www.elsevier.com/locate/freeradbiomed

Free Radical Biology &

Original Contribution

Hypoxia-inducible factor 1 proteomics and diving adaptations

in ringed seal

Peter Johnsona, Robert Elsnerb, Tania Zenteno-Savınc,*

aDepartment of Biomedical Sciences, Ohio University, Athens, OH 45701, USAbInstitute of Marine Science, University of Alaska Fairbanks, Fairbanks, AK 99775, USA

cPlaneacion Ambiental y Conservacion, Centro de Investigaciones Biologicas del Noroeste, Mar Bermejo 195, Playa Palo Santa Rita,

La Paz, BCS, 23090, Mexico

Received 21 December 2004; revised 8 March 2005; accepted 8 March 2005

Available online 5 April 2005

Abstract

The putative amino acid sequence of ringed seal (Phoca hispida) hypoxia-inducible factor 1a (HIF-1a) derived from DNA sequence

analysis of the single-copy gene has been investigated. The rationale for these studies was to determine the reasons for the presence of HIF-

1a at relatively high levels in seal tissues, and its possible role in protection against diving-related oxidative damage. Sequence analysis

indicated that the bHLH/PAS and TAD functional domains are very similar to those in terrestrial mammals, although there were significant

sequence differences between the mouse and seal proteins in a region of the ODD domain. Some of these results indicate that seal HIF-1a

protein can bind HIF-Ih, DNA, transcriptional coactivators, and von Hippel–Lindau protein (pVHL). The presence of HIF-1a in seal tissues

was not related to the absence of pVHL, which was found to be present in all seal tissues examined. It is concluded that seal HIF-1a may act

as a transcriptional activator and that its presence in seal tissues is probably not caused by its inability to interact with pVHL. It is suggested

that seal HIF-1 may serve two functions in the postdiving period, namely, to attenuate ischemia/reperfusion-induced oxidative stress and to

allow efficient lung reinflation.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Hypoxia-inducible factor; Ringed seal (Phoca hispida); Von Hippel–Lindau protein; Ischemia– reperfusion injury; Breath-hold diving; Free radical

damage

Introduction

The intracellular content of the protein known as

hypoxia-inducible factor 1a (HIF-1a) is increased under

conditions of low oxygen tension (hypoxia) in terrestrial

animals that have been studied [1,2]. This can occur

because critical proline [2,3] and asparagine [4,5]

residues in the protein are not hydroxylated at low Po2levels, and the absence of such hydroxylation prevents

0891-5849/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.freeradbiomed.2005.03.008

Abbreviations: DIG, digoxigenin-dUTP; HIF, hypoxia-inducible factor;

PCR, polymerase chain reaction; pVHL, von Hippel–Lindau protein; ROS,

reactive oxygen species.

* Corresponding author. Fax: +52 612 125 3625.

E-mail address: tzenteno04@cibnor.mx (T. Zenteno-Savın).

intracellular proteolysis of the protein. However, in

normoxia, HIF-1a is hydroxylated at those specific

proline and asparagine residues, causing it to be bound

by the von Hippel–Lindau protein (pVHL), thereby

triggering its ubiquitination and intracellular proteolysis

by the proteasome [6–10]. In hypoxia, where HIF-1a is

present in cells, the nonhydroxylated protein can interact

with another cellular protein (HIF-1h), which is

expressed constitutively in hypoxia and normoxia. The

dimer thus formed (called hypoxia-inducible factor, HIF-

1) can then act as a nuclear transcription factor by

binding to the hypoxia response element (HRE) which

causes their transcriptional upregulation [3,11,12]. As

many as 90 genes have been identified that can be

upregulated in response to hypoxia [13], with these genes

being typically involved in angiogenesis, erythropoiesis,

Medicine 39 (2005) 205 – 212

P. Johnson et al. / Free Radical Biology & Medicine 39 (2005) 205–212206

energy metabolism, apoptosis, and cell proliferation [14–

16]. Recent studies have also shown that HIF-1 plays

important roles in the pathophysiology of preeclampsia,

intrauterine growth retardation, hypoxia-mediated pulmo-

nary hypertension, and cancer [13,17–20], and that

defects in pVHL expression can also have pathological

consequences [21–23].

The diving habits of marine mammals and birds mean

that these organisms are exposed to wide variations of O2

tension, and avoidance of the deleterious effects of

exposure to metabolic hypoxia and reoxygenation is of

great importance as these species depend on successful

breath-hold diving to obtain food and escape predators.

Breath-hold diving, which results in hypoxia, hyper-

capnia, and acidosis [24], requires a number of cardio-

vascular adaptations, including a large capacity for

storage of O2 in blood and tissues and a redistribution

of blood flow during diving. Such redistribution produces

a regional ischemia in diving and a prompt reperfusion

on emersion [25]. This dive-associated ischemia–reperfu-

sion theoretically would be expected to cause marked

oxidative stress through production of ROS on reperfu-

sion, but diving mammals exhibit little evidence of such

pathological damage [26]. This suggests that the diving

mammals have evolved specialized mechanisms for

coping with the chronic pattern of ischemia–reperfusion,

and although it has not been possible to measure tissue

oxidative stress before, during, and after diving episodes

in living animals, recent studies have indicated that

diving mammal tissues possess protective adaptations

against undue oxidative stress. Studies on antioxidant

enzymes and antioxidant defenses in tissue samples in

vitro of an arctic marine mammal, the ringed seal (Phoca

hispida), have indicated that its tissues can eliminate ROS

more effectively than similar tissues in terrestrial mam-

mals [26–28]. Additionally, we have established that the

seal genome contains a gene for HIF-1a and that this

protein and HIF-1h are expressed in seal tissues such as

lung, skeletal muscle, myocardium, and kidney [29],

suggesting that the HIF-1 system may be involved in

the ability of the seal to avoid oxidative damage on

return to normoxia.

Previous work [30] on HIF-1a in rainbow trout

(Oncorhynchus mykiss) has shown that its amino acid

sequence differs in many ways from sequences of

terrestrial mammal HIF-1a proteins, although it does

contain the characteristic oxygen-dependent degradation

(ODD) domain sequence, which would permit interaction

with pVHL and subsequent intracellular proteolysis. This

study also showed that the content of HIF-1a is

maximal at normoxic venous Po2 (38 torr) in trout

gonad cells, in contrast with the situation in terrestrial

mammals, where maximum HIF-1a cellular content is

generally reached at much lower Po2 values (10 torr or

less). As we have already established [29] that HIF-1a

is normally present in several seal tissues, with the

highest expression level in lung, we investigated aspects

of the seal HIF-1 system to explain why such a

phenomenon might occur. Specifically, we have

addressed the issues of gene copy number for HIF-1a,

its gene structure and amino acid sequence, as well as

the presence of pVHL in seal tissues. In this article, we

report that the seal genome contains a single copy of

the HIFA gene for HIF-1a protein, and that there are

some major similarities and differences in the partial

amino acid sequence of HIF-1a and HIF-1a proteins

between seal, terrestrial mammals, and rainbow trout.

We also show that the expression of pVHL in seal

tissues closely parallels the content of HIF-1a and HIF-

1h proteins, a situation different from that in terrestrial

mammal tissues.

Materials and methods

DNA and protein preparations from seal tissue

Seal tissues (kidney, liver, lung, muscle, and heart)

were obtained incidental to subsistence hunting through

collaboration with the North Slope Borough Department

of Wildlife Management and Inupiat Eskimo hunters

near Barrow, Alaska. DNA and protein extracts from

these tissues were prepared as described previously

[31].

PCR amplimer preparation and purification

Primers were custom-synthesized by IDT (Coralville,

IA, USA). Some amplimers were generated using

primers described previously [29], and other amplimers

were made using the following new primer sets which

were based on either known seal or mouse HIF-1a

DNA sequences. For amplimer generation and sequen-

cing between exons 2 and 3, two primer sets were used,

5VGTTGCCACTYCCMCAYAA (forward primer in exon 2)

5VAAACATACAGAAGGGCTCTCA (reverse primer in

exon3) and5VCVCATCTTGATAARGCYTCTGTT(forwardprimer in exon 2) 5VCACACTGTGTCCAGTTAGTT

(reverse primer in exon 3). For amplimer generation and

sequencing between exons 6 and 7, the primer set

5VTGCTTGGTGCTGATTTGTGAA (forward primer in

exon 6) 5VTRTCATGRTGAGTTTTGGTCAGA (reverse

primer in exon 7) was used, and additional sequence

information was obtained using the forward sequenQ

cing primer 5VAGGATGAACCCAGAGGACTT (seal

intron 6–7 sequence). For amplimer generation and

sequencing between exons 14 and 15, the primer set

5VCYTGGAAACGWGTRAAAGGA (forward primer in

exon 14) 5VCTTGATCCAAAGCTCTGAGTA (reverse

primer in exon 15) was used. Prior to sequence analysis,

amplimers were purified using the Promega Wizard PCR

Preps DNA Purification System, except for the small (99-

Table 1

Lengths of intron sequences in the seal and mouse HIF-1a genes

Intron location between exons Intron length (bp)

Seal Mouse

2 and 3 379 1242

3 and 4 84 92

5 and 6 569 1535

6 and 7 4800a 3717

9 and 10 664 594

10 and 11 900a 905

11 and 12 131 109

14 and 15 544 516

Seal and mouse sequence data are from GenBank accession numbers

AY843289–AY843295 and GenBank Y09085, respectively.a Indicates an estimated length based on amplimer sizes, as complete

intron sequence data have not been obtained.

P. Johnson et al. / Free Radical Biology & Medicine 39 (2005) 205–212 207

bp) amplimer from exon 15, which was purified using a

Centricon YM-30 Centrifugal Filter Device.

DNA sequence analysis and sequence comparisons

Bidirectional sequencing on an ABI Prism-310

Genetic Analyzer was performed using the ABI BigDye

Terminator Original or v3.1 Cycle Sequencing kit. DNA

sequences and translated seal DNA sequence data were

compared with mouse HIF-1a (GenBank Y09085) and

rainbow trout (GenBank AF304864) DNA and protein

sequences using the Gene Runner program. The seal

DNA sequence data obtained in this work, which also

identify the positions of some of the introns in the

seal gene, are found under GenBank Accession Nos.

AY843289–AY843295.

Dot-blot protein detection

The presence of HIF and pVHL proteins in the nuclear

protein preparations was detected immunologically by a

dot-blot procedure with the methods described previously

[29] using antibodies from Santa Cruz Biotechnology

(Santa Cruz, CA, USA). In these studies, the same

primary and secondary antibodies for HIF proteins were

used as before, and for pVHL detection, the primary

antibody used was a rabbit polyclonal IgG antibody to

full-length human pVHL (Item sc5575), and the secon-

dary antibody was a bovine anti-rabbit IgG-HRP (Item

sc2370). For each protein, the same dilutions of primary

and secondary antibodies were used in the assays (1:100

and 1:1000, respectively). Visualization and quantitation

of dot intensities were performed by densitometry using

the Bio-Rad Quantity One software system. Analyses

were performed on three different preparations in each

case, and the Minitab 12.22 program was used for one-

way ANOVA of the data to determine if significant

differences between the data sets existed at the 95%

confidence level (P < 0.05).

HIF-1a gene copy number analysis

The Roche nonradioactive DIG (digoxigenin-dUTP)

OMNI System for PCR probes was used to determine copy

number for the HIF-1a gene in seal DNA using the

Southern blot procedure described in the kit protocol.

Restriction digests of the seal lung DNA preparation were

performed separately with EcoRI and PstI as there were no

cleavage sites for these enzymes in the seal sequence of the

PCR products between exons 9 and 12. Digestions were

performed at a DNA concentration of 20 ng/AL and at two

different enzyme:substrate ratios for each enzyme (13 and

26 units/Ag DNA). After electrophoresis in 1% agarose, the

samples were treated with DIG-labeled probes which were

synthesized using PCR primer sets for exons 9–10 and 9–

12 as described previously [29].

Results

General organization of the seal HIF-1a gene

The results of the sequence analysis in these studies

reveal that the exon sequences in seal and mouse are

generally very similar, with only a few deletion/insertions

occurring, and relatively few substitutions present as

discussed in more detail in the following subsections.

Furthermore, the dispositions of exons in the seal and

mouse HIF-1a genes are identical, although the sizes of the

introns separating the exon sequences differ significantly in

some cases (Table 1).

Sequence analysis of the putative bHLH/PAS domain

The basic helix–loop–helix (bHLH)/PAS domain is a

component of a number of proteins that permits DNA

binding and is involved in regulation of gene expression

[12]. This domain is located within exons 2 to 5 of the

mouse gene. Partial sequence analysis of the seal genome

spanning exons 2 to 7 was performed, and the translation of

the seal exon sequences (Fig. 1A) shows that these sequence

regions are strongly conserved between mouse and seal.

Only four substitutions occur in the compared sequences, all

of which are in exon 3 (residues 77–124). In contrast, there

are several substitutions and a two-residue insertion between

the mammalian sequences and the rainbow trout sequence.

Sequence analysis of the putative ODD domain

The ODD domain of HIF proteins is involved in the ability

of the protein to interact with pVHL followed by ubiquiti-

nation and degradation by the proteasome complex [3,4].

This domain is located within exons 9 to 12 of the mouse

gene. Partial sequence analysis was performed on seal PCR

amplimers, and translation of the exon sequences (Fig. 1B)

shows that between the mouse and seal amino acid

sequences, there are only seven substitutions and a single

residue insertion in seal within the residues compared from

P. Johnson et al. / Free Radical Biology & Medicine 39 (2005) 205–212208

exon 9 to the end of exon 11 (residue 578). In contrast, the

trout sequence exhibits a number of major differences

compared with the mammalian sequences in this region,

including a 21-residue insertion and a 27-residue deletion. A

proline residue (Pro 402) that has been implicated as a site for

posttranslational hydroxylation and regulation of the inter-

action with pVHL [2,3] in terrestrial mammalian HIF-1a is

also present in the seal and trout sequences.

Sequence analysis of the putative TAD domain

The transactivational (TAD) domain of HIF proteins

contains sequences that permit the protein to interact with

cotranscriptional activator proteins, thereby enhancing the

effectiveness of HIF as a transcriptional activator [5,6]. The

N-terminal region of the TAD domain (TAD-N) is located

within exons 11 and 12 of the mouse gene, and partial

sequence analysis was performed on PCR amplimers

obtained from seal DNA by the use of primer sets from

exons 11 and 12. Translation of the seal exon sequences (Fig.

1B) shows that there is very close similarity of sequence

between the mouse and seal proteins in exon 11 (residues

511–578), with only a single substitution being present.

ig. 1. Partial amino acid sequence alignments for mouse, ringed seal, and rainbow trout HIF-1a proteins obtained from DNA sequencing and translation. (A)

equences from exons 2 to 7, (B) sequences from exons 9 to 12; (C) sequences of exons 14 and 15. The numbering of residues is that of the amino acid

equence of the mouse protein (GenBank Y09085). The trout sequence is from GenBank AF304864 and the seal sequence is from GenBank Accession Nos.

Y843289–AY843295. Where there are no letters or dashes in the seal sequence, this indicates that sequence information was not obtained for those residue

ositions. Known specific functional residues (Pro 402, Pro 563, Leu 573, Cys 796, and Asn 799) are indicated by asterisks.

F

S

s

A

p

Within the exon 11 sequence, both sequences (and the trout

sequence) contain a proline at position 563, which, like Pro

402, has been implicated in HIF-1a interaction with pVHL.

Furthermore, the surrounding sequences of Pro 563 are also

identical, including the critical residue Leu 573, which has

been implicated in pVHL recognition and subsequent prolyl

hydroxylation [31]. However, comparison of the mouse and

seal sequences in exon 12 (residues 579–694) reveals 26

substitutions and a single deletion in seal within the 100-

residue positions compared. In exon 12, the trout sequence is

very different from those of the mammalian proteins, with

only 15 residue identities in 117 residue positions with the

mammalian proteins, including two large and one small

deletion.

The C-terminal region of the TAD domain (TAD-C) is

located within mouse HIF-1a exons 14 and 15. This

domain contains the hydroxylatable asparagine residue

(Asn 799 in mouse), which is involved in the inhibition of

gene activation [1,7], and a cysteine residue (Cys 796 in

mouse), which has been implicated in transcriptional

upregulation [32]. Partial analysis of the exon 14–15 seal

gene sequence was performed and translation of the exon

sequences revealed almost complete identity between the

Fig. 1 (continued).

P. Johnson et al. / Free Radical Biology & Medicine 39 (2005) 205–212 209

mouse and seal sequences (Fig. 1C). Only four substitu-

tions were found in the exon 14 sequence (residues 731-

772), and in exon 15, the mouse and seal sequences were

completely identical. The trout sequence again exhibited

some sequence differences from the seal and mouse

sequences, but it did contain both the Cys 796 and Asn

799 residues.

Copy number analysis for the HIF-1a gene

The copy number of the seal HIF-1a gene was determined

to establish if the high levels of HIF-1a in seal tissues could

be related to the presence of multiple copies of the gene. As

described under Materials and methods, the region between

exons 9 and 12 was chosen as the site for restriction enzyme

Fig. 2. HIF-1a gene copy number in ringed seal determined by Southern

blotting. Seal DNA was prepared and digested separately with EcoRI and

PstI, and then treated with DIG-labeled DNA probes after electrophoresis

as described under Materials and methods. The two lanes for each

restriction enzyme are digestions performed at lower (left lane) and higher

(right lane) enzyme:substrate ratios. The E-HindIII lane is a DNA fragment

size standard. The sizes of single DNA fragments detected in the EcoRI and

PstI digests are 5.5 and 6.7 kb, respectively.

Table 2

Relative percentile expression levels of HIF-1a, HIF-Ih, and pVHL in seal

tissues as determined by dot-blot analysis

Tissue HIF-1a HIF-Ih pVHL

Kidney 39 T 5a 15 T 4 14 T 4

Liver 22 T 7 10 T 5 2 T 2

Lung 100 77 T 14 92 T 9

Muscle 91 T 4 82 T 8 67 T 8

Heart 49 T 7 31 T 5 24 T 5

a Values are expressed as the mean percentage T SD for three separate

analyses normalized to the expression level of HIF-1a in lung (=100%).

P. Johnson et al. / Free Radical Biology & Medicine 39 (2005) 205–212210

analysis, and primer sets for exons 9–10 and 9–12 were

used. When restriction digests were performed with EcoRI

and PstI, single bands were detected when primers for exons

9–10 were used (Fig. 2). Single bands of identical size were

also seen when the primers for exons 9–12 (results not

shown) were used. These results indicate that the seal

genome contains a single copy of the HIF-1a gene.

Expression of pVHL in seal tissues

Because of the apparent conservation of functionality in

essential sequences between seal and terrestrial mammal

HIF-1a proteins, and the presence of only a single copy of

the HIF-1a gene in the seal genome, the expression of

pVHL in seal tissues was investigated to establish if the

presence of HIF-1a protein in seal tissues could be the result

of a lack of pVHL in the same tissue. As shown in Fig. 3,

dot-blot analyses of seal tissue extracts using specific

antibodies to HIF-1a, HIF-1h, and pVHL revealed that

pVHL was detected in seal tissues that contained HIF-1a,

and Table 2 shows that relative amounts of pVHL generally

paralleled those of both HIF-1a and HIF-1h, with the

highest relative levels of all three proteins occurring in lung.

Fig. 3. Representative dot-blot analyses of HIF and pVHL protein

expression in ringed seal tissues. Nuclear protein fractions from kidney,

liver, lung, muscle, and heart were prepared, and 5-Ag protein samples were

used in the assay with primary antibodies against mouse HIF-1a, goat HIF-

1h, and human pVHL as described under Materials and methods.

A lack of pVHL in seal tissues therefore cannot be the

explanation for the presence of HIF-1a in these tissues.

Discussion

The results reported in this article show that in the exon

sequences examined, the seal HIF-1a gene contains

sequences that are closely identical to exon sequences in

mouse HIF-1a, and that these exons appear to be arranged

similarly to those in the mouse gene. There is also

similarity to the rainbow trout HIF-1a sequence, but the

sequence similarity is not as strong and exhibits many of

the differences that also exist between mouse and rainbow

trout sequences. These results show that the seal_sadaptation to an aquatic environment (with routine bouts

of hypoxia followed by reperfusion) does not involve the

sequence changes to the HIF-1a protein that are seen in a

teleost that normally exists in a more aerobic aquatic

environment and has different physiological responses to

emerging asphyxia [33].

The exon sequences selected for this study were those

known to be involved in the ability of HIF-1a to interact

with HIF-1h, with DNA (both involving the bHLH/PAS

domain), with pVHL (the ODD domain), and with tran-

scriptional coactivators (the TAD domain). All of the

residues previously shown to be critical for these functions

in terrestrial mammal HIF-1a were demonstrated to be

present in the seal protein (viz. Pro 402, Pro 563, Leu 573,

Cys 796, and Asn 799 of the mouse sequence), and the

immediate sequences surrounding these residues were either

identical or very strongly conserved. The only exception to

this conservatism was in the C-terminal region of the TAD-

N domain, where there was considerable sequence variation

between seal and mouse HIF-1a in approximately 25% of

the residue positions beginning 12 residues on the C-

terminal side of Leu 573. Although it is possible that these

differences may be related to changes in the function or

stability of seal HIF-1a, it should be noted that this

sequence region differs even more between the mouse and

human proteins, and this sequence in rainbow trout region is

also very different from that in the mammalian proteins.

These comparisons suggest that this part of the sequence

may not be essential for the critical properties of HIF-1a,

and may be a hypervariable region subject to genetic drift.

P. Johnson et al. / Free Radical Biology & Medicine 39 (2005) 205–212 211

The experiments performed in these studies on HIF-1a

gene copy number and pVHL expression in seal tissues

clearly show that the presence of HIF-1a protein in seal

tissues cannot be explained by multiple gene copies for HIF-

1a or by a lack of expression of pVHL which would prevent

degradation of HIF-1a. These results, in combination with

the sequence conservation in HIF-1a indicating that HIF-1a

can interact with pVHL, suggest that there must be other

factors that permit HIF-1a to exist in seal tissues despite the

presence of pVHL and exposure to normoxic conditions for a

considerable period. In this context, it is interesting to note

that recent studies [34] have shown that HIF-1a expression in

human lung epithelial tissue is downregulated by the

presence of a natural antisense HIF-1a whose production is

enhanced by HIF-1a. The question as to whether alterations

in such a mechanism could explain the high levels of HIF-1a

protein expression in seal tissues remains to be examined.

It is intriguing from a physiological point of view that seal

lung tissue, well-oxygenated during nondiving periods,

appears to have the highest relative expression of both HIF-

1a and pVHL. The alveolar ducts of seals, in contrast to those

of terrestrial mammals, are reinforced with cartilage, allow-

ing for sequential airway collapse during dives. Therefore,

lung alveolar gas exchange is prevented in dives deeper than

approximately 2 atm equivalent pressure, and lung tissue

remains compressed and anoxic until surfacing [35]. In

consequence, seal lungs are exposed to cycles of acute

ischemia–reperfusion and the potential for considerable

oxidative stress, and other tissues, notably skeletal and

cardiac muscle, are subjected to frequent periods of inter-

mittent ischemia during dives [25]. The levels of oxidative

damage that occur in seal tissues as a consequence of diving

have not been measured, but are assumed to be relatively

minor given that the diving lifestyle is not associated with an

abnormally short life span. Hypoxia-tolerant species, such as

the ringed seal, must have evolved special mechanisms that

confer protection in preparation to avoid the ischemia–

reoxygenation-induced oxidative stress [36,37], and in

particular lung tissue has special problems associated with

elevated free radical levels because of their known adverse

effects on lung surfactants, which are critical components in

lung function [38]. It has been shown that in mouse, the HIF

protein known as HIF-2a [39] is involved in lung surfactant

function as levels of lung surfactant are abnormally low in

premature mice when this protein is not present in lung tissue

[40]. In this situation, the absence of HIF-2a causes

decreased levels of VEGF in alveolar cells and prevents the

normal conversion of glycogen to surfactant through the

consequent downregulation of enzymes of carbohydrate

metabolism. These effects, which were shown to be

prevented by administration of VEGF, result in decreased

production of surfactant and fatal respiratory distress

syndrome. In the case of diving mammals, the presence and

possible role of HIF-2a in lung tissue have not yet been

investigated, but it is tempting to speculate that in seal lung,

HIF-1a may play a similar role as it is a transcription factor

for VEGF and enzymes of carbohydrate metabolism in

animal tissues. It may therefore be possible that HIF in ringed

seal lung not only contributes to the protection against rapid

cyclic bouts of ischemia–reoxygenation, which would not be

temporally possible with the relatively slow response of a

protein degradation and induction system, but also helps to

maintain the highly developed surfactant system that is

required to allow reinflation of the collapsed lungs following

a dive.

Acknowledgments

This work was possible thanks to the members of the

North Slope Borough, the Alaska Department of Wildlife

Management and the Barrow Arctic Science Consortium,

who provided logistic support and assistance in obtaining

ringed seal samples. Sampling was performed under terms

of a marine mammal scientific permit issued to R. Elsner

by the Office of Protected Species, U.S. National Marine

Fisheries Service. Import permits were issued by Instituto

Nacional de Ecologıa, Mexico. We thank Dr. K.T.

Coschigano of the Department of Biomedical Sciences,

Ohio University, for valuable advice on the design of the

PCR primers, and M.Sc. Jesus Neftali Gutierrez Rivera of

the Molecular Biology Laboratory, CIBNOR, for valuable

assistance in sample preparation and extraction. Research

was funded by grants from the Office for Naval Research

(N00014-00-1-0314), UC Mexus, CONACYT, CIBNOR

(T.Z.S.), and Ohio University (P.J.).

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