Transduced human PEP-1–heat shock protein 27 efficiently
protects against brain ischemic insult
Jae J. An
1,
*, Yeom P. Lee
1,
*, So Y. Kim
1
, Sun H. Lee
1
, Min J. Lee
1
, Min S. Jeong
1
, Dae W. Kim
1
,
Sang H. Jang
1
, Ki-Yeon Yoo
2
, Moo H. Won
2
, Tae-Cheon Kang
2
, Oh-Shin Kwon
3
, Sung-Woo Cho
4
,
Kil S. Lee

Correspondence
S. Y. Choi, Department of Biomedical
Science and Research Institute for
Bioscience and Biotechnology, Hallym
University, Chunchon 200-702, Korea
Fax: +82 33 241 1463
Tel: +82 33 248 2112
E-mail:
W. S. Eum, Department of Biomedical
Science and Research Institute for
Bioscience and Biotechnology, Hallym
University, Chunchon 200-702, Korea
Fax: +82 33 241 1463
Tel: +82 33 248 2112
E-mail:
*These authors contributed equally to this
work
(Received 30 October 2007, revised 10
January 2008, accepted 15 January 2008)
doi:10.1111/j.1742-4658.2008.06291.x
Reactive oxygen species contribute to the development of various human
diseases. Ischemia is characterized by both significant oxidative stress and
characteristic changes in the antioxidant defense mechanism. Heat shock
protein 27 (HSP27) has a potent ability to increase cell survival in response
to oxidative stress. In the present study, we have investigated the protective
effects of PEP-1–HSP27 against cell death and ischemic insults. When
PEP-1–HSP27 fusion protein was added to the culture medium of astrocyte
and primary neuronal cells, it rapidly entered the cells and protected them
against cell death induced by oxidative stress. Immunohistochemical analy-
sis revealed that, when PEP-1–HSP27 fusion protein was intraperitoneally

into living cells. These include carrier peptides derived
from the HIV-1 Tat protein, Drosophila Antennapedia
(Antp) protein and herpes simplex virus VP22 protein
[17]. To increase the biological activity of transduced
proteins in cells, a novel carrier is required to trans-
duce the target protein in its active native structural
form. Morris et al. [18] have designed a PEP-1 peptide
carrier, which consists of three domains: a hydropho-
bic tryptophan-rich motif, a spacer, and a hydrophilic
lysine-rich domain. When they mixed PEP-1 peptide
and a target protein (GFP or b-galactosidase) and then
overlaid them on cultured cells, they found that non-
denatured target protein was transduced.
In a previous study, we have shown that a Tat–
Cu,Zn-superoxide dismutase (SOD) fusion can be
transduced into HeLa cells, and protects the cells from
oxidative stress-induced destruction [19]. PEP-1–SOD
was efficiently transduced into neuronal cells across
the blood–brain barrier and protected against ischemic
insults [20]. Recently, we reported the protective effects
of transduced PEP-1–SOD in neuronal cell death and
paraquat-induced Parkinson’s disease in mice models
[21]. In addition, we demonstrated that the PEP-1–
ribosomal protein S3 (rpS3) fusion protein efficiently
transduces into skin cells ⁄ tissues and protects against
UV-induced skin cell death [22].
In the present study, we designed a PEP-1–HSP27
fusion protein expression vector (Fig. 1) for direct
transduction in vitro and in vivo in its native active
form. The results show that the PEP-1–HSP27 fusion

PEP-1–HSP27
PEP-1–HSP27
Control HSP27
His-Tag
His-Tag
HSP27
HSP27
PEP-1
His-Tag Lac O
T7 Prom
Xho I
Fig. 1. The expression vector for the PEP-1–HSP27 fusion protein.
(A) Construction of the PEP-1–HSP27 expression vector system
based on the vector pET-15b. A synthetic PEP-1 oligomer was
cloned with into the NdeI and XhoI sites, and human HSP27 cDNA
was cloned into the XhoI and BamHI sites of pET-15b. (B) Diagram
of the expressed control HSP27 and PEP-1–HSP27 fusion proteins.
Each contains a His tag consisting of six histidine residues. Expres-
sion was induced by adding isopropyl thio-b-
D-galactoside (IPTG).
J. J. An et al. Protective effects of PEP-1–HSP27 against brain ischemia
FEBS Journal 275 (2008) 1296–1308 ª 2008 The Authors Journal compilation ª 2008 FEBS 1297
analysis using antibody against rabbit polyhistidine
(Fig. 2B).
Transduction of PEP-1–HSP27 fusion protein into
astrocyte and neuronal cells
The intracellular delivery of PEP-1–HSP27 fusion
proteins into astrocytes was confirmed by direct
fluorescence analysis. As shown in Fig. 3A, almost all
cultured cells were found to be transduced with PEP-1–

astrocytes in a concentration-dependent manner.
Figure 4A shows that PEP-1–HSP27 fusion protein
was efficiently transduced into astrocytes in a time-
and dose-dependent manner. However, control HSP27
was not transduced into the cells (data not shown).
We also assessed the transduction of PEP-1–HSP27
fusion protein into primary neuronal cells. As shown
(kDa)
AB
12
150
75
50
37
25
3123
Fig. 2. Expression and purification of the PEP-1–HSP27 fusion
protein. Protein extracts of cells and purified fusion proteins were
analyzed by 12% SDS–PAGE (A) and subjected to western blot
analysis with antibody against rabbit polyhistidine (B). Lane 1,
non-induced PEP-1–HSP27; lane 2, induced PEP-1–HSP27; lane 3,
purified PEP-1–HSP27.
A
a
b
c
d
a
b
c

of transduced HSP27 fusion protein persisted in the
cells for 12 h. The same patterns were obtained when
we used primary neuronal cells (data not shown).
Effect of transduced PEP-1–HSP27 fusion proteins
on the viability of cells under oxidative stress
To determine whether the transduced fusion protein
has a functional role in cells under oxidative stress, we
examined the viability of cells containing transduced
fusion proteins after administration of hydrogen per-
oxide. When cells were exposed to 1.2 mm hydrogen
peroxide, only 35% of the cells were viable. The viabil-
ity of cells pre-treated with PEP-1–HSP27 fusion pro-
teins and then exposed to hydrogen peroxide was
markedly increased up to 95% (Fig. 5).
Next, we examined the effect of PEP-1–HSP27
transduction on DNA fragmentation induced by
hydrogen peroxide. Biological macromolecules are
known to be major targets of oxidative stress. As
shown in Fig. 6, DNA fragmentation was considerably
induced by hydrogen peroxide in astrocytes; however,
the levels of DNA fragmentation were significantly
decreased by transduction of the PEP-1–HSP27 fusion
protein. We also measured cell viability and DNA
fragmentation using hydrogen peroxide in primary
neuronal cells. Transduced PEP-1–HSP27 efficiently
protects the neuronal cell viability (data not shown),
as seen for astrocytes. These results indicate that trans-
duced PEP-1–HSP27 fusion protein plays a defensive
role against cell death induced by oxidative stress in
the cells.

Cell viability (% of control)
100
80
60
40
20
0
C 0.5H
2
O
2
12
*
+
+
+
3(µM)
Fig. 5. Effect of transduced PEP-1–HSP27 on cell viability. Hydro-
gen peroxide (1.2 m
M) was added to astrocytes pretreated with
0.5–3 l
M PEP-1–HSP27 for 1 h. Cell viabilities were estimated using
an MTT colorimetric assay. Each bar represents the mean ± SEM
obtained from five experiments. Asterisks and crosses denote
statistical significance at P < 0.05 and P < 0.01, respectively.
J. J. An et al. Protective effects of PEP-1–HSP27 against brain ischemia
FEBS Journal 275 (2008) 1296–1308 ª 2008 The Authors Journal compilation ª 2008 FEBS 1299
30 min before ischemia. At 4 and 7 days following
ischemic insult, PEP-1–HSP27-treated, vehicle-treated
and sham-operated control animals were killed and the

However, the PEP-1–HSP27-treated group showed
M1 2
3
Fig. 6. Transduced PEP-1–HSP27 fusion protein inhibits stress-
induced DNA damage. Astrocytes were exposed to hydrogen per-
oxide in the absence or presence of 3 l
M PEP-1–HSP27. After
hydrogen peroxide exposure, DNA fragmentation was analyzed by
agarose gel electrophoresis. M represents DNA molecular mass
markers (100 bp DNA ladder). Lane 1, control cells; lane 2, hydro-
gen peroxide-exposed cells; lane 3, PEP-1–HSP27-treated hydrogen
peroxide-exposed cells.
ba
dc
fe
hg
B
A
120
Sham
Vehicle
4 days
7 days
100
80
60
Relative density (%)
40
20
0

and their iba-1 immunoreactivities were markedly
decreased in the CA1 regions (Fig. 10E,G).
Under the same experimental conditions, we
performed Fluoro-Jade B (F-JB) histofluorescence
staining. In the sham-operated control group, no
F-JB-positive neurons were detected in the hippocam-
pal CA1 region (Fig. 10B). F-JB-positive neurons were
abundant in the hippocampal CA1 region 4 days after
ischemic insult because of neuronal death in this region
(Fig. 10D). However, the numbers of F-JB-positive
neurons in the hippocampal CA1 region in the PEP-1–
HSP27-treated group after 4 and 7 days were signifi-
cantly decreased (Fig. 10F,H).
Discussion
Heat shock proteins (HPSs) have very important func-
tions, such as acting as molecular chaperones under
physiological conditions or in response to stress. The
most common inducible HSPs in the nervous system
are HSP70 and HSP27, and they have been shown to
be neuroprotective. In particular, HSP27 belongs to
the family of small heat shock proteins, which protect
against apoptotic cell death triggered by various stim-
uli such as oxidative stress, and increase the anti-
oxidant defense of cells by decreasing the levels of
reactive oxygen species (ROS) [23–25]. HSPs have been
implicated as modulators of disease pathology in many
neurological conditions [10–13]. Moreover, studies
have demonstrated marked differences for each HSP
A
B

unclear. Although HSP27 has been considered as hav-
ing potential as a therapeutic protein, its inability to
enter cells hinders its use for this purpose. Therefore,
in an effort to deliver HSP27 protein to cells and tis-
sues, we investigated the possibility of protein trans-
duction. As the HSP27 has multiple roles, it may be
considered as a potential therapeutic protein against
various neuronal diseases if the protein can be deliv-
ered into cells. Morris et al. [18] have designed a
21-residue peptide carrier, the PEP-1 peptide, that
allows transduction of proteins in their native condi-
tion.
To express the cell-permeable PEP-1–HSP27 protein,
the human HSP27 gene was fused to a PEP-1 peptide
in a bacterial expression vector to produce a genetic
in-frame PEP-1–HSP27 fusion protein. The PEP-1–
HSP27 fusion protein was a major component of the
total soluble proteins in cells, and was found to be
nearly homogeneous and more than 95% pure by
SDS–PAGE analysis. The identity of the expressed
and purified PEP-1–HSP27 fusion proteins was con-
firmed by western blot analysis using an anti-rabbit
polyhistidine antibody.
B
A
iba-1 F-JB
Sham
Vehicle
4 days
7 days

avoided in studies of protein transduction into living
cells [26]. However, in this study, we were unable to
detect any differences in the distribution of the fluores-
cence of transduced PEP-1–HSP27 fusion proteins in
non-fixed and fixed cells. These results demonstrate
that cell fixation with paraformaldehyde is not
required for PEP-1–HSP27 transduction. Similar
observations have been reported indicating that arti-
facts of protein transduction are not induced by para-
formaldehyde fixation [27]. Our previous studies
showed that transduction of PEP-1–SOD and PEP-1–
rpS3 fusion proteins into neuronal and skin cells was
not affected by paraformaldehyde fixation [21,22].
Purified PEP-1–HSP27 fusion proteins were effi-
ciently transduced into astrocytes in a time- and dose-
dependent manner. The fusion protein was transduced
into cells within 10 min, and levels gradually increased
up to 60 min after transduction. Morris et al. [18]
showed that PEP-1 peptide ⁄ green fluorescent protein
(GFP, 30 kDa) or b-Gal (b-galactosidase, 119 kDa)
mixtures can be transduced into a human fibroblast
cell line (HS-68) and into Cos-7 cells by incubation
with a PEP-1 peptide carrier and the GFP or b-Gal
proteins for 30 min at 37 °C. These differences in the
time courses of transduction may depend on whether
the target protein is fused to the PEP-1 vector or
mixed with the PEP-1 peptide. Fusion with the PEP-1
vector may alter the conformation, polarity or molecu-
lar shape of the target protein, improving transduction
of the fusion proteins into cells.

ischemia. At 4 and 7 days following ischemia, the pro-
tective effects of the fusion proteins were confirmed
by immunohistochemistry. The magnitude of the pro-
tective effect of PEP-1–HSP27 fusion protein was
indicated by the 78% and 70% survival of CA1
neurons, respectively, after 4 and 7 days. In addition,
we observed that the PEP-1–HSP27 fusion protein
crossed the blood–brain barrier and the protein levels
significantly increased throughout the brain. Recently,
Cho et al. [30] demonstrated that PEP-1–cargo fusion
proteins can be efficiently delivered into neurons in
the ischemic hippocampus, and that PEP-1–SOD treat-
ment of animals with ischemic damage (induced prior
to treatment) reduces that damage.
Oxidative stress is an important underlying factor in
delayed neuronal death induced by ischemic insult.
Release of ROS and increases in lipid peroxidation can
be detected at a very early stage [8,31,32]. We observed
a significant increase in brain MDA levels, a marker of
lipid peroxidation, 3 h after an ischemic insult, similar
to that reported previously [33]. However, increased
MDA levels were significantly reduced by pretreatment
with transduced PEP-1–HSP27.
Neuronal death induced by injury of the central ner-
vous system causes activation of microglia. It has been
reported that activated microglia contribute to various
neurodegenerative diseases via the production of cyto-
toxic molecules such as free radicals, proinflammatory
prostaglandins and cytokines [34–36]. Ionized calcium-
binding adaptor molecule 1 (iba-1) is a calcium-bind-

ischemic ⁄ reperfusion animal models, and demonstrated
that cell damage is reduced in hippocampus neurons
by approximately 50% in HSP27 transgenic animal
models [39,40]. Recently, Kwon et al. reported that
transduced Tat–HSP27 protein reduces infarct volume
(29.5%) compared with controls (39.1%) in ische-
mic ⁄ reperfusion animals [41]. In addition, Badin et al.
[42] demonstrated that, in animal ischemia models,
herpes simplex virus carrying HSP27 reduced neuronal
cell death by 44%. These results indicate that HSP27
protected against neuronal cell death induced by ische-
mia and stroke.
In summary, we demonstrate here for the first time
that human HSP27 fused with PEP-1 peptide (PEP-1–
HSP27) can be efficiently transduced in vitro and
in vivo in its native conformation. Moreover, PEP-1–
HSP27 fusion protein markedly protected against
stress-induced cell death and ischemic insults.
Although the detailed mechanism remains to be fur-
ther elucidated, our success in protein transduction of
PEP-1–HSP27 may provide a new strategy for protect-
ing against cell destruction resulting from ischemic
damage, and therefore may provide an opportunity for
development of therapeutic agents for the treatment of
various human diseases including stroke.
Experimental procedures
Materials
Restriction endonuclease and T4 DNA ligase were
purchased from Promega Co. (Madison, WI, USA). Oligo-
nucleotides were synthesized from Gibco BRL custom

CATCGGA-3¢, contains a BamHI restriction site. PCR was
performed and the PCR product was excised with XhoI
and BamHI, eluted, ligated into a pPEP-1 vector using
T4 DNA ligase, and transformed into E. coli DH5a cells.
The PEP-1–HSP27 sequences were confirmed by sequence
analysis.
To produce the PEP-1–HSP27 fusion proteins, the plas-
mid was transformed into E. coli BL21 cells. The trans-
formed bacterial cells were grown in 100 mL of LB media
at 37 °CtoaD
600
value of 0.5–1.0 and induced with
0.5 m m IPTG at 37 °C for 4 h. Harvested cells were lysed
by sonication at 4 °C in a binding buffer (5 mm imidazole,
500 mm NaCl, 20 mm Tris ⁄ HCl, pH 7.9). The recombinant
PEP-1–HSP27 was purified by loading clarified cell extracts
onto a Ni
2+
-nitrilotriacetic acid Sepharose affinity column
(Qiagen) under native conditions. After washing the column
with 10 volumes of binding buffer and six volumes of a
wash buffer (25 m m imidazole, 500 mm NaCl, and 20 mm
Tris ⁄ HCl, pH 7.9), the fusion proteins were eluted using an
eluting buffer (0.25 m imidazole, 500 mm NaCl, 20 mm
Tris ⁄ HCl, pH 7.9). The fractions containing the PEP-1–
HSP27 fusion proteins were combined, and salts were
removed using PD-10 column chromatography (Amersham,
Braunschweig, Germany). The protein concentration was
Protective effects of PEP-1–HSP27 against brain ischemia J. J. An et al.
1304 FEBS Journal 275 (2008) 1296–1308 ª 2008 The Authors Journal compilation ª 2008 FEBS

3
, 10% fetal bovine serum and antibiotics
(100 lgÆmL
)1
streptomycin, 100 unitsÆmL
)1
penicillin) at
37 °C under humidified conditions (95% air and 5% CO
2
).
For transduction of PEP-1–HSP27, the primary neuronal
cells and astrocytes were grown to confluence on a 6-well
plate. Then the culture medium was replaced with 1 mL of
fresh solution. After the cells had been treated with various
concentrations of PEP-1–HSP27 for 1 h, the cells were trea-
ted with trypsin ⁄ EDTA (Gibco) and washed with NaCl ⁄ P
i
.
The cells were harvested for the preparation of cell extracts
for western blot analysis.
Fluorescence analysis
For direct detection of fluorescein-labeled protein, purified
PEP-1–HSP27 was labeled using an EZ-Label fluorescein
isothiocyanate (FITC) protein labeling kit (Pierce, Rock-
ford, IL, USA). The FITC labeling was performed
according to the manufacturer’s instructions. Cultured
cells were grown on glass coverslips and treated with
3 lm PEP-1–HSP27 fusion proteins. Following incubation
for 1 h at 37 °C, the cells were washed twice with
NaCl ⁄ P

forebrain ischemia
This study used the progeny of Mongolian gerbils (Meriones
unguiculatus) obtained from the Experiment Animal Center
at Hallym University. The animals were housed at constant
temperature (23 °C) and relative humidity (60%) with a fixed
12 h light ⁄ dark cycle and free access to food and water. Pro-
cedures involving animals and their care conformed to the
institutional guidelines, which are in compliance with current
NIH Guidelines for the Care and Use of Laboratory Ani-
mals, and were approved by the Hallym Medical Center
Institutional Animal Care and Use Committee.
Male Mongolian gerbils weighing 65–75 g were placed
under general anesthesia using a mixture of 2.5% isoflurane
(Abbott Laboratories, Abbott Park, IL, USA) in 33%
oxygen and 67% nitrous oxide. To determine whether
transduced PEP-1–HSP27 protects from ischemic damage,
gerbils were intraperitoneally injected with PEP-1–HSP27
fusion protein (2 mgÆkg
)1
) 30 min before occlusion of com-
mon carotid arteries. A midline ventral incision was made
in the neck. The common carotid arteries were isolated,
freed of nerve fibers, and occluded with non-traumatic
aneurysm clips. Complete interruption of blood flow was
confirmed by observing the central artery in the eyeball
using an ophthalmoscope. After 5 min occlusion, the
aneurysm clips were removed. Restoration of blood flow
J. J. An et al. Protective effects of PEP-1–HSP27 against brain ischemia
FEBS Journal 275 (2008) 1296–1308 ª 2008 The Authors Journal compilation ª 2008 FEBS 1305
(reperfusion) was observed directly under the ophthalmo-

Lipid peroxidation was measured according to the method
described by Zhang et al. [46]. An aliquot (100 lL) of brain
supernatant was added to a reaction mixture containing
100 lL of 8.1% SDS, 750 lL of 20% acetic acid (pH 3.5),
750 lL of 0.8% thiobarbituric acid and 300 lL distilled
water. Samples were then boiled for 1 h (95 ° C) and centri-
fuged at 4000 g for 10 min. The absorbance of the superna-
tant was measured by spectrophotometry at 532 nm.
Fluoro-Jade B histofluorescence staining
To examine the effect of PEP-1–HSP27 on ischemic dam-
age, the sections were stained using F-JB, a marker for neu-
rodegeneration [47]. The sections were first immersed in a
solution containing 1% sodium hydroxide in 80% alcohol,
followed by a solution with 70% alcohol. They were then
transferred to a solution of 0.06% potassium permanga-
nate, and then to a 0.0004% F-JB staining solution (Histo-
chem, Jefferson, AR, USA). After washing, the sections
were placed on a slide warmer, and examined using an epi-
fluorescent microscope (Carl Zeiss). Using this method,
neurons that undergo degeneration are brightly stained in
comparison to the background [48].
Immunohistochemistry for iba-1
To confirm the neuroprotective effects and reactive gliosis
after PEP-1–HSP27 treatment, immunohistochemistry was
performed according to the method previously described
[49]. The sections were incubated with rabbit anti-ionized
calcium-binding adapter molecule 1 (iba-1) (1 : 500; Wako,
Osaka, Japan) and subsequently exposed to biotinylated
goat anti-rabbit IgG and streptavidine peroxidase complex
(diluted 1 : 200; Vector (Burlingame, CA, USA)). They

Biology and Medicine. Oxford University Press, Oxford.
3 Dore S, Otsuka T, Mito T, Sugo N, Hand T, Wu L,
Hurn PD, Traystman RJ & Andreasson K (2003) Neu-
ronal overexpression of cyclooxygenase-2 increases cere-
bral infarction. Ann Neurol 54, 155–162.
4 Petito CK, Torres-Munoz J, Roberts B, Olarte JP,
Nowak TS Jr & Pulsinelli WA (1997) DNA
Protective effects of PEP-1–HSP27 against brain ischemia J. J. An et al.
1306 FEBS Journal 275 (2008) 1296–1308 ª 2008 The Authors Journal compilation ª 2008 FEBS
fragmentation follows delayed neuronal death in CA1
neurons exposed to transient global ischemia in the rat.
J Cereb Blood Flow Metab 17, 967–976.
5 Frantseva MV, Carlen PL & Perez Velazquez JL (2001)
Dynamics of intracellular calcium and free radical
production during ischemia in pyramidal neurons. Free
Radic Biol Med 31, 1216–1227.
6 Numagami Y, Sato S & Ohnishi ST (1996) Attenuation
of rat ischemic brain damage by aged garlic extracts: a
possible protecting mechanism as antioxidants. Neuro-
chem Int 29, 135–143.
7 Won MH, Kang TC, Jeon GS, Lee JC, Kim DY, Choi
EM, Lee KH, Choi CD, Chung MH & Cho SS (1999)
Immunohistochemical detection of oxidative DNA dam-
age induced by ischemic–reperfusion insults in gerbil
hippocampus in vivo. Brain Res 836, 70–78.
8 Chan PH (2001) Reactive oxygen radicals in signaling
and damage in the ischemic brain. J Cereb Blood Flow
Metab 21, 2–14.
9 Yenari MA, Giffard RG, Sapolsky RM & Steinberg
GK (1999) The neuroprotective potential of heat shock

active proteins into mammalian cells. Nat Biotechnol 19,
1173–1176.
19 Kwon HY, Eum WS, Jang HW, Kang JH, Ryu J, Lee
BR, Jin LH, Park J & Choi SY (2000) Transduction of
Cu,Zn-superoxide dismutase mediated by an HIV-1 Tat
protein basic domain into mammalian cells. FEBS Lett
485, 163–167.
20 Eum WS, Kim DW, Hwang IK, Yoo KY, Kang TC,
Jang SH, Choi HS, Choi SH, Kim YH, Kim SY et al.
(2004) In vivo protein transduction: biologically active
intact PEP-1–superoxide dismutase fusion protein effi-
ciently protects against ischemic insult. Free Radic Biol
Med 37, 1656–1669.
21 Choi HS, An JJ, Kim SY, Lee SH, Kim DW, Yoo KY,
Won MH, Kang TC, Kwon HJ, Kang JH et al. (2006)
PEP-1–SOD fusion protein efficiently protects against
paraquat-induced dopaminergic neuron damage in a
Parkinson disease mouse model. Free Radic Biol Med
41, 1058–1068.
22 Choi SH, Kim SY, An JJ, Lee SH, Kim DW, Ryu HJ,
Lee NI, Yeo SI, Jang SH, Won MH et al. (2006)
Human PEP-1–ribosomal protein S3 protects against
UV-induced skin cell death. FEBS Lett 580, 6755–
6762.
23 Jakob U & Buchner J (1994) Assisting spontaneity: the
role of HSP90 and small HSPs as molecular chaper-
ones. Trends Biochem Sci 19, 205–211.
24 Kiang JG & Tsokos GC (1998) Heat shock protein
70 kDa: molecular biology, biochemistry, and physiol-
ogy. Pharmacol Ther 80, 183–201.

FEBS Journal 275 (2008) 1296–1308 ª 2008 The Authors Journal compilation ª 2008 FEBS 1307
damage in different brain regions following transient
cerebral ischemia in gerbils. Neurosci Res 41, 233–241.
32 Wang Q, Sun AY, Simonyi A, Jensen MD, Shelat PB,
Rottinghaus GE, MacDonald RS, Miller DK, Lubahn
DE, Weisman GA et al. (2005) Neuroprotective mecha-
nisms of curcumin against cerebral ischemia-induced
neuronal apoptosis and behavioral deficits. J Neurosci
Res 82, 138–148.
33 Sharma SS, Dhar A & Kaundal RK (2007) FeTPPS
protects against global cerebral ischemic–reperfusion
injury in gerbils. Pharmacol Res 55, 335–342.
34 Kreutzberg GW (1996) Microglia: a sensor for patho-
logical events in the CNS. Trends Neurosci 19, 312–318.
35 Araham H, Losonczy A, Czeh G & Lazar G (2001)
Rapid activation of microglial cells by hypoxia, kainic
acid, and potassium ions in slice preparations of the rat
hippocampus. Brain Res 906, 115–126.
36 Teismann P & Schulz JB (2004) Cellular pathology of
Parkinson’s disease: astrocytes, microglia and inflamma-
tion. Cell Tissue Res 318, 149–161.
37 Kato H, Tanaka S, Oikawa T, Koike T, Takahashi A
& Itoyama Y (2000) Expression of microglial response
factor-1 in microglia and macrophages following cere-
bral ischemia in the rat. Brain Res 882, 206–211.
38 Ito D, Tanaka K, Suzuki S, Dembo T & Fukuuchi Y
(2001) Enhanced expression of Iba1, ionized calcium-
binding adapter molecule 1, after transient focal cere-
bral ischemia in rat brain. Stroke 32, 1208–1215.
39 Brar BK, Stephanou A, Wagstaff MJ, Coffin RS, Mar-

M, Noguchi T et al. (1996) Cytokine-induced apoptotic
cell death in a mouse pancreatic beta cell line: inhibition
by Bcl-2. Diabetologia 39, 530–536.
46 Zhang DL, Zhang YT, Yin JJ & Zhao BL (2004) Oral
administration of Crataegus flavonoids protects against
ischemia ⁄ reperfusion brain damage in gerbils. J Neuro-
chem 90
, 211–219.
47 Candelario-Jalil E, Alvarez D, Merino N & Leon OS
(2003) Delayed treatment with nimesulide reduces mea-
sures of oxidative stress following global ischemic brain
injury in gerbils. Neurosci Res 47, 245–253.
48 Schmued LC & Hopkins KJ (2000) Fluoro-Jade B: a
high affinity fluorescent marker for the localization of
neuronal degeneration. Brain Res 874, 123–130.
49 Hwang IK, Yoo KY, Kim DW, Choi SY, Kang TC,
Kim YS & Won MH (2006) Ionized calcium-binding
adapter molecule 1 immunoreactive cells change in the
gerbil hippocampal CA1 region after ischemia ⁄ reperfu-
sion. Neurochem Res 31, 957–965.
Protective effects of PEP-1–HSP27 against brain ischemia J. J. An et al.
1308 FEBS Journal 275 (2008) 1296–1308 ª 2008 The Authors Journal compilation ª 2008 FEBS