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J Physiol 589.10 (2011) pp 2585–2596
The Journal of Physiology
Dehydroepiandrosterone protects against oxidative
stress-induced endothelial dysfunction in ovariectomized
Jo˜ ao Paulo Gabriel Camporez1, Eliana Hiromi Akamine2, Ana Paula Davel1,3, Celso Rodrigues Franci4,
Luciana Venturini Rossoni1and Carla Roberta de Oliveira Carvalho1
1Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of S˜ ao Paulo, SP, Brazil
2Department of Pharmacology, Institute of Biomedical Sciences, University of S˜ ao Paulo, SP, Brazil
3Department of Anatomy, Cellular Biology and Physiology & Biophysics, State University of Campinas, SP, Brazil
4Department of Physiology, Medical School of Riber˜ ao Preto, University of S˜ ao Paulo, SP, Brazil
Non-technical summary
in postmenopausal than in premenopausal women. Moreover, it has been shown that
dehydroepiandrosterone (DHEA), a steroid hormone secreted by adrenal glands, reduces during
ageing. Its reduced plasma level has been related to increased prevalence of obesity, insulin
resistance and cardiovascular disease. We show that DHEA treatment in ovariectomized rats,
an experimental model of menopause, reduces blood pressure and improves vascular function.
Furthermore, DHEA reduced reactive oxygen species (ROS), correcting the reduced protein
expression of Cu/Zn-SOD, an antioxidant protein, and increased protein expression of NADPH
oxidase, a pro-oxidant protein. This work shows the potential effect of DHEA upon correction of
endothelial dysfunction observed on oestrogen deprivation.
It is well known that cardiovascular disease is more frequent
Abstract Cardiovascular disease is less frequent in premenopausal women than in age-matched
men or postmenopausal women. Moreover, the marked age-related decline in serum
dehydroepiandrosterone (DHEA) level has been associated to cardiovascular disease. The aim of
this study was to evaluate the effects of DHEA treatment on vascular function in ovariectomized
and 8weeks after surgery both groups were treated with vehicle or DHEA (10mgkg−1week−1)
for 3weeks. Aortic rings were used to evaluate the vasoconstrictor response to phenylephrine
(PHE) and the relaxation responses to acetylcholine (ACh) and sodium nitroprusside (SNP).
Tissue reactive oxygen species (ROS) production and SOD, NADPH oxidase and eNOS protein
expression were analysed. PHE-induced contraction was increased in aortic rings from OVX
compared to SHAM, associated with a reduction in NO bioavailability. Furthermore, the
relaxation induced by ACh was reduced in arteries from OVX, while SNP relaxation did
not change. The incubation of aortic rings with SOD or apocynin restored the enhanced
PHE-contraction and the impaired ACh-relaxation only in OVX. DHEA treatment corrected
the increased PHE contraction and the impaired ACh-induced relaxation observed in OVX by
an increment in NO bioavailability and decrease in ROS production. Besides, DHEA treatment
NADPH oxidase protein expression in the aorta of OVX rats. The present results suggest an
important action of DHEA, improving endothelial function in OVX rats by acting as an anti-
oxidant and enhancing the NO bioavailability.
(Received 24 November 2010; accepted after revision 11 March 2011; first published online 14 March 2011)
Corresponding author C. R. O. Carvalho: Department of Physiology and Biophysics, Institute of Biomedical Sciences,
University of S˜ ao Paulo, 05508-900 S˜ ao Paulo, S.P., Brazil. Email: email@example.com
Abbreviations ACh, acetylcholine; DHEA, dehydroepiandrosterone; NO, nitric oxide; ORT, oestrogen replacement
therapy; OVX, ovariectomized rats; PHE, phenylephrine; ROS, reactive oxygen species; SNP, sodium nitroprusside.
C ?2011 The Authors. Journal compilation C ?2011 The Physiological SocietyDOI: 10.1113/jphysiol.2011.206078
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2586 J. P. G. Camporez and others
J Physiol 589.10
It is known that oestrogen has profound effects on
the cardiovascular system (White, 2002). Indeed, cardio-
vascular disease is much less frequent in premenopausal
women than in age-matched men or postmenopausal
women (Burt et al. 1995).
Endothelial dysfunction is a common factor in
the development of many cardiovascular diseases. In
line with this, endothelial function was shown to be
impaired in ovariectomized women (Virdis et al. 2000)
and hypertensive female rats (Wassmann et al. 2001;
Widder et al. 2003). Indeed, it has been observed that
ovariectomy reduces vascular response to acetylcholine in
Sprague–Dawley rats (Squadrito et al. 2000; Lam et al.
2006), female hypertensive rats (Wassmann et al. 2001;
Widder et al. 2003), and Wistar rats (Paredes-Cabejal
et al. 1995). Ovariectomy also increases vascular response
to phenylephrine in Sprague–Dawley rats (Lam et al.
2006), and Wistar rats (Paredes-Cabejal et al. 1995). This
increment in superoxide anion production (Wassmann
et al. 2001; Lam et al. 2006) as well as a reduction in
eNOS protein expression (Widder et al. 2003). Moreover,
ovariectomy promoted an increment in blood pressure of
normotensive women (Mercuro et al. 2004), as well as in
female Wistar rats (Mendonc ¸a et al. 2007), and in hyper-
tensive rats (Ito et al. 2006).
Oestrogen replacement
ovariectomized womenand
the deleterious effect of ovariectomy by reducing
blood pressure and increasing antioxidant defence and
eNOS protein expression. However, large controlled
clinical trials have not shown benefits of ORT, or
have even shown deleterious effects, such as an
increment of venous thromboembolic disease, stroke
and breast cancer incidence (Prentice et al. 2009).
oestrogen deprivation-induced deleterious effects on the
cardiovascular system will be valuable.
Dehydroepiandrosterone (DHEA) has been considered
a potential alternative hormone for ORT, due to its
apparent safe, antioxidant and metabolic effects (Labrie
et al. 2005). DHEA and its sulfated form (DHEAS) are the
most abundant cholesterol-derived hormones in humans
and their plasma concentrations progressively decline
with age. An inverse relation between plasma DHEA
concentration and obesity, diabetes and incidence of
et al. 1986; Nestler et al. 1988; Schriock et al. 1988).
Moreover, DHEA treatment protects heart of diabetic rats
protects endothelial cells against apoptosis (Liu et al.
2007), and stimulates ser1177 eNOS phosphorylation
(Formoso et al. 2006).
endothelial dysfunction and high blood pressure levels
(Wassmann et al. 2001; Widder et al. 2003). Therefore,
DHEA evokes beneficial mechanisms that could reduce
endothelial dysfunction and reduce the cardiovascular
risk present after surgical menopause in rats. Thus,
the present work hypothesizes that DHEA could be an
alternative hormonal therapy, and provide benefits to the
vascular system of postmenopausal women. To evaluate
this hypothesis, the aim of the present study was to assess
relaxation, focusing on the mechanisms involved, mainly
related to NO and oxidative stress, in an experimental
menopause models in rats.
Animal care and protocol
All experimental procedures were approved by and
conducted in accordance with the guidelines of the
Animal Care and Use Committee of the Biomedical
Institute of Sao Paulo University, according to the
guidelines of the Brazilian Societies of Experimental
obtained from colonies maintained at the Institute of
Biomedical Sciences of the University of S˜ ao Paulo.
At 8weeks of age, the rats were anaesthetized with a
ketamine–xylazine–acepromazine mixture (64.9, 3.2 and
0.78mgkg−1, respectively, I.P.) and were ovariectomized
(OVX) or sham-operated (SHAM). After 8weeks of
surgery, OVX and SHAM rats were treated with vehicle
(soybean oil) or DHEA (10mgkg−1week−1, S.C.) for
3weeks. The DHEA administration protocol was based
on studies published by us and others (Campbell et al.
2004; Medina et al. 2006; Jacob et al. 2009). During
all the procedures rats were housed in a constant room
temperature environment, with 12:12h light–dark cycle,
with free access to standard rat chow and tap water. Rats
were randomly divided into four groups: (1) SHAM, (2)
SHAM+DHEA, (3) OVX, and (4) OVX+DHEA.
female Wistarratswere
Biometrics characteristics
At the end of the treatment period, the body weight
gain, retroperitoneal fat pad and uterus weight were
Arterial blood pressure measurement
Rats were anaesthetized with a ketamine–xylazine–
acepromazine mixture (64.9, 3.2 and 0.78mgkg−1,
I.P.) and allowed to breathe room air
spontaneously. The right carotid artery was cannulated
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Dehydroepiandrosterone reduces reactive oxygen species in aorta 2587
with a polyethylene catheter (PE-50 with heparinized
saline) that was exteriorized in the mid-scapular region.
After 24h, arterial pressure and heart rate were measured
021, PanLab, Barcelona, Spain) and recorded by using
an interface and software for computer data acquisition
(Power Lab 4/25; ADInstruments, Sydney, Australia).
Heart rate was determined from the interbeat intervals.
Vascular reactivity study
Vascular function was evaluated in aortic rings from all
groups studied. We used the aortic rings as a model to
study vascular function because in this artery the main
endothelium-dependent vasodilator factor is NO. Thus,
since the aim of the present study is to assess the role of
NO and reactive oxygen species on the DHEA treatment
in OVX rats, the aortic rings are the best model to answer
our questions without misinterpretation. Segments of the
thoracic aorta (4mm in length), free of connective tissue,
were mounted in an isolated tissue chamber containing
Krebs–Henseleit solution (containing (in mM): 118 NaCl,
4.7 KCl, 25 NaHCO3, 2.5 CaCl2.2H2O, 1.2 KH2PO4, 1.2
MgSO4.7H2O, 11 glucose, and 0.01 EDTA), gassed with
95% O2–5% CO2, and maintained resting tension of 1g
at 37◦C at pH 7.4 as previously published by our group
(Davel et al. 2008). In order to analyse the influence of
the endothelium on vascular responses, the endothelial
rubbing the lumen with a needle. Isometric tension was
recorded by using an isometric force transducer (Letica
TRI 210, Barcelona, Spain) connected to an acquisition
system (MP100, Biopac Systems, Goleta, CA, USA).
After 30min equilibration, concentration–response
curves to the α1-adrenoceptor agonist phenylephrine
(PHE, 10−10–10−4moll−1),
10−11–10−5moll−1) and to the nitric oxide donor
sodium nitroprusside (SNP, 10−11–10−5moll−1) were
obtained. At the end of the experiment, the presence
or absence of functional endothelium was verified in all
aortic rings by observing whether relaxation occurred on
exposure to ACh (10−5moll−1) or not. The endothelium
was considered intact if the aortic ring relaxed more than
by less than 10% relaxation. At the end of experiments,
aortic rings were allowed to dry for at least 24h and
then were weighed. Vasoconstrictor responses to PHE are
expressed as gram (g) of tension per milligram (mg) of
tissue. Vasodilator concentration–response curves were
obtained in the aortic rings pre-contracted with PHE
(∼10−6moll−1), which is approximately 80% of maximal
contraction. Relaxation induced by ACh and SNP was
expressed as the percentage of the tonus obtained with
the endothelium-
The role of NO and superoxide anion in the
response to PHE and ACh was evaluated by incubating
someaortic ringswith
inhibitor L-NG-nitro-L-arginine methyl ester (L-NAME;
dismutase (SOD; 150Uml−1) or the inhibitor of NADPH
oxidase, apocynin (100μmoll−1), added 30min before
the construction of the concentration–response curves to
PHE or ACh.
For each concentration–response curve to PHE and
ACh, the maximal response (Rmax) and the agonist
concentration log resulting in 50% of the Rmax (log
(GraphPad Prism software, USA).
thenon-selective NOS
Reactive oxygen species measurement in aorta
Thoracic aortas were carefully dissected out and cleaned
of connective tissue. Aortas were then divided into
cylindrical segments 4mm in length and were first
immersed in an embedding medium (tissue freezing
medium) and then frozen and kept at −80◦C until super-
oxide anion was measured. The oxidative fluorescent
dye hydroethidine was used to evaluate the in situ
production of reactive oxygen species (ROS) as pre-
viously described (Hernanz et al. 2004). Transverse aortic
sections (10μm) were obtained in a cryostat from
previously frozen aorta and collected on glass slides
and left to reach equilibrium for 30min at 37◦C in
phosphate-buffered saline (PBS). Fresh buffer containing
hydroethidine (5μmoll−1) was topically applied to each
tissue section and coverslipped. Slides were incubated
in a light-protected, humidified chamber at 37◦C for
30min. Control sections received the same volume of
PBS. Images were obtained with a Nikon E1000 micro-
scope equipped for epifluorescence (excitation at 488nm;
emission at 610nm). Fluorescence was detected with a
585nm long-pass filter. The fluorescence intensity was
as arbitrary units.
Western blot analysis
For analysis of eNOS, ser1177 phosphorylated eNOS,
nitrogen and kept at −70◦C until the day of analysis.
They were homogenized in RIPA lysis buffer containing
protease inhibitor cocktail (1:5000 dilution), sodium
fluoride (100mM), sodium pyrophosphate (10mM),
sodium orthovanadate (100mM), and PMSF (10mM).
Proteins from homogenized aorta (75μg of protein
extracts) were electrophoretically separated by 7.5% or
12% SDS-PAGE and then transferred to polyvinylidene
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2588J. P. G. Camporez and others
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Table 1. Body weight gain, retroperitoneal fat pad and uterus weight from ovariectomized
(OVX) or sham-operated (SHAM) rats treated with vehicle or DHEA for 3 weeks
49.2 ± 2.6
0.6 ± 0.06
79.3 ± 4.5∗
1.1 ± 0.10∗
Body weight gain (g)
Retroperitoneal fat pad
(mg (100 g BW)−1)
Uterus weight (mg)
47.8 ± 3.0
0.5 ± 0.04
83.3 ± 4.3∗
1.1 ± 0.09∗
613 ± 103 594 ± 50176 ± 14∗
178 ± 24∗
The values are expressed as means ± SEM. Two-way ANOVA:∗P < 0.01 in comparison to
SHAM and SHAM+DHEA.
Table 2. Changes in systolic (SBP) and diastolic blood pressure (DBP) and in heart
ratefromovariectomized (OVX)orsham-operated (SHAM)ratstreated withvehicle
or DHEA for 3 weeks
130.5 ± 2.6
90.2 ± 2.6
365.2 ± 14.6
128.4 ± 2.7‡
94.0 ± 1.6‡
354.5 ± 10.0
SBP (mmHg)
DBP (mmHg)
Heart rate (bpm)
129.2 ± 2.5
91.5 ± 1.0
356.8 ± 9.1
139.6 ± 1.8∗
100.2 ± 2.3∗
384.0 ± 11.9
The values are expressed as mean ± SEM. Two-way ANOVA:∗P < 0.05 in comparison
to SHAM and ‡P < 0.05 in comparison to OVX.
difluoride membranes overnight at 4◦C using a Mini
Trans-Blot Cell system (Bio-Rad) containing 25mM Tris,
190mM glycine, 20% methanol and 0.05% SDS.
After blockade of non-specific sites with 5% non-fat
dry milk, membranes were incubated overnight at
4◦C with the following primary antibodies: anti-eNOS
(1:1000, BD Transduction Laboratories, Lexington, KY,
USA), anti-phospho eNOSser1177(1:1000, Cell Signaling
Technology, Inc., Danvers, MA, USA), anti-Cu/Zn
SOD (1:2000, Sigma-Aldrich, Germany), anti-Mn SOD
(1:2000, Sigma-Aldrich), anti-gp91phox (1:1000, Upstate
Biotechnology, Inc., Lake Placid, NY, USA) and
anti-p22phox (1:500, Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA, USA). After washing (10mM Tris,
100mM NaCl, and 0.1% Tween 20), membranes
were incubated with horseradish peroxidase-conjugated
anti-rabbit IgG antibody (1:1500, Bio-Rad Laboratories,
Hercules, CA, USA) for gp91phox, p22phox, Mn-SOD
and phopho eNOS, and with anti-mouse IgG (1:1500,
Bio-Rad) for Cu/Zn-SOD and eNOS. The same
membranes were used to determine α-actin protein
expression using a mouse monoclonal antibody (1:1500,
Sigma-Aldrich). Membranes were thoroughly washed,
enhanced luminol chemiluminescence system (ECLPlus,
Amersham GE Healthcare, UK) and subjected to
MF-Chemibis-Bio-Imageing System (BioAmerica Inc.,
Miami, FL, USA). Signals on the immunoblot were
quantified with Scion Image software.
Total expression of the proteins is given as the ratio
between the optical density for the specific protein and
the signal for α-actin protein expression.
weredetected using an
Dehydroepiandrosterone, phenylephrine hydrochloride,
acetylcholine hydrochloride, sodium nitroprusside, SOD
(bovine erythrocyte),
L-NAME and apocynin were
purchased from Sigma-Aldrich (USA); stock solutions
(10mmoll−1) were prepared in distilled water, except for
Statistical analysis
Results were expressed as means±SEM, and n represents
the numbers of animals used in each experiment. All
results were analysed by two-way ANOVA followed by
the Bonferroni post hoc test. A value of P <0.05 was
considered significant.
Biometrics and haemodynamic parameters
OVX rats displayed elevated body weight gain (SHAM:
47.8±3.0 vs. OVX: 83.3±4.3g; n=10, P <0.01)
and retroperitoneal fat pad (SHAM: 0.5±0.04 vs.
OVX: 1.1±0.09mg (100g BW)−1; n=10, P <0.01)
(Table1). However, they presented reduced uterus
weight (SHAM: 613±103 vs. OVX: 176±14mg;
n=10, P <0.01) as compared to SHAM rats (Table1).
DHEA treatment did not modify these parameters;
body weight gain (SHAM+DHEA: 49.2±2.6 and
OVX+DHEA: 79.3±4.5g; n=10), retroperitoneal fat
pad (SHAM+DHEA: 0.6±0.06 and OVX+DHEA:
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Dehydroepiandrosterone reduces reactive oxygen species in aorta2589
weight (SHAM+DHEA: 594±50 and OVX+DHEA:
178±24mg; n=10), compared to respective groups
treated with vehicle (Table1).
Table2 shows that OVX rats presented higher systolic
and diastolic blood pressure as compared to SHAM rats,
while heart rate was not different in different groups.
in both groups (Table2).
Vascular reactivity
The contraction induced by PHE was increased in aorta
from OVX rats compared with SHAM (Fig.1A) (Rmax:
SHAM 0.84±0.03 vs. OVX 1.27±0.07g of tension per
mg of tissue; n=11, P <0.01). On the other hand, the
endothelium-dependent relaxation induced by ACh was
reduced in aorta from OVX rats (Fig.1B) (Rmax: SHAM
91.6±2.7 vs. OVX 74.1±2.7% of relaxation; n=9,
P <0.01), while there was no change in the relaxation
in OVX rats corrected both the increased PHE-induced
contraction (Fig.1A) (Rmax: OVX+DHEA 0.90±0.04g
of tension per mg of tissue; n=11) and the impaired
ACh-induced relaxation (Fig.1B) (Rmax: OVX+DHEA
90.3±2.4% of relaxation; n=9); while DHEA treatment
in SHAM rats did not change either PHE-induced
contraction(Fig.1A) (Rmax: SHAM+DHEA 0.86±0.06g
of tension per mg of tissue; n=11) or ACh-induced
relaxation; n=9). There were no changes in EC50values
among groups for each drug studied (data not shown).
Effects of DHEA treatment on the endothelial and NO
modulation of phenylephrine-induced contraction
in OVX rats
Endothelium and NO modulation of PHE-induced
contraction were evaluated by endothelium denudation
or L-NAME incubation, respectively. An increase in Rmax
to PHE was observed in all groups of animals after
endothelium removal (Table3) or L-NAME incubation
(Table3). It is important to note that in the absence of
(L-NAME) there were no differences among groups. EC50
values were similar in all conditions (data not shown).
Effects of DHEA treatment on the role of superoxide
anion in the phenylephrine-induced contraction
in OVX rats
To evaluate the possible role of superoxide anion
modulation, derivedfrom
PHE-induced contraction, SOD and apocynin were
incubated in some aortic rings 30min before the
concentration–response curves to PHE. Both SOD and
apocynin significantly reduced the Rmaxto PHE in aorta
from OVX rats, whereas they did not have any effects in
Figure 1. Vascular reactivity to phenylephrine, acetylcholine
and sodium nitroprusside
Concentration–response curve to phenylephrine (A), acetylcholine
(B) and sodium nitroprusside (C) in endothelium-intact aortic rings
from SHAM, OVX and DHEA-treated rats. Results are expressed as
means ± SEM. Two-way ANOVA:∗P < 0.01 in comparison to SHAM
(n = 9–13).
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2590J. P. G. Camporez and others
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Table 3. Effect of endothelium denudation (E−), NG-nitro-L-arginine methyl
ester (L-NAME), SOD and apocynin (Apo) on the maximal response (Rmax) to
0.86 ± 0.06
2.71 ± 0.25‡
2.37 ± 0.07‡
0.80 ± 0.02
0.74 ± 0.09
as mean ± SEM
0.90 ± 0.04†
2.66 ± 0.16‡
2.42 ± 0.10‡
0.84 ± 0.04
0.82 ± 0.07
0.84 ± 0.03
2.69 ± 0.17‡
2.53 ± 0.13‡
0.84 ± 0.05
0.85 ± 0.09
†P < 0.01 in comparison to OVX, ‡P < 0.01 in comparison to E+
(endothelium intact).
1.27 ± 0.07∗
2.54 ± 0.10‡
2.33 ± 0.11‡
0.90 ± 0.02‡
0.88 ± 0.11‡
expressed ofper mg
∗P < 0.01toSHAM,
Table 4. Effect of SOD and apocynin (Apo) on the maximal
response (Rmax) to acetylcholine
95.7 ± 3.7
95.7 ± 1.6
94.4 ± 1.6
90.3 ± 2.4†
94.9 ± 1.6
90.5 ± 2.3
91.6 ± 2.7
93.4 ± 2.5
92.5 ± 3.9
74.1 ± 2.7∗
96.5 ± 2.2‡
93.4 ± 1.7‡
The values are expressed as mean ± SEM (% of relaxation).
Two-way ANOVA:∗P < 0.01 in comparison to SHAM, †P < 0.01
in comparison to OVX, ‡P < 0.01 in comparison to E+rings (end-
othelium intact).
(data not shown).
Effects of DHEA treatment on the role of superoxide
anion in the acetylcholine-induced relaxation
in OVX rats
The involvement of superoxide anion in ACh-induced
relaxation was also evaluated. Both SOD and apocynin
significantly enhanced the Rmax to ACh in aortic rings
from OVX rats, whereas they did not have any effect in
aorta from SHAM and DHEA-treated rats (Table4). In
addition, there were no differences among groups in EC50
to ACh in the presence of SOD or apocynin (data not
Reactive oxygen species production
In order to evaluate tissue reactive oxygen species
(ROS) production, dihydroethidium (DHE) staining
wasperformedin aorta.
higher in aorta from OVX rats than in SHAM, indicating
for 3weeks did not change ROS production in aorta
Asobserved inFig.2
production in aorta from OVX rats (Fig.2).
Western blot analysis of endothelial nitric oxide
synthase, superoxide dismutase and subunits
of NADPH oxidase
Ovariectomy or DHEA treatment did not change end-
to SHAM (Fig.3B). Furthermore, DHEA treatment in
OVX rats corrected this reduction (Fig.3B).
In addition, Cu/Zn-SOD protein expression was
reduced in aorta from OVX rats and corrected by
DHEA treatment (Fig.4A), while there was no change
in Mn-SOD protein expression among groups (Fig.4B).
Besides reduction of Cu/Zn-SOD protein expression, the
aorta from OVX rats displayed an increased protein
expression of gp91phox and p22phox (Fig.5) NADPH
corrected the increased of both NADPH oxidase subunits
in aorta (Fig.5).
It has been shown that oestrogens have protective
effects on the vascular system (White, 2002), based
on epidemiological, clinical and molecular findings. In
line with this affirmation, the loss of oestrogens may
have profound effects on the cardiovascular system, with
increase in blood pressure and endothelial dysfunction,
in experimental models of menopause (Wassmann et al.
against oxidative stress-induced endothelial dysfunction
in ovariectomized rats.
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Dehydroepiandrosterone reduces reactive oxygen species in aorta2591
The favourable biological effects of oestrogen in the
cardiovascular system have been recognized, in spite
of the fact that several randomized clinical trials have
failed to observe a benefit of hormone replacement
therapy on the incidence of cardiovascular events in post-
menopausal women (Hulley et al. 1998; Rossouw et al.
2002; Prentice et al. 2009). On the other hand, oestrogen
endothelial dysfunction (Virdis et al. 2000) and increased
blood pressure (Mercuro et al. 2004). It is well known
that ovariectomized rats display endothelial dysfunction,
showing reduction in vascular response to ACh and
increase in vascular response to PHE (Wassmann et al.
2001; Wong et al. 2006). Furthermore, Mendonc ¸a et al.
(2007) found increased blood pressure after 11weeks of
seems to be important not only for females, since
response to ACh (Kimura et al. 2003). In line with
those observations, our study shows an enhancement in
blood pressure, increased vascular contraction to PHE
and reduced endothelium-dependent relaxation to ACh
following ovariectomy in rats.
Supporting the hypothesis of the present study, 3weeks
of DHEA treatment reduced the systolic and diastolic
blood pressure, and corrected vascular responses to PHE
and ACh in ovariectomized rats. The plasma DHEA level
reduces during the ageing process and this has been
related to the prevalence of body weight gain, obesity
and insulin resistance, and mortality by cardiovascular
disease (Barrett-Connor et al. 1986; Nestler et al. 1988;
Schriock et al. 1988). Both aged men and aged women
submitted to DHEA treatment for 6months displayed
insulin sensitivity (Villareal & Holloszy, 2004). Moreover,
in a double-blind study, DHEA-treated postmenopausal
women for 12weeks showed an enhancement of end-
othelial function (Williams et al. 2004). Considering that
the adrenal steroid DHEA is the unique sexual steroid
hormone source in postmenopausal women (Labrie,
2010) and the risks associated with ORT (Prentice et al.
2009), the data presentedhere concerningthe mechanism
of DHEA action suggest that DHEA could be used as a
successful alternative hormone replacement therapy for
postmenopausal women.
Indeed, somestudies
demonstrated that DHEA treatment induces several
rats (Aragno et al. 2008), delay atherosclerotic plaque
formation in rabbits (Hayashi et al. 2000), reduce weight
gain and fat deposition in rats (Han et al. 1998), increase
insulin sensitivity (Campbell et al. 2004) and insulin
secretion in rats (Medina et al. 2006), and reduce blood
pressure in ovariectomized rats (Bhuiyan & Fukunaga,
2010). In addition, DHEA increases in vitro endothelial
proliferation (Williams et al. 2004), and protects end-
othelial cells against apoptosis (Liu et al. 2007), both
effects independently of oestrogen receptors. Besides, the
present study has for the first time shown that DHEA
treatment in vivo corrects both contractile and relaxation
has several effects on the cardiovascular system, which
Figure 2. Reactive oxygen species in aorta
Representative fluorescence photomicrographs of microscopic sections of thoracic aorta from SHAM, OVX and
DHEA-treated rats. Vessels were labelled with the oxidative dye hydroethidine, which produces a red fluorescence
when oxidized to ethidium bromide by superoxide anion. Quantitative analysis for fluorescence photomicrographs
of microscopic sections of thoracic aorta from SHAM, OVX and DHEA-treated rats. Results are expressed as
means ± SEM. Two-way ANOVA:∗P < 0.01 in comparison to SHAM and #P < 0.01 in comparison to OVX (n = 7).
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2592J. P. G. Camporez and others
J Physiol 589.10
could be direct or indirect, which reduce cardiovascular
risk factors such as body weight gain or insulin resistance.
The present results suggest that the correction of
increase in PHE-induced contraction and decrease
in ACh-induced relaxation observed in aortas from
Figure 3. eNOS protein expression and phosphorylation in
Representative Western blots (top) and quantitative analysis
(bottom) for total eNOS protein expression (A) and ser1177 eNOS
phosphorylation (B) in thoracic aorta from SHAM, OVX and
DHEA-treated rats. Results are expressed as means ± SEM. Two-way
ANOVA:∗P < 0.01 in comparison to SHAM and #P < 0.01 in
comparison to OVX (n = 9).
production, since the eNOS phosphorylation on ser-1177
enhances the NO production by eNOS (Dimmeler et al.
1999). In line with the present results obtained in
aorta, Bhuiyan & Fukunaga (2010) showed a decrease
in eNOS phosphorylation in kidney from ovariectomized
Wistar rats. Moreover, Widder et al. (2003) demonstrated
Figure 4. Cu/Zn-SOD and Mn-SOD protein expression in aorta
Representative Western blots (top) and quantitative analysis
(bottom) for Cu/Zn-SOD (A) and Mn-SOD (B) protein expression in
thoracic aorta from SHAM, OVX and DHEA-treated rats. Results are
expressed as means ± SEM. Two-way ANOVA:∗P < 0.01 in
comparison to SHAM and #P < 0.01 in comparison to OVX (n = 8).
C ?2011 The Authors. Journal compilation C ?2011 The Physiological Society
Page 9
J Physiol 589.10
Dehydroepiandrosterone reduces reactive oxygen species in aorta 2593
that ovariectomy is related to a reduction of eNOS
protein expression in aorta from SHR rats, which was
not observed in our study using aorta from Wistar
rats. Recently, consistent with our results, Bhuiyan &
phosphorylation in kidney from ovariectomized rats,
leading to blood pressure reduction. In addition, there
are some in vitro studies which have demonstrated that
DHEA directly stimulates eNOS phosphorylation in end-
othelial cells (Liu & Dillon, 2004; Formoso et al. 2006).
Furthermore, our results exclude a possible role of DHEA
treatment on impaired ability of vascular smooth muscle
to relax in response to exogenous NO donor, since the
similar in all groups and was not changed by DHEA
Besides the reduction of NO production by eNOS,
the increased superoxide anion in vascular cells provokes
a decline in NO bioavailability, leading also to end-
othelial dysfunction (Cai & Harrison, 2000). The main
source of superoxide anion in the vascular system is
the enzyme NADPH oxidase (Cai & Harrison, 2000).
Similar to other studies (Wassmann et al. 2001; Tsuda
et al. 2005), here we observed that beyond decrease in
eNOS phosphorylation, oxidative stress is also involved
in the reduced NO bioavailability in aorta from OVX
rats. Therefore, endothelial dysfunction in OVX rats is
stress. In fact, PHE and ACh response of aortic rings
from OVX rats were also corrected by scavenging super-
oxide anion and inhibiting NADPH oxidase. Reinforcing
our results, aorta from OVX rats displayed an increase
in ROS production. Oxidative stress-induced endothelial
dysfunction by oestrogen withdrawal has already been
endothelial dysfunction resulting from acute oestrogen
deprivation in women was caused by oxidative stress.
In SHR OVX rats reduction in relaxation response to
carbachol in aortic rings associated with increased super-
oxide anion production was demonstrated (Wassmann
et al. 2001). Moreover, oestrogen protects female OVX
mice against increase in ROS production in aorta, and
angiotensin II-induced intracellular ROS production in
vascular smooth muscle (Strehlow et al. 2003).
Several studies have shown a potential antioxidant
effect of DHEA. The present study also demonstrates
a role of DHEA in reducing superoxide anion content
in aorta from OVX rats. This effect may represent
the mechanism by which DHEA treatment corrects the
vascular response to PHE and ACh in aorta from OVX
rats. Yorek et al. (2002) observed that DHEA reduced
plasma thiobarbituric acid-reactive substances (TBARS)
and superoxide anion production in arterioles from
diabetic rats, and Aragno et al. (2006) also demonstrated
reduction in ROS content in hearts from diabetic rats
treated with DHEA. It seems that DHEA has an anti-
since it reduced oxidative stress-induced skeletal muscle
oxidative stress parameters in plasma and in peri-
pheral blood mononuclear cells in diabetic subjects were
significantly decreased by DHEA treatment (Brignardello
et al. 2007). Besides, in vitro studies have shown direct
Figure 5. NADPH oxidase subunits protein expression in aorta
Representative Western blots (top) and quantitative analysis
(bottom) for gp91phox (A) and p22phox (B) protein expression in
thoracic aorta from SHAM, OVX and DHEA-treated rats. Results are
expressed as means ± SEM. Two-way ANOVA:∗P < 0.01 in
comparison to SHAM and #P < 0.01 in comparison to OVX (n = 8).
C ?2011 The Authors. Journal compilation C ?2011 The Physiological Society
Page 10
2594J. P. G. Camporez and others
J Physiol 589.10
effects of DHEA on oxidative stress parameters. DHEA
reduced ROS production and NF-κB activation in end-
othelial cells treated with TNF-α (Guti´ errez et al. 2007),
2007) and stimulated their proliferation (Williams et al.
As discussed above and demonstrated in the pre-
sent study, menopause and ovariectomy induce oxidative
stress, mainly in the cardiovascular system. One reason
for this outcome could be the modulation of the activity
or expression of antioxidant enzymes. Tsuda et al. (2005)
demonstrated that aorta from female mice displayed less
NADPH oxidase activity and expression than aorta from
male mice, while ovariectomy showed an increase in
NADPH oxidase activity and expression in females. This
observed in OVX rats, since NADPH oxidase is the
main source of superoxide anion associated with end-
othelial dysfunction (Cai & Harrison, 2000). In addition,
antioxidant systems are also depressed by oestrogen
decreased SOD mRNA expression in aorta from OVX
mice, which was related to an increase in the presence
of the superoxide anion, and oestrogen replacement
therapy prevented this downregulation (Strehlow et al.
In our study we also observed a modulation of
SOD and NADPH oxidase protein expression following
ovariectomy, displaying reduced Cu/Zn-SOD protein
expression and increased gp91phox and p22phox (sub-
units of NADPH oxidase) protein expression, which
would explain the oxidative stress-induced endothelial
dysfunction. Besides, we observed a correction of
Cu/Zn-SOD downregulation and gp91phox and p22phox
increment by DHEA treatment, which could be the
main result of DHEA treatment, leading to reduced
ROS production and consequently enhanced vascular
function in OVX rats. Besides the vascular antioxidant
effect by modulating expression of enzymes involved
in oxidant/antioxidant activity, the present work shows
that DHEA treatment also enhances SOD activity in
aorta from aged rats (Wu et al. 2007), prevents oxidative
injury in obstructive jaundice in rats by increasing SOD
activity in liver (Celebi et al. 2004), and also raises SOD
activity in liver from diabetic rats (Aragno et al. 1999).
However, the present study is the first to demonstrate
that DHEA treatment can modulate Cu/Zn-SOD,
gp91phox and p22phox (NADPH oxidase) protein
In summary, this study confirms the pivotal role
of oxidative stress-induced endothelial dysfunction in
OVX rats. Furthermore, it indicates the potential role of
DHEA therapy, instead of oestrogen replacement therapy,
as an antioxidant, improving endothelial function,
reducing systolic and diastolic blood pressure, and
modulating eNOS phosphorylation, and Cu/Zn-SOD,
gp91phox and p22phox protein expression in aorta from
OVX rats.
Aragno M, Mastrocola R, Alloatti G, Vercellinatto I, Bardini P,
Geuna S, Catalano MG, Danni O & Boccuzzi G (2008).
Oxidative stress triggers cardiac fibrosis in the heart of
diabetic rats. Endocrinology 149, 380–388.
Aragno M, Mastrocola R, Catalano MG, Brignardello E, Danni
O & Boccuzzi G (2004). Oxidative stress impairs skeletal
muscle repair in diabetic rats. Diabetes 53, 1082–1088.
Aragno M, Mastrocola R, Medana C, Graziella M, Vercellinatto
I, Danni O & Boccuzzi G (2006). Oxidative stress-dependent
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Aragno M, Tamagno E, Gatto V, Brignardello E, Parola S,
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Brignardello E, Runzo C, Aragno M, Catalano MG, Cassader
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Hernanz R, Briones AM, Alonso MJ & Vila E, Salaices M
(2004). Hypertension alters role of iNOS, COX-2, and
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& Vittinghoff E (1998). Randomized trial of estrogen plus
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Labrie F (2010). DHEA, important source of sex steroids in
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& Labrie C (2005). Is dehydroepiandrosterone a hormone? J
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Liu D, Si H, Reynolds KA, Zhen W, Jia Z & Dillon JS (2007).
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Pharmacol 38, 73–80.
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Fritzemeier K, Hegele-Hartung C, Neyses L & Bauersachs J
(2003). Improvement of endothelial dysfunction by selective
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Funder JW, Sudhir K & Komesaroff PA (2004).
Dehydroepiandrosterone increases endothelial cell
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vivo by mechanisms independent of androgen and estrogen
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Vanhoutte PM & Huang Y (2006). Raloxifene prevents
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age-related changes in the nitric oxide pathway in the arteries
of rats and the intervention effect of
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DD & Dillon JS (2002). Effect of treatment of diabetic rats
with dehydroepiandrosterone on vascular and neural
function. Am J Physiol Endocrinol Metab 283, E1067–E1075.
Author contributions
J.P.G.C., E.H.A. and C.R.O.C. contributed to the conception
and design of the experiments. J.P.G.C. performed and analysed
most of the experiments in this study, with technical assistance
from E.H.A., A.P.D., C.R.F. and L.V.R. All authors analysed and
interpreted data. J.P.G.C., L.V.R. and C.R.O.C. wrote the article.
There were no conflicts of interest.
We are grateful to Dr Ronald D. P. K. Ranvaud for helpful
suggestions on the manuscript. This work was funded by
research grants from FAPESP and CNPq, Brazil.
C ?2011 The Authors. Journal compilation C ?2011 The Physiological Society


text:
Keyboard hinted for Rock Band 3
Why have four instruments when you can bust out five? Rumour has it the next instalment of Rock Band 3 adds the electric keyboard to the mix.
Joystiq reports that the Green Day: Rock Band as downloaded from Xbox Live Marketplace teases the introduction of the keyboard in the series' next iteration.
An line of icons appearing at the end of the demo mirrors the Rock Band logo, albeit with one addition: the black and white keys of a keyboard situated to the right of a big "3."
Developer Harmonix has publicly admitted work on Rock Band 3, though refuses to discuss details. With speculation already mounting about vocal harmonies and now the infusion of the keyboard, expect a big debut next month during E3.
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- F1 2015 coming to PC, PS4 and Xbox One this summer, first screenshots released
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subset: commoncrawl


text:
|Social media helps drive demand for FaithWords devotional
|Written by Jeremy Burns
|Monday, 21 April 2014 02:04 PM EDT
FaithWords is increasing its first printing run for Jesus Daily to 100,000. The larger print run for the book by Aaron Tabor, M.D., is based on demand from booksellers and shows the power of social media.
Jesus Daily is a 365-day devotional based on themes from Facebook's No. 1 "most engaging page" in the world, the publisher said. Nearly 26 million people have "liked" the Facebook page.
Using creative design elements and interactive activities, Tabor's book challenges readers to respond to each daily reflection using a variety of social media tools.
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text:
Massachusetts | Medicaid.gov
Medicaid-Marketplace Overview. Massachusetts is operating a State-based Marketplace, known as the Massachusetts Health Connector. The state has expanded Medicaid ...From:http://www.medicaid.gov › Medicaid
Medical Assistance MA Minnesota's Medicaid Program for ...
Medical Assistance. Home > Health care. Overview. Medical Assistance (MA) is Minnesota's Medicaid program. It is the largest of Minnesota's publicly funded health ...From:http://www.dhs.state.mn.us/main/idcplg?IdcService=GET_DYNAMIC_CONVERSION&...
Medicaid Financing: An Overview of the Federal Medicaid ...
2 00 Overview of the FMAP Formula The Medicaid program is jointly funded by states and the federal government. In states that choose to participate—and all states ...From:http://https://kaiserfamilyfoundation.files.wordpress.com/2013/01/8352.pdf
Vocational Rehabilitation - Medicaid (MA) Maximum ...
Vocational Rehabilitation; Medicaid (MA) Maximum Allowable Fee Schedules; Contact Us. Offices / Personnel; Contacts & Resources; Content Feedback; How to ApplyFrom:http://dwd.wisconsin.gov › Vocational Rehabilitation
Medicaid Liens and Estate Recovery in Massachusetts
INTRODUCTION. Massachusetts, like many states, is contending with the dilemma of ever-increasing Medicaid costs in times of increased fiscal constraint.From:http://aspe.hhs.gov/daltcp/reports/MAliens.htm
2015 Advance Notice - Centers for Medicare & Medicaid Services
CMS uses two trend factors to calculate county rates. CMS plans to rebase the county FFS rates for 2015 as part of the calculation of the rates for 2015.From:http://www.cms.gov/Medicare/Health-Plans/MedicareAdvtgSpecRateStats/...
Medicaid Estate Recovery Collections - ASPE
U.S. Department of Health and Human Services. Medicaid Estate Recovery Collections Thomson/MEDSTAT September 2005. PDF VersionFrom:http://aspe.hhs.gov/daltcp/reports/estreccol.htm
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Introduction. Discussions between states and managed care plans about capitation rates have become increasingly contentious. In numerous markets, health plans are ...From:http://www.urban.org/publications/309064.html
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GIS 13 MA/022: 2014 Medicaid Only Income and Resource Levels and Spousal Impoverishment StandardsFrom:http://www.health.ny.gov › 2013 General Information System Messages
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by: Brian E. Barreira, Esq. Under federal Medicaid law and MassHealth regulations, spouses have the right to transfer assets to each other at any time without causing ...From:http://masshealth.info


text:
Eric Cantor Is Resigning From Congress Early
Published 4:22 am, Friday, August 1, 2014
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Just hours after stepping down as House majority leader, the once-powerful Rep. Eric Cantor (R-Virginia) announced he won't even complete his term as a rank-and-file lawmaker.
In an interview with the Richmond Times-Dispatch, Cantor said he made the decision so his constituents could have his replacement immediately after the November election instead of January, when his term would have ended.
"I want to make sure that the constituents in the 7th District will have a voice in what will be a very consequential lame-duck session," Cantor said in an article published Friday morning. "That way he will also have seniority, and that will help the interests of my constituents."
Cantor, who had previously been considered the leading candidate to replace John Boehner as House Speaker, lost his own Republican primary in a shocking June upset to the previously unknown college professor David Brat. He announced he would step down from his party leadership post at the beginning of August and was replaced as majority leader by former House Majority Whip Kevin McCarthy (R-California).
Since the loss, Cantor has reportedly been a near-invisible presence in House politics.
"Cantor has participated in floor debate only twice, offering brief remarks before passage of uncontroversial bills to revamp worker training programs and extend adoption incentives," Roll Call reported Wednesday.
Cantor's next plans aren't immediately clear.
Join the conversation about this story »
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text:
The Quip and Kale's Treehut experience, haughtily pouring in the evening candlelight
Nobody really knows what the point of Quip and Kale's Treehut is. It at one time had things to play on, but a union representing particularly fragile children had it removed to reduce their paperwork load.
Shun Princess Mode
© copyright 2011–2015
A Pants Aflame production


text:
I'm sorry... I tipped West Ham to go down and Sam Allardyce has proved me wrong. His imminent sacking is disappointing
- The club and Sam Allardyce have proved me wrong, it shows a disappointing lack of loyalty to sack Sam Allardyce
- By Sunday night West Ham will be comfortably clear from relegation
- The football has not always been pretty, but he masterminded huge wins
- Yaya Toure does not get the recognition he deserves in the Premier League
Dear West Ham fans,
In my very first column of the season, I wrote that I feared for your team. I was worried that the inconsistencies of the previous campaign, the quality of the performances and the value of the summer signings meant you would flirt with relegation and I predicted you would finish fourth from bottom.
After all the stick I got over the years at Upton Park I find it very hard to apologise to any West Ham fan but, I am happy to admit just this once, that I was wrong about your team.
Hitting form: West Ham's run of four wins in February saved them from relegation
By Sunday night West Ham will still be in the Premier League and comfortably clear of the relegation zone. I could not see that happening at the start of the season. The club and Sam Allardyce have proved me wrong.
Even though his imminent sacking may not come as a surprise, it shows a disappointing lack of loyalty from the West Ham board. What kind of message does it send to a prospective new manager when you ditch a man who has achieved everything asked of him?
At times the football has not been pretty but Allardyce masterminded three big wins over Tottenham and a crucial 0-0 draw at Chelsea. That result sent West Ham on a four-match winning streak — their first in seven years — and was the run that saved their season and kept them up.
It is admirable Allardyce got the chance to see things through, especially with other clubs sacking managers for fun — the bottom three have had seven between them this season – and he has managed in a bullish manner throughout.
On his way: Sam Allardyce is understood to be nearing an exit from Upton Park
They have survived quite comfortably in the end, but there have been problems. Andy Carroll, who Allardyce signed for record money, has scored only twice and his injury saga was a real concern. The away form has barely improved since last season, either.
But Sam is a troubleshooter, the kind of man clubs turn to for a quick fix. Now West Ham's problem of survival has been solved, the fans and the club are ready for a change. Allardyce will feel aggrieved he lost his job over the brand of football rather than results, but all is not lost.
Tony Pulis was sacked by Stoke in similar circumstances but has evolved and enhanced his reputation with Crystal Palace. If this is the end for Sam, he can easily do the same thing at another top club.
West Ham fans are purists and want their team to play with flair, but we have not seen that for a few generations and any transition will not happen overnight.
Out! West Ham fans let their frustration out on the manager after defeat to West Brom
The team are set up for Matt Jarvis and Stewart Downing to find Carroll with crosses. The new boss will have to make the most of the players he has, making tweaks to the way they play rather having a radical overhaul.
For all the desire to play more attractive football, sacking Allardyce is a huge gamble. The club will want to be a Premier League side when they move into the Olympic Stadium in 2016 and Sam could have provided that stability.
He achieved all that was asked and usually in business if you meet your objectives, you keep your job. But for West Ham fans football is an entertainment business and they do not feel entertained.
It is not a decision that will be taken lightly, but when Allardyce was booed by his own fans, it was the beginning of the end. It was never an easy relationship but now it's up to the board to find a new man who fits the bill.
Fresh new start: A new manager could drive West Ham up the league next season
In the spotlight: Yaya Toure, Age 30, Manchester City
He may not be the most original pick for Spotlight but when Yaya Toure said he did not receive enough recognition earlier this season, I could not agree more.
There is a lot of talk at the moment about the development of young English players but any youngster will only take inspiration from what Toure is doing on and off the pitch.
He is one of the most humble people I've met in football, he is a team player first and foremost and his individual ability is unquestioned.
Underrated: Yaya Toure has not received the praise he deserves after another superb season
It is rare to find a player who so perfectly mixes pace, power and technique. He can finish like a forward and defend like a centre half, as he proved for Barcelona in the 2009 Champions League final.
If City are struggling to score on Sunday, it will probably be Yaya who pops up with a winner. If he keeps it up I might say he is better than my old team-mate Patrick Vieira. But he's still got some work to do to match the fantastic Frenchman.
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MM Friday Finds
MM blogger Robin Bielman's novel Yours At Midnight is on special at 99c at the moment. Get it at:
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MM bloggers Edie Ramer, Mary Hughes, Dale Mayer & Michelle Diener have stories in a boxed set ENTRANCED which is on special at 99c for a very limited time. Get it at:
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Coming Up
On March 5th we welcome Coreene Callahan, author of the Dragonfury Series, to MM. Coreene is a rabid hockey fan with a love of the great outdoors. More comfortable on a ski slope than in a shopping mall, she spends her days dreaming up and writing high-octane, action-packed paranormal romances.
On March 12th we have romantic comedy, paranormal mysteries and romantic mysteries author Darlene Gardner as our guest.
We are excited to have Australian romance author Jennie Jones visiting Magical Musings on March 25th.
Contemporary romance author Maggie Kelley will be here on April 2nd to talk about her love of New York and her debut novel, Breaking the Bachelor.
Jeffe Kennedy, author of the Twelve Kingdoms fantasy romance series and other books, will drop by Magical Musings on May 7.
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Tag Archives: Miracle Interrupted
My First Monthly Giveaway
We have a winner! Random.org picked Sue C to win her choice of an ecopy of Galaxy Girls or Mo's Heart. Congrats, Sue, and thank everyone for commenting! We do a lot of giveaways at Magical Musings, and today is … Continue reading
Miracle Lane – with Giveaway!
The giveaway winner of MIRACLE LANE is Leigh Morgan. Congratulations, Leigh! While I was posting the stories of my fellow Magical Musings bloggers every other Monday, MIRACLE LANE, the third book of my Miracle Interrupted series, was published. A couple … Continue reading
Edie Ramer's Story
Today, we have our very own Edie Ramer's story on her writer's journey. Edie is one of the founding members of MagicalMusings and like all of us, followed a difficult road to publication. Deciding to go the indie route, Edie … Continue reading
Title Help!
I'm excited about getting such a great title for my new book from StrangeAngel, STARDUST MIRACLE. The book should be published in May, I hope, along with a novella. StrangeAngel will be the recipient of a book. A lot of … Continue reading


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- The Clinton Chronicles . . . unbelievable! ~J
- Take Your Word? CNN Blames State Department Hack on Russia Without Evidence / Sputnik International
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Tag Archives: Party leaders of the United States Senate
Five Justices Vow to Protect Felon Senators
Posted by Gordon Duff Veterans Today March 10, 2015 Scalia to McConnell: "We've got your back." Justice Scalia, a Logan Act plotter? The Logan Act is not a joke. 47 Republican members of the US Senate are now officially felons, … Continue reading
Posted in Financial/economic information, Illuminati/Terrorism/Corruption, Political Tagged Antonin Scalia, Attorney general, Barack Obama, First Amendment to the United States Constitution, Mitch McConnell, Party leaders of the United States Senate, Republican Party (United States), United States, United States Constitution, United States Department of Justice Leave a comment
PressTV: Americans seek treason charges against 47 Republican senators
Thu Mar 12, 2015 12:40AM From left: Ted Cruz, Rand Paul, Mitch McConnell Tens of thousands of Americans have signed a petition to the White House demanding treason charges against 47 Republican senators who attempted to sabotage President Barack Obama's … Continue reading
RT: President Obama vetoes Keystone pipeline bill
Published time: February 24, 2015 20:42 Edited time: February 24, 2015 21:37 A depot used to store pipes for Transcanada Corp's planned Keystone XL oil pipeline is seen in Gascoyne, North Dakota (Reuters/Andrew Cullen) As promised, US President Barack Obama … Continue reading
Posted in Financial/economic information, Illuminati/Terrorism/Corruption, Political Tagged Associated Press, Barack Obama, Capitol Hill, Democratic Party (United States), Fiscal year, Funding, Mitch McConnell, Party leaders of the United States Senate, United States Congress, United States Department of Homeland Security 1 Comment
PressTV: US House approves Keystone XL oil pipeline despite Obama veto threat
Fri Jan 9, 2015 9:23PM Critics argue that the proposed pipeline is a death warrant to the environment and to the people. Despite President Barack Obama's veto threat, the US House of Representatives has overwhelmingly approved the construction of the … Continue reading
Posted in Financial/economic information, Illuminati/Terrorism/Corruption, Political Tagged Barack Obama, Capitol Hill, Democratic Party (United States), John Boehner, Keystone Pipeline, Mitch McConnell, Party leaders of the United States Senate, Republican Party (United States), United States Congress, United States House of Representatives 2 Comments
RT: 'Death warrant to our environment & people': Native Americans say no to Keystone XL (PHOTOS)
Published time: January 04, 2015 01:31 Edited time: January 04, 2015 02:24 Image from Facebook (No to KXL rally) Dozens of Native Americans have descended on Washington DC on the first weekend of 2015 to stage a protest in front … Continue reading
Posted in Financial/economic information, Illuminati/Terrorism/Corruption, Political, Spiritual Tagged Alberta, Athabasca oil sands, Barack Obama, Canada, Gulf Coast of the United States, Keystone Pipeline, Mitch McConnell, Native Americans in the United States, Party leaders of the United States Senate, United States 9 Comments
USA TODAY: Democrat leader reaped $1.1 million from sale of land he didn't own
This story from 2006 has now gone viral! It's all over the internet. How nice!!! ~J BY CROOK Source: CrookedAmerica APRIL 16, 2014 Apparently not much has changed in the past decade in regards to the crook Harry Reid and his … Continue reading
PressTV: US Senate fails to pass extension of jobless benefits
Fri Feb 7, 2014 1:19AM GMT The US Senate has failed to extend long-term unemployment benefits for 1.7 million Americans for three months. On Thursday, Republicans for the second time in less than a month blocked a bill to restore … Continue reading


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Public Bids & Legal Notices
To receive RFQs, RFPs or bid documents (listed above, when available), please complete and submit the form below with the project name, and details/files will be emailed to you.
Legal Notice: 2015 Heavy Equipment Rental
Steep Stream
Yellow Creek has the steepest grade in the Cuyahoga River watershed.
Leapin' Lizards!
The five-lined skink is the only known lizard species in the Metro Parks. It is found near the Mustill Store among the Cascade Locks.
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Jessica A Bruno (waybeyondfedup)Shared publicly -
Part Swan, Part Goose: An Uncommon Memoir of Womanhood, Work, and Family (Signed Book) , Swoosie Kurtz with Joni Rodgers
Myths & Meanings: Support For Depression
A Note From The Editor: As we've recently seen in the news, depression can have devastating consequences. It's a real, far-reaching struggle
Tools of the Job Trade: Your LinkedIn Profile
I first joined LinkedIn because I felt like I had to. It was 2007, I had just graduated from college and started my first job, and LinkedIn
When Your Past Is A Harsh Winter
We see it all around us with the slow and steady arrival of spring. New life and a new day. There's something sacred about the spring, and I
4 Ways To Serve A Pixie
Don't call it a cutie. Or a baby orange. Pixies hold their own in the citrus world, and next month specifically kicks off their appreciation
Paranormal Forums
Paranormal Forums is a forum based supernatural community to find or share info on aliens, ghosts, psychic, secrets, the Illuminati and many
Myths & Meanings: Treating Depression
A Note From The Editor: With weighty topics there is a great measure of uncertainty and discomfort, yet also a great call to stand together
Ahu Tongariki
Ahu Tongariki Theme For Google Chrome. Stunning Image Quality, HD Custom Designed Theme, packed with Easter Island detail.…
Are We Addicted to Stress?
Stress. Whether you wear it physically or emotionally, it is never a pleasant state of being. Stress announces itself in a variety of forms.
The Beautician Embodied: Osmia Organics (+ A Giveaway!)
Imagine an innovative entrepreneur in the skincare and beauty industry, and an ER physician might not be the first thing that comes to mind.
Darling x The Tig: Ginger Berry Crumble
A Note From The Editor: Already fans of Meghan Markle thanks to her role on Suits, we're even more impressed by the down-to-earth approach s
Two Left Feet: Chapter VI
Losing My Balance, Keeping My Time When I set out to learn to dance, it was to do so as a layman — to integrate dance into my everyday life.
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Sign the Petition: Turn it Off!
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CDC Urges Measles Vaccinations as Number of Cases Exceeds 100 – The Vacc...
Director Dr. Thomas Frieden blamed a lack of vaccinations for the resurgence of a disease that the CDC said was eliminated in the U.S. in 20
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More Labrador Retriever Puppies and Dogs for sale.
TX.- FIELD PREPPED "WORKING" LAB PUPS - ... WyngMaster Labradors (Est. January, 2000) in Tulsa, Oklahoma
AKC Championship Pedigree Chocolate... DR Kennels in Harrisonville/ S. KC, Missouri
4xGMPR,MH,QAA Sired AKC Yellow/Blac... Badger Canyon Labrador Retrievers in Benton City, Washington
AKC CHOCOLATE LITTER - MASTER HUNTE... Kinnaman Retrievers in Sisters, Oregon
black lab pups EIC/CNM clear Crumpler Kennels in Canton, Texas
LABS AKC BLK&YELLOW - MASTER HUNTER SIRE... Kinnaman Retrievers in Sisters, Oregon
STARGATE CHOCOLATE & BLACK PUPPIES Champion Chocolate Labs in Collinsville, Texas
Three Sisters Labrador Puppies due ... Three Sisters Labradors in Powell Butte, Oregon
QAA AKC Yellow Lab Puppies Birch Run Premier Labradors in Minnesota
Sporting Life Kennels Started Briti... Sporting`Life British Kennels in Oxford, Mississippi
Master Hunter sired blacks ready to... Norse Retrievers in Dassel, Minnesota
3 STUDS AVAILBLE - BLACK - CHOCOLATE - Y... WyngMaster Labradors (Est. January, 2000) in Tulsa, Oklahoma
4XGMPR x GMPR Advantage Pointing Labs breeding champions in Yuma, Colorado
started akc british female lab $3,2... kadens labs in saint paul, Minnesota
Sire 4XGMPR x Dam CPR, APR Advantage Pointing Labs breeding champions in Yuma, Colorado
Chocolate British Lab Puppies For S... Rock Springs Lab in Blountsville, Alabama
Mud MH Blacks and Chocolates due la... Erins Edge Labradors in Richfield, Wisconsin
MARKED AS SOLD WyngMaster Labradors (Est. January, 2000) in Tulsa, Oklahoma
Sporting Life Kennels Started Labra... Sporting`Life British Kennels in Oxford, Mississippi
Adult female Lab/house trained Korkys Inspiration Lovin Labs in New Lothrop, Michigan
6 Month Old AKC Master Hunter And J... Johnson's Labrador Retrievers in Murfreesboro, Tennessee
4 Years Old AKC Champion Bloodline ... Johnson's Labrador Retrievers in Murfreesboro, Tennessee
AKC Lab Puppies Upcoming Litter!!! ... Johnson's Labrador Retrievers in Murfreesboro, Tennessee
AKC Master Hunter And Junior Hunter... Johnson's Labrador Retrievers in Murfreesboro, Tennessee
AKC Lab Puppies Upcoming Litter!!! ... Johnson's Labrador Retrievers in Murfreesboro, Tennessee
HRCH MH x CGCA THDN Bred Yellow Lab... SageCreek Kennels in Wolfe City, Texas
HRCH Black (Yellow Factored) Stud D... Lincoln Creek Labs in Waco, Nebraska
CKC Reg' Black/Choc- Hunting/Hunt test prospects - Labrador Retrievers in Quesnel, British Columbia - Eromit Labrador Retrievers
Born August 16th 11 pups- 5 chocolate males, 3 chocolate females, and 3 black males.
Sire: Eromit's Tennessee Trickshot SGDC (aka Chester)- FC HRCH Watermark's Texas Welcome MH x Lockfive's April Foolya MH
Dam: Eromit's Prairiestorm Garbi (aka Garbi) - Merganser's Ferris Bueller MH WC x SHR Gras-land's Sweet Brandy JH WC
This is a very strong pedigree loaded with field champions and master hunters (see complete pedigree here ). Both sire and dam are hunted regularly and sire competes in agility, rally and is training for SH. Pups from his previous litters have been hunting as early as 5 months of age and are training for hunt tests, obedience, agility, and dog diving careers. Chester is a big, handsome HIGH drive dog no fear kind of dog who is an excellent marker and has a great off-switch- quiet and well mannered in the house and in the blind. Garbi is a smaller chocolate female who lives to retrieve. Both are very willing to please and easy to train. Pups will make terrific hunting dogs, competitors, and companions for active families.
Both parents are certified free of hip/elbow dysplasia, eye disease and EIC/CNM. Health clearances, photos and more are on our website www.eromit.com
Puppies will be ready for their new homes October 11th. Prior to going home they will be exposed to birds, water, gunfire- handled daily and started on socializing and training. Sold with an extensive30 month health guarantee, titling incentives & Canadian Kennel Club registration. References from past puppy buyers are available.
Ad location: Quesnel, British Columbia
Ad created: Aug 18, 2012
Contact Information
Contact Name: Erin and Tim Lynes
250-992-5253Contact breeder through email Click here to visit website
Eromit Labrador Retrievers Breeder Information


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BP terminated contracts for two deepwater oil drilling rigs in the Gulf of Mexico as the British oil company slashes its exploration budget due to fallen oil prices.
MacGregor reorganized into seven operative divisions in the beginning of 2015 to improve customer focus.
Offshore Mooring and Loading Division drives the
Dutch offshore contractor Heerema Offshore Services has signed a letter of intent (LOI) with Sembcorp Marine's Jurong Shipyard in Singapore for the construction
The Shipping Corporation of India Ltd. (SCI) accepted delivery of a Very Large Crude Oil Carrier (VLCC) on 28 March, 2015. The vessel has been named "Desh Vibhor".


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What Do You Think of Designer Beauty Collaborations?
by Beauty
Like us on Facebook
This beautifully packaged lipstick is Pixel Pink, the shade created for Thakoon's spring 2008 runway show. As of today, it's available from Lancôme, and in just about a month, the brand will release limited-edition glosses from designer Christopher Kane. Then, in late March, the Heatherette boys are launching a huge collection for MAC. And Avon has a cute Cynthia Rowley beauty line. Phew!
What do you think about fashion designers creating beauty products? And if you made the big cosmetic-executive decisions, who would you tap for the next designer collection?
Read More ThakoonBella PollLancome
chezmadame 7 years
Anyone know where I can find Behnaz Red and Proenza Pink?
misstiffie 7 years
i love it.. i bought the MAC Barbie & McQueen line.. and I just bought the whole MAC Fafi line today :)
ms_dior_cherie 7 years
cool concept
chiquita29 7 years
Does AVON still make the Cyntiha Rowley line? I couldn't find it.
vanilla19 7 years
I love designer beauty collaborations, the packaging is always so creative and pretty. I really want the Christopher Kane juicy tubes!
fancy818 7 years
this is interesting to me.
TrickyTara627 7 years
i think marc jacobs should have a makeup line!!
Yorkdale_Girl 7 years
Pucci did have a line - Guerlain did it last summer, I think. The colors and packaging were GORGEOUS, but I couldn't justify keeping the items, so back they went. It was a shame, too - they were GORGEOUS!
almost-famous 7 years
I'm not as picky, however, I love the packaging!
Dana18 7 years
It is just another way to attract customers.
Lovely_1 7 years
I am picky too...
aimeeb 7 years
I'm super picky so they better be amazing colors...
emalove 7 years
I have to like the colors, otherwise, I don't get very excited.
annebreal 7 years
Great idea especially when they're affordable! And yeah, TGIF!
betsy-scott 7 years
i'm with you on TGIF!! i like the idea but it has to be quality. i hear armani's beauty line is unbelievable. the packaging would sell me tho...could you imagine a PUCCI beauty line!? ugh. i'd buy the whole thing just to decorate my apartment with the boxes :o) xxoo to all of you bellas
brittanyk 7 years
I'm agreeing with the TGIF thing, McNasty. Anyway, I love the idea of designers collaborating. Generally the packaging is a little bit more special and they come up with some great colors. I love the Cynthia Rowley line Avon did. Cute colors, cute packaging, and I was happy with the quality of the product. I'm also loving the color of this lipstick. I'd definitely consider buying it if I liked it :)
McNasty 7 years
Neat color! Have never been the first one here. tgif everyone!
Link Love
- The gorgeous pale pink shade is only $11 — The Cut
- Skin care secrets from a hand model — Byrdie
- How to create a custom-made lash curler with your selfies — InStyle
- Beauty tricks to look bright-eyed and well-rested — Real Simple
- Could this makeup ingredient cause cancer? — Beauty High
- A closer look at the science behind facial workouts — Daily Makeover
- Proof that salt is an essential ingredient for your skin care DIYs — StyleList
- This common beauty tool could stave off bunions — xoVain


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Win A 96.1 Seize The Deal Golf Card!<Classic Rewards Club
If golf is part of your spring and summer, then the Oldiez 96.1 2014 Golf Card is the perfect thing for you to WIN! The card includes 12 of Central New York's golf courses all for just $50. We are giving one away this weekend!
Click HERE for a complete list of courses and more details
If you're not a golfer, this would make the perfect gift for someone who is! Father's Day is coming next weekend.


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OMA Monarch Speakers
We don't even know where to begin with the OMA Monarch speakers. Hidden behind that grill are two 15-inch speakers that are said to deliver bass that "no high end design using small woofers can even hope to match". Those large wood baffles are designed to help load the woofers so that you can place them in any part of the room and still enjoy what we can only hope is fulfilling and rich sound. Lastly, the horns are crafted from a proprietary cast aluminum alloy, and a compression driver covers the midrange and high frequencies.
There's no word on a price and they're available in a variety of wood finishes.
Christen Costa
Grew up back East, got sick of the cold and headed West. Since I was small I have been pushing buttons - both electronic and human. With an insatiable need for tech I thought "why not start a blog focusing on technology, and use my dislikes and likes to post on gadgets."
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Eero: Blanket Your Home With Reliable, Fast WiFi


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One thing that surprises me right now is how many Members ask me to travel with them. It is a good thing. Both Democrats and Republicans want me to travel with them to their districts or states this summer. I interpret their invitation to mean that they find value in visiting our agencies and my being along. I like that. What I don't care for is the partisan rhetoric I experience almost every day. It has to take a toll on the populace, who must get tired of it. It must take a toll on the candidates as they get bashed all the time. And I tell you, it takes a toll on me. I like Congress. I want it to work but I just do not see that happening right now.
I predict that post November elections, we will have two extremely productive years in Congress. It will be different than what we have had for a number of years, regardless of which party wins. I won't forecast who is going to control the House, Senate or White House but I do predict that Congress will be more productive. When pushed to the limit, when facing a midnight deadline, Congress tends to do the right thing. For a number of problems America is facing, midnight is fast approaching. Debt, deficit, spending priorities, tax overhaul, energy policy, entitlement reform. The time is approaching when Congress will have to deal with them and I still believe Congress will do the right thing. The urgency of our problems demands bipartisan action. It may be ugly getting there, but I believe both parties will do the right thing in the end.
May 4, 2012
Midnight Approaching
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More German Wirehaired Pointer Puppies and Dogs for sale.
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AKC German Wirehair Pointer Puppies - German Wirehaired Pointers in Walla Walla, Washington - TnC Kennels
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Ad location: Walla Walla, Washington
Ad created: Nov 21, 2013
Contact Information
Contact Name: Ty Dangerfield
509-520-9043Contact breeder through email
TnC Kennels Breeder Information


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Villino Castellano Apartments Accommodation & Rooms
Situated in Meta, Villino Castellano Apartments is close to Museo Correale di Terranova, Port of Sorrento, and Piazza Tasso. Also nearby are Deep Valley of the Mills and Sedile Dominova.
Property Features.
Villino Castellano Apartments has a marina. Wireless Internet access is available in public areas (surcharges apply). The staff can provide concierge services, tour/ticket assistance, and limo/town car service. Additional amenities include a rooftop terrace, a terrace, and multilingual staff. For a surcharge, guests receive transportation including a shuttle from the hotel to the airport at scheduled times, a beach shuttle, and a ferry terminal shuttle. Guest parking is available on a limited first-come, first-served basis (surcharge).
The 12 guestrooms at Villino Castellano Apartments include air conditioning. All units have kitchenettes with refrigerators. Housekeeping is offered once per stay.
Very close to the local railway and the drop off point for the airport bus service to Naples. The apartments are set away from the tourist spot. When you exit the door you are…
Enter dates to check prices
Hotel Info: 800 1205131
- Superior Double Room
- Standard Double Room
- Superior Quadruple Room
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At a glance
Key facts
Hotel size
- This hotel has 12 rooms
- Check-in time 3:30 PM-8 PM
- Check-out time is 10:30 AM
Required at check in
- Credit card deposit required
- Government-issued photo ID required
Travelling with others
- Pets allowed *
- 1 per room (up to 12 kg)
- WiFi in public areas *
- WiFi in rooms *
- Airport drop off at scheduled times *
- Ferry terminal shuttle *
- Self parking (limited) *
- Discounted offsite parking
- Beach shuttle *
Nearby One Star Meta hotels
In the hotel
- Beach shuttle (surcharge)
- Winery attached
- Marina on site
- 24-hour front desk
- Concierge services
- Tours/ticket assistance
- Limo or Town Car service available
- Laundry facilities
- Rooftop terrace
- Garden
- Terrace
In the room
- Climate control
- Air conditioning
- Refrigerator
- Kitchenette
- Housekeeping - once per stay
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Villino Castellano Apartments Accommodation & Rooms's small print
Cash transactions at this property cannot exceed EUR 999.99, due to national regulations. For further details, please contact the property using information in the booking confirmation.
Only registered guests are allowed in the guestrooms.
For transfers, guests must contact the property before arrival at the number on the booking confirmation.
Mandatory fees
You'll be asked to pay the following charges at check in or check out:
A tax is imposed by the city and collected at the property in cash. The tax applies to stays from 1 April to 31 October. This tax does not apply to residents of the city, or children under 18 years of age. Please note that further exemptions may apply. For further details, please contact the property using the information contained in the confirmation received after booking.
- A tax is imposed by the city: EUR 1 per person, per night, up to 7 nights. This tax does not apply to children under 18 years of age.
- Cleaning fee: EUR 25 per accommodation, per stay
- Seasonally adjusted heating fee: EUR 8 per accommodation, per night
- Resort fee (April - October): EUR 1.00 per person, per night
Optional extras
Self parking costs EUR 5 per day
Air conditioning is available for an extra charge of EUR 8 per day
Rollaway beds are available for EUR 10 per night
Cribs/Infant beds are available for EUR 5 per night
Pets are allowed for an extra charge of EUR 25 per accommodation, per stay (maximum EUR 25 per stay)
Airport shuttle service is offered for an extra charge of EUR 80 per vehicle (one way)
WiFi is available in guestrooms for EUR 5 per day (rates may vary)
WiFi is available in public areas for EUR 5.00 per day (rates may vary)
We have included all charges provided to us by this hotel. However, charges can vary, for example, based on length of stay or the room you book.


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Published 10:23 am, Wednesday, July 23, 2014
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Shares are down 0.1% though are actually rising on the news.
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