Báo cáo khoa học: Antibacterial peptides in stimulated human granulocytes Characterization of ubiquitinated histone H1A doc

Antibacterial peptides in stimulated human granulocytes
Characterization of ubiquitinated histone H1A
Yuqin Wang, William J. Grif®ths, Hans JoÈ rnvall, Birgitta Agerberth and Jan Johansson
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden
Antibacterial peptides were isolated from human peripheral
granulocytes of a healthy d onor who had been treated with
granulocyte-colony stimulating factor (G-CSF) and cortisol.
Peptides were solubilized in acidi®ed chloroform/methanol,
and partitioned in chloroform/methanol/water. Water-
soluble polypeptides were separated by cation-exchange and
reversed-phase chromatography. Several previously char-
acterized antibacterial polypeptides were identi®ed; defen-
sins 1±3, defensin 4, lysozyme, eosinophil cationic protein,
and calgranulin A. In addition, several histone fragm ents
were isolated and exhibited activity against the Gram-
positive bacterium Bacillus megaterium strain Bm11. These
fragments included two C-terminal fragments of histone
H1A, three C-terminal fragments of histone H1D, one
fragment of histone H1B, and two fragments of histone H4.
The molecular masses of both histone H1A fragments, as
determined by electrospray (ES) MS, were 270 Da higher
than those calculated from their amino acid sequences. The
two histone H1A fragments corresponded to Lys152±
Lys222 (75 27  1 Da) and Lys167±Lys222 (6023  1 Da).
Tandem MS (MS/MS) of the 7.5 kDa and 6.0 kDa frag-
ments indicated that the post-translational modi®cation is
on Lys222, the e-amino group of which was conjugated with
the a-carboxyl group of the tripeptide Arg-Gly-Gly. This
®nding was substantiated by digestion of the 7.5-kDa poly-
peptide with trypsin and analysis of the resulting peptides by
ES MS and MS/MS. T he tripeptide Arg-Gly-Gly c orre-

sponded uniquely to the t hree C-terminal residues of
ubiquitin, demonstrating the presence of ubiquitinated
histone H1A.
Keywords: antibacterial peptides; histone; post-translational
modi®cation; ubiquitination; tandem mass spectrometry.
In innate immunity, antibacterial peptides are important
effector molecules, and exhibit broad-spectrum antimicro-
bial activities [1 ]. Over the last 2 0 y ears, more t han 500
peptides with bactericidal properties have been isolated
from pla nts, insects and mammals, as well as from bacteria
[2]. Despite apparent diversity in structure, most of these
peptides are a mphipathic and c ationic, and can therefore
interact with p hosp holipid membranes, in particular with
membranes rich i n anionic phospholipids. Differing from
classical antibiotics, these peptide antibiotics are gene-
encoded, and the mature active peptides are derived from
inactive precursors by proteolysis. Post-translational m od-
i®cations can occur during processing, and include
N-terminal formation o f p yroglutamate or C-terminal
amidation [3,4].
Many mammalian antibacterial peptides, like a-defensins
and cathelicidins [5], were initially found in granulocytes; of
these the a-defensins are the most studied. The a-defensins
are 29±35 residues long, have th ree disul®de bridges, and
constitute > 5% of t he total cellular protein in human
neutrophil granulocytes [6].
Histones are the major proteins in chromatin, where their
function is toassist in DNA folding a nd packaging. However,
the presence o f histones on t he plasma membrane of activated
human peripheral blood lymphocytes suggests t hat histones

are not con®ned to the nucleus [7]. Similarly, histones H2A
and H2B have been found extracellularly in preparations of
homeostatic thymus hormone [8]. Furthermore, the histone-
derived antibacterial peptides parasin and buforin have been
isolated from the epithelial layer of t he cat®sh [9], and the
stomach tissue of the Asian toad [10], respectively.
All h istones are basic proteins, containing a relatively large
amount of lysine and/or arginine. Several post-translational
modi®cations of histones have been found, including acetyl-
ation, methylation, phosphorylation, ADP-ribosylation,
glycosylation and ubiquitination [11]. Histone H2A was the
®rst histone found to be modi®ed by covalent linkage to
ubiquitin; the carboxyl end of ubiquitin is linked to the
e-amino group of Lys119 in H2A [12]. Subsequently, histone
H2B was found to be mo di®ed in the same manner o n
Lys120, but to a l esser extent (1±2%) than in histone H2A
(10%) [13,14]. Recently, ubiquitination of histone H3 was
reported [15]. The covalent linkage of ubiquitin targets most
proteins for proteasome degradation, whereas the ubiquiti-
nation of histones has been suggested to be i mportant for
chromatin r emodelling during transcription [11]. In this
study, we isolated antibacterial peptides from human gran-
ulocytes obtained from a donor after t reatment with
granulocyte-colony stimulating factor (G-CSF) and cortisol,
a treatment which gives a m aximum number of peripheral
Correspondence to Y. Wang, Department of Medical, Biochemistry
and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
Fax: + 46 8337462, Tel.: + 46 87287681,
E-mail:
Abbreviations: G-CSF, granulocyte-colony stimulating factor; ES,

electrospray; TFA, tri¯uoroacetic acid; CID, collision induced disso-
ciation; MS/MS, tandem mass spectrometry; uH1A, ubiquitinated
histone H1A; D2S, dextrin-2-sulfate.
(Received 21 May 2001, revised 14 November 2001, accepted 15
November 2001)
Eur. J. Biochem. 269, 512±518 (2002) Ó FEBS 2002
blood granulocytes. In addition to several known antibac-
terial peptides, histone fragments were isolated and showed
activity against the Gram-positive bacterium, Bacillus mega-
terium Bm11. Here we present evidence for ubiquitination of
histone H1Ausing electrospray ( ES) MS. The tripeptide A rg-
Gly-Gly, corresponding to the three C-terminal residues of
ubiquitin, was found to be linked to Lys222 of histone H1A.
MATERIALS AND METHODS
Extraction and puri®cation of proteins from stimulated
human granulocytes
Granulocytes from a healthy donor treated w ith G -CSF
(Neupogen, 5 lgákg
)1
body weight, 1 day before harvest)
and c ortisol (100 mg Solucortef, 15 min before start of
harvest) were obtained from the Department of Transfusion
Medicine at the Karolinska Hospital.
Harvested human peripheral blood granulocytes
(4.5 ´ 10
10
cells, 8 2% granulocytes) were centrifuged at
3000 g for 30 min and the pellet (69 g) was homogenized in
1330 mL chloroform/methanol (2 : 1 , v/v), containing
0.1% (v/v) tri¯uoroacetic acid. Next 332 mL 0.1

M
NaCl
was added, yielding a two-phase system of chloroform/
methanol/0.1
M
NaCl, 8 : 4 : 3 (v/v). After phase separa-
tion, the aqueous phase was lyophilized, redissolved in 30%
(v/v) acetic acid, and desalted by RP chromatography using
aSep-PakC
18
cartridge (Waters), which had been equili-
brated in 0.1% (v/v) aqueous tri¯uoroacetic acid. Salts were
removed by washing with 10% (v/v) acetonitrile containing
0.1% (v/v) tri¯uoroacetic acid, after which proteins were
eluted with 80% (v/v) a cetonitrile containing 0.1% (v/v)
tri¯uoroacetic acid. The e luate was lyophilized and r edis-
solved in 0.02
M
sodium phosphate buffer pH 6.4, and
applied to a cation-exchange column (Express-ion exchang-
er C, Whatman) that had b een equilibrated in the same
buffer. Proteins were eluted with: (a) 0.02
M
phosphate
buffer pH 6.4; (b) 0.2
M
NaCl in 0.02
M
phosphate buffer
pH 6.4; and (c) 0.2

M
HCl. The contents of the three eluates
from the cation-exchange column were further separated by
RP-HPLC on a Resource column (10 ´ 2.0 cm, Pharma-
cia), with a water/acetonitrile gradient (see Fig. 1A). Each
fraction was lyophilized and redissolved in 100 lL0.1%
(v/v) a queous tri¯uoroacetic a cid, and screened for a ntibacte-
rial activity. F ractions containing activity were puri®ed by
RP-HPLC on a C
18
column (Vydac, 250 ´ 4.6 mm) with a
water/acetonitrile gradient and an inhibition zone assay.
Components showing activity were characterized.
Antibacterial assay
An inhibition zoneassay [16] with B. megaterium strain Bm11
or Escherichia coli strain D21 was used to detect antibacterial
activity. Lyophilized samples were redissolved in 100 lL
0.1% (v/v) aqueous tri¯uoroacetic acid, and 3-lL a liquots
were loaded into wells (3 mm) punched into agarose plates
seeded with the test bacteria. After incubation at 30 °C
overnight,thed iametersofth ei nhibitionzo neswererec orded.
Protein analysis
The HPLC-puri®ed polypeptides demonstrating activity
were characterized by Edman degradation on an Applied
Biosystems 494 Prosice sequencer and M ALDI-TOF MS.
Some peptides were further analysed by ES MS and MS/
MS. Amino acid analysis was performed on a 4151 Alpha
Plus amino a cid analyser (LKB Biochrom) using the
ninhydrin method. Tryptic digestion was performed as
described previously [4].

Mass spectrometry
ForMALDI-TOFMS,aliquotsofthesamplesweremixed
with 0.5 lL matrix (saturated a-cyano-4-hydroxy-cinnamic-
acid in 70% acetonitrile containing 0.1% tri¯uoroacetic
acid) on the target plate and left to dry. Mass spectra were
Fig. 1. Chromatography data. (A) RP chromatography (Resource
column 10 ´ 2.0 cm) of the 0.2
M
HCl e luate from cation-exchange
chromatography. The ¯ow rate w as 6 mLámin
)1
and fractions of 6 mL
were collected. The dashed line indicates the gradient of acetonitrile
containing 0.1% (v/v) tri¯uoroacetic a cid. The line with asterisks
indicates the activity (inhibition z one diameter) against B. megaterium
Bm11 and the line with circles indicates the activity a gainst E. coli D21.
(B) Analytical RP-HPLC (Vydac C
18
) of the fraction containing
histone fragments from Resource column (indicated in A). The ¯ ow
rate was 1 mLámin
)1
. The two uH1A fragments (Lys152±Lys222 and
Lys167±Lys222) we re eluted in peaks 2 a nd 5, respectively. Thr ee
histone H1D fragments (A179±K212, K175±K212 and T166±K212)
were found in peaks 1, 3 and 4, respectively.
Ó FEBS 2002 Ubiquitination of histone H1A (Eur. J. Biochem. 269) 513
recorded on a Finnigan MAT Lasermat 2000 operated in
the positive ion mode.
Nano-ES mass spectra were recorded on an AutoSpec-

OATOFFPD (Micromass) hybrid high re solution double
focusing magnetic sector-orthogonal acceleration TOF tan-
dem mass spectrometer [17]. Mass spectra were recorded at
both low (3500 resolution, 10% valley de®nition) and high
resolution (12500 resolution, 10% valley de®nition) as
magnet and voltage scans, respectively. Accurate mass
measurements were made as voltage scans at high resolution.
Nano-ES mass and MS/MS spectra were also recorded
on a Q-TOF (Micromass), quadrupole (Q)-TOF instru-
ment [18]. Both mass and collision induced dissociation
(CID) spectra were recorded. For the r ecording o f m ass
spectra, the quadrupole w as operated in the radio
frequency only mode and all transmitted ions accelerated
orthogonally into the TOF. For the recording of MS/MS
spectra the quadrupole was operated as a mass ®lter and
used to select the d esired precursor ion isotope cluster.
Precursor ions were directed into a hexapole collision cell
preceding the orthogonal acceleration region. The collision
gas was argon and the collision voltage was in the range
10±40 V.
RESULTS
Identi®cation of antibacterial polypeptides in stimulated
granulocytes
Water-soluble polypeptides were obtained from G-CSF-
and cortisol-treated human granulocytes, by homogeniza-
tion in chloroform/methan ol followed by phase separation
in chloroform/methanol/0.1
M
NaCl. Polypeptides in the
water phase were separated by cation-exchange chroma-

tography. Three fractions were obtained from the cation-
exchange colu mn by elution with 0.02
M
phosphate buffer
pH 6.4; 0.2
M
NaCl in 0.02
M
phosphate buffer pH 6.4; and
®nally 0.2
M
HCl. The antibacterial activity of each fraction
was determined by an inhibition zone assay. While all three
fractions exhibited antibacterial ac tivity, most antibacterial
polypeptides were found after elution with 0.2
M
HCl
(Table 1). Components i n the fractions were subjected to
RP-HPLC on a Resource column and fractions that had
antibacterial activity were puri®ed by RP-HPLC on a
Vydac C18 column. Fig. 1 shows the Resource RP separa-
tion of the 0.2
M
HCl eluate and the C18 RP separation of
fractions eluting with  20% acetonitrile during Resource
chromatography and showing a ntibacterial activity. Each
fraction was tested by an antibacterial assay, and active
peptides were identi®ed by amino acid sequence analysis.
Several known antibacterial polypeptides w ere identi®ed
(Table 1). Most of the a-defensins (1±3) were eluted from

the cation-exchange column in 0.2
M
NaCl in 0.02
M
phosphate buffer pH 6.4, while a-de fensin 4, l ysozyme,
eosinophil cationic protein and calgranulin A and several
fragments of histones H1 and H4 were found in the fraction
eluted with 0.2
M
HCl.
Puri®cation and identi®cation of ubiquitinated histone
H1A (uH1A) fragments
Two h istone H1A f ragments and t hree histone H1D
fragments were puri®ed by C18 R P-HPLC (Fig. 1B) and
identi®ed by Edman degradation for 20 cycles. As indicated
in Fig. 1B, peak 2, eluting at 16% acetonitrile, contained a
polypeptide corresponding to Lys167±Lys222 of histone
H1A, and peak 5, eluting at 18% acetonitrile, contained a
longer f ragment o f h istone H1A (Lys152±Lys222). How-
ever, the masses o f both polypeptides ( 6023  1 Da and
7527  1 Da, respectively) determined by ES MS (Fig. 2),
were 270 Da higher than predicted from the primary
structure of histone H1A. Three C-terminal histone H1D
fragments were contained i n p eaks 1, 3 and 4 (Fig. 1B),
respectively, corresponding to Ala179±Lys212, Lys175±
Lys212 and Thr166±Lys212. ES mass spectra con®rmed
these assignments and showed no covalent modi®cations.
Tandem m ass spectra were recorded on a Q-TOF
instrument of the [M + 10H]
10+

±[M+12H]
12+
ions
from the mod i®ed histone H1A fragments. The resultant
CIDspectrumofthe[M+10H]
10+
ion from the shorter
histone H1A(Lys167±Lys222) polypeptide is shown i n
Fig. 3. Prominent fragment ion peaks were observed as a
result of peptide bond cleavage N-terminal to a proline
residue. Table 2 describes the fragment ions observed i n this
CID spectrum.
Series of b-type and y-type ions were observed
(Scheme 1 ), which, combined, cover the complete
sequence of t he polypeptide. T his means that it was
possible to localize the modi®cation to the C-terminal
lysine residue. The interpretation of the CID spectrum
presented in Fig. 3 was con®rmed by comparison w ith
spectra of the [M + 11H]
11+
and [M + 12H]
12+
ions,
and also with the CID spectra of the longer histone H1A
polypeptide (data not shown). T he two histone H1A
polypeptides gave similar y-type ion series, however, the
b-type ion series was displaced b y 15 residues in t he larger
polypeptide, as expected from its N -terminal elongation
by 15 residues.
Thus MS/MS spectra of the H1A polypeptides indicated

that the 270 Da modi®cation was at the C-terminal lysine
residue. Histones H2A, H2B, H3 are known to be reversibly
ubiquitinated by c ovalent a ddition o f ubiquitin, where the
carboxyl end of ubiquitin is linked to t he e-amino group
of a lysine residue. A 270-Da modi®cation could correspond
to the conjugation of the tripeptide Arg-Gly-Gly to the
Lys222 side chain e-amino group. Signi®cantly, a database
Table 1. Identi®ed polypeptides with antibacterial activity contained in
the three cation-exchange column fractions. Eluate a, 0.02
M
phosphate
buer pH 6.4; e luate b, 0.2
M
NaCl in 0.02
M
phosphate b uer p H 6.4;
eluate c, 0.2
M
HCl.
Cation-exchange
column eluates Polypeptides
Elution from
RP-HPLC
(% acetonitrile)
Eluate a a-Defensins 1±3 33%
Eluate b a-Defensins 1±3 33%
Eluate c Histone H1A, H1D, H4 20%
fragments
Eluate c a-Defensin 4 40%
Eosinophil cationic protein

Histone H1B fragment
Eluate c Lysozyme 48%
Calgranulin A
Eluate c Histone H4 fragment 50%
514 Y. Wang et al. (Eur. J. Biochem. 269) Ó FEBS 2002
(Swissprot v ersion 39) search revealed that i n humans, a
C-terminal tripeptide segment Arg-Gly-Gly corresponds
uniquely to the three C-terminal residues of ubiquitin.
Thus, it would seem probable that histone H1A is
ubiquitinated, and that the 270 Da modi®cation corre-
sponds to the addition of A rg-Gly-Gly to the Lys222 side
chain amino group (Scheme 2). To con®rm this hypo-
thesis, a modi®ed histone fragment was digested with
trypsin and the resultant tryptic peptides were characterized
by ES MS.
Fig. 2. ES mass spectra. (A) ES mass spec-
trum of the polypeptide in peak 2 (see
Fig. 1B). (B) ES mass spectrum of the poly-
peptide in peak 5 (see Fig. 1B). The large
panels show the raw data, and the insets show
the deconvoluted s pectra on a true mass scale.
Fig. 3. MS/MS spectra. MS/MS spectrum of
the [M + 10H]
10+
ion at m/z 603 s hown in
Fig. 2A. The inset shows the y
1
ion corre-
sponding to the Arg-Gly-Gly-eLys fragment.
See Table 2 for description of all fragment

ions identi®ed.
Ó FEBS 2002 Ubiquitination of histone H1A (Eur. J. Biochem. 269) 515
The tryptic digest of modi®ed histone H1A(Lys152±
Lys222) was analysed by ES MS on both Q-TOF and
AutoSpec-OATOFFPD instruments. Tryptic digestion was
not complete, resulting in both the regularly expected
peptides and those in which a cleavage site had been
missed. When both sets of data were taken into account,
100% cov erage of the polypeptide was a chieved ( Fig. 4).
By recording accurate mass spectra at high resolution
(12500, 10% valley de®nition) on the AutoSpec-OAT-
OFFPD it was also possible to con®rm the elemental
composition o f the T
24
peptide, which contains the Arg-
Gly-Gly modi®cation of the C-terminal lysine residue.
Further e xperiments were performed i n w hich tryptic
peptide T
24
containing the Arg-Gly-Gly modi®cation was
subjected to CID. To achieve suf®cient product ion
sensitivity it was necessary to record these spectra on the
Q-TOF instrument, which is not capable of high resolution
precursor ion selection. The unit mass resolution available
was not suf®cient to uniquely select the desired precursor,
and thus a m ixture of precursor i ons was fragmented.
However, the immonium ions characteristic of Arg and
Lys were o bse rved, and t his, in combination with
the accurate mass data, identi®ed the modi®cation as
Arg-Gly-Gly.

Quantitation and antibacterial activity of uH1A
fragments
The concentration of uH1A fragments measured by amino
acid analysis corresponded to 2.5 pmol per 10
8
cells.
Antibacterial assays showed that the uH1A fragments were
effective a gainst B. megaterium Bm11, but inactive against
E. coli D21. The diameter o f t he inhibition zone against
B. me gaterium Bm11 was 8 mm when 67 l
M
uH1A frag-
ment (Lys152±Lys222) was applied. The corresponding
Table 2. Fr agment ions observed in the CID spectrum of the [M + 10H]
10+
ion at m/z 603 of modi®ed histone H1A fragment Lys167±Lys222. K* is modi®ed by the addition of 270 Da corresponding to Arg-
Gly-Gly.
b
1
+
b
2
+
b
3
+
b
4
+
b

5
+
b
6
+
b
7
+
b
 2 
8
b
9
2+
b
10
2+
b
11
2+
b
12
2+
b
14
2+
b
15
2+
b

17
3+
b
3  4
18
K1 KVAKSPKKAKAAAKPKKAT20
y
55
9+
y
50
8+
y
41
7+
b
3  4
21
b
22
4+
b
24
4+
b
4  5
25
b
27
5+

b
30
5+
b
32
5+
b
5  6
35
K21 S PAKPKAVKPKAAKPKAAK40
y
34
6+
b
45
7+
y
5  6 
31
y
26
5+
b
8  9 
52
y
21
3+
y
19

3+
P41 KAAKPKAKKAAAKKK*
y
2  3  4 
16
y
14
3+
y
2  3 
11
y
10
2+
y
9
2+
y
5
+
y
4
+
y
2
+
y
1
+
Scheme 1. Pe ptide fragmentation nomenclature. Protonated peptides

fragment in low energy CID reactions predominantly at amide bonds
to give b ions with the charge resid ing on the N-terminus of the pep-
tide, and y ions with the c harge residing on t he C-termin us of the
peptide.
Scheme 2. Sc hematic structure of uH1A fragment. The a-carboxy
group of Gly is linked to the e-amino group of Lys222 of hi stone H1A.
516 Y. Wang et al. (Eur. J. Biochem. 269) Ó FEBS 2002
data for 10-fold concentrated (670 l
M
) human antibacterial
peptide LL-37 was 10 mm.
DISCUSSION
Several histone fragments were puri®ed from human
granulocytes from a donor stimulated with G-CSF and
cortisol and found to exhibit strong antibacterial activity
against the Gram-positive bacterium B. me gate rium Bm11.
The mass spectrometric results showed that the t wo
C-terminal histone H1A fragments Lys152±Lys222 and
Lys167±Lys222 were modi®ed by Arg-Gly-Gly at Lys222
via an isopeptide bond between that e-amino group and the
a-carboxyl group of the terminal Gly of Arg-Gly-Gly. This
conclusion was arrived at upon consideration of the MS/
MS spectra of the 6.2- and 7.5-kDa histone H1A polypep-
tides, and o f the accurate mass and MS/MS spectra of the
tryptic digest of the 7.5-kDa polypeptide. The con®rmation
of the primary structures of large polyp eptides by MS/MS is
comparatively rare [19]. In the present study the interpre-
tation of the CID spectra was greatly facilitated by the
tendency of the protein to f ragment at amide bonds
N-terminal to proline, giving intense y-type ions and

complimentary b-type ions. T his made it possible t o locate
the m odi®cation of Lys222 of h istone H1A. This so called
Ôproline effectÕ has been noted previously [20,21] and is a n
important factor in the interpretation of C ID spectra of
polypeptides.
Ubiquitin contains 76 residues and adopts a stable
globular conformation with the evolutionarily conserved
three C-terminal residues, Arg-Gly-Gly, ¯exibly disordered
[22]. I nterestingly, both of the two histone H1A fragments
now found were derived by c leavage of a Val±Lys peptide
bond. A similar site between an aliphatic residue and a basic
residue (i.e. the peptide segment Leu±Arg) must be cleaved
in ubiquitin to release the C-terminal tripeptide Arg-Gly-
Gly. This suggests that the modi®ed histone fragments were
derived from ubiquitinated histone H1A by proteolysis
taking place between one hydrophobic and one basic
residue. Ubiquitination has been found in histones H2A,
H2B and H3, and the amount of ubiquitinated histone was
found to vary during the cell cycle [23]. At present, it is not
known whether the presence of uH1A in human granulo-
cytes after treatment with G-CSF and cortisol is an effect of
the stimulation, or if h istone H1A is also ubiquitinated i n
nonstimulated cells. It is also not known whether histone
H1A i s ubiquitinated so as t o target it t o a degradation
pathway or for other reasons.
Several recent studies have indicated extra-nuclear loca-
tions of histones, with some studies suggesting immunolog-
ical roles of histones, in addition to the classical function of
histones in chromatin conformation. In phytothaemagglu-
tinin-activated human peripheral lymphocytes, histone H2B

was found to move from the nucleus to the plasma
membrane and interact with d extrin-2-sulfate ( D2S) [7].
D2S is a sulfated polysaccharide that binds to histone H2B
on the cell surface and there it can inhibit human
immunode®ciency virus type 1 infection [7]. Moreover,
nonacetylated histone H2A was found in the cytoplasm of
the gastric gland c ells of Asian toads, from which the
antibacterial peptide buforin I is derived by pepsin-mediated
cleavage between Tyr39 and Ala40 [10]. Whether the uH1A
fragments d escribed in this paper are derived from the
nucleus, or from another cellular compartment remains to
be established.
ACKNOWLEDGEMENTS
We thank E. Cederlund and C. Palmberg for technical assistance a nd
O
È
. Wrange and J. Lundahl for constructive comments on this
manuscript. This study was supported by the Swedish Medical
Research Council, Hagberg s stiftelse Ane
Â
rs stiftelse, and Magn.
Bergvalls stiftelse, and by the Research funds of the Karolinska
Institutet.
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24
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