Moens L
(2004) Reactivity studies of the Fe(III) and Fe(II)NO
forms of human neuroglobin reveal a potential role
against oxidative stress. J Biol Chem 279, 22841–22847.
Goldfish globins under hypoxia A. Roesner et al.
3642 FEBS Journal 275 (2008) 3633–3643 ª 2008 The Authors Journal compilation ª 2008 FEBS
[...]... (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding Anal Biochem 72, 248–254 Supplementary material The following supplementary material is available online: Goldfish globins under hypoxia Fig S1 Sequence of C auratus hemoglobin a cDNA (accession number AM933143) Fig S2 Sequence of C auratus hemoglobin b cDNA (accession... AM933144) Fig S3 Sequence of the C auratus myoglobin 1 cDNA (accession number AM747267) Fig S4 Sequence of C auratus myoglobin 2 cDNA (accession number AM747268) Fig S5 Partial sequence of C auratus neuroglobin cDNA (accession number AM933145) Fig S6 Partial sequence of C auratus cytoglobin 1 cDNA (acc no AM933146) Fig S7 Western blot quantification of neuroglobin and myoglobin under hypoxia Fig S8 Western... quantification of neuroglobin and myoglobin in goldfish and zebrafish Table S1 Oligonucleotide primer sequences used for cloning of goldfish globins Table S2 Oligonucleotide primer sequences used for quantitative real-time RT-PCR This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing are not responsible for the content or functionality... Mathews AJ, Moens L, Dewilde S & Brittain T (2006) The reaction of neuroglobin with potential redox protein partners cytochrome b5 and cytochrome c FEBS Lett 580, 4884– 4888 54 Wakasugi K, Nakano T & Morishima I (2003) Oxidized human neuroglobin acts as a heterotrimeric Galpha protein guanine nucleotide dissociation inhibitor J Biol Chem 278, 36505–36512 55 Hoppeler H & Vogt M (2001) Muscle tissue adaptations... (2001) Muscle tissue adaptations to hypoxia J Exp Biol 204, 3133–3139 56 Levine BD & Stray-Gundersen J (2001) The effects of altitude training are mediated primarily by acclimatization, rather than by hypoxic exercise Adv Exp Med Biol 502, 75–88 57 Sollid J, De Angelis P, Gundersen K & Nilsson GE (2003) Hypoxia induces adaptive and reversible gross morphological changes in crucian carp gills J Exp Biol... note: Blackwell Publishing are not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 275 (2008) 3633–3643 ª 2008 The Authors Journal compilation ª 2008 FEBS 3643 . Mb and Ngb protein and mRNA levels increased significantly. The data suggest that these globins are involved in conveying hypoxia tolerance to zebrafish. Here we investigate the response of globin. [9,10]. Cyprinid fishes of the genus Carassius (the crucian carp Carassius carassius and its domestic Asian form, the goldfish C. auratus) routinely experience hypoxia and even anoxic phases in their. protein expression in goldfish as well as in zebrafish brain, suggesting that nonmuscle expression of Mb emerged before the diver- gence of D. rerio and Cyprininae within the lineage of the Cypriniformes.