text:
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Biomaterials 24 (2003) 873–882
The effect of Gu-Sui-Bu (Drynaria fortunei J. Sm) immobilized
modified calcium hydrogenphosphate on bone cell activities
Jui-Sheng Suna, Guo-Chung Dongb, Chun-Yu Linc, Shiow-Yunn Sheuc, Feng-Huei Linb,*,
Li-Ting Chend, Walter Hong-Shong Changd, Yng-Jiin Wange
aDepartment of Orthopedic Surgery, National Taiwan University Hospital, 7 Chung-Shan South Road, Taipei, Taiwan, ROC
bInstitute of Biomedical Engineering, College of Medicine, College of Engineering, National Taiwan University, No. 1, Jen–Ai Road, 100 Taipei,
Taiwan, ROC
cSchool of Pharmacy, Taipei Medical University, Taiwan, ROC
dDepartment of Biomedical Engineering, Chung- Yuan Christian University, Chung-Li 32023, Taiwan, ROC
eInstitute of Biomedical Engineering, National Yang-Ming University, Taipei, Taiwan, ROC
Received 5 October 2001; accepted 8 August 2002
In our previous study, we have validated the efficacy and the safety of Gu-Sui-Bu [Drynaria fortunei (Kunze) J. Sm.] by the bone
cells culture. However, a satisfactory delivery system for Gu-Sui-Bu must be developed before it can be used in clinical medicine. In
this study, we try to use modified calcium hydrogenphosphate (MCHP) bioceramic as a carrier to transport Gu-Sui-Bu into the bone
cell culture system. Toward this goal, we evaluated the effect of a Gu-Sui-Bu-immobilized modified calcium hydrogenphosphate
(GI-MCHP) on the bone cells activities.
The Chinese medicine: Gu-Sui-Bu [Drynaria fortunei (kunze) J. Sm] was extracted and then immobilized on the surface of MCHP.
The rat osteoblasts–osteoclasts co-culture system was used as the experimental model. After the cells grew to 80% confluence,
different sizes of GI-MCHP particles were tested. The mitochondria activity of the bone cells after exposure was determined by
colorimetric assay. Biochemical markers such as lactate dehydrogenase (LDH), alkaline phosphatase (ALP), acid phosphatase
(ACP) and prostaglandin E2titer were analyzed to evaluate the bone cells activities. Histomorphometric study of osteoclasts
activities and the phenotype expression of osteoblasts were also evaluated.
There is no detectable titer of LDH secretion into the medium and no significant change in the intracellular ALP content. The
ALP titer in the culture medium did increase significantly at 3 days' culture, while there is a significant decrease in the intracellular
ACP content and significant increase in the ACP titer in the medium. The concentrations of PGE2in tested medium are always
significantly higher than that of control medium during the 7 days' culture. At the end of 7 days' culture, the PGE2concentrations in
the tested medium were still 4.74 times that of the control medium. After GI-MCHP treatment on bone cells, the size of the
osteoclasts seems decreased and their cell integrity seems lost, while the osteoblasts phenotype expression was relatively preserved.
From this study, we demonstrated that Gu-Sui-Bu [Drynaria fortunei (Kunze) J. Sm.] immobilized MCHP has well preserved the
potential beneficial effects of Gu-Sui-Bu on the bone cells culture.
r 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Gu-sui-bu; Calcium hydrogenphosphate; Bone cells; Prostaglandin; RTPCR
1. Introduction
Throughout the world, humans have traditionally
used plants both as food and medicine. In Europe,
chemistry developed rapidly after the influence of
Paracelsus. Active ingredients were isolated from plants,
and drugs were prepared in the salt form to use as
medication. In China, several drugs isolated from plants
have been prepared in the salt form in recent years, but
herbal medication, developed in the ancient tradition,
continues to be widely used in Chinese populations [1].
Traditional Chinese medicine has shown effective
clinical responses. Reports of efficacy of traditional
Chinese medicine are increasing in numbers [2–5]. The
traditional Chinese medicines: Gu-Sui-Bu [Drynaria
fortunei] was commonly used to manage musculoskeletal
*Corresponding author. Tel.: +886-2-23660390; fax: +886-2-2394-
E-mail address: firstname.lastname@example.com (F.-H. Lin).
0142-9612/02/$-see front matter r 2002 Elsevier Science Ltd. All rights reserved.
Page 2
traumatic disorders of orthopedics with satisfactory
results [6]. Specifically, through tissue culture and
isotope tracing, Ma et al. found that Gu-Sui-Bu
injection significantly promoted calcification of the
cultivated chick embryo bone primordium, increased
ALP activity in the cultivated tissue, and accelerated the
synthesis of proteoglycan. It was also confirmed that the
promotion of proteoglycan synthesis was an important
factor in the promotion of calcification [7]. Later, by
using ovariectomized rat osteoporotic model, Ma et al.
demonstrated that treatment of Gu-Sui-Bu can increase
the bone volume of ovariectomized rat [6]. In our
previous study, we have validated the efficacy and the
safety of Gu-Sui-Bu [Drynaria fortunei (Kunze) J. Sm.]
by the bone cells culture [8]. One of the major effects of
Gu-Sui-Bu on the bone cells is probably mediated by its
effect on the osteoclasts activities. However, a satisfac-
tory delivery system for Gu-Sui-Bu must be developed
before it can be used in animal model or clinical medicine.
Biomaterial composites are often tested for osteoin-
ductive and osteoconductive properties using animal
bone induction and repair models. The material to be
used requires that the material itself can be resorbed or
dissolved as bone growth occurs, and the end result is a
new remolded bone. This requires the resorbability and
the lack of toxicity or other harmful effects arising from
the release and metabolism of the degeneration products
[9,10]. The ultimate goal of implantation of biomaterials
in skeleton is to reach full integration of non-living
implant with living bone. Some investigators have
described a tricalcium phosphate carrier for rhTGFs
[11]; collagen and hydroxyapatite have also been used as
the bone-filling materials in orthopedic surgery [12].
It is known that the organic molecules can provide an
effective means to manipulate the surface properties of
the biodegradable ceramic. There are two ways to
modify the surface of the biodegradable ceramic by
organic molecules. The first one is through surface
adsorption but organic molecules will easily be washed
out in the physiological environment. The second
approach is to graft organic molecules through covalent
bond to the hydroxyl groups that are available on the
surface of the ceramics. In our institute, hexamethylene
diisocyanate (HMDI) group has been reported as a
coupling agent for calcium hydrogenphosphate (CaH-
PO4, CHP) was the candidate ceramic. The studies
showed that the HMDI could react with hydroxyl
groups of hydrogenphosphate and form a covalent bond
between HMDI and hydrogenphosphate [13]. In this
study, we try to use modified calcium hydrogenpho-
sphate (MCHP) bioceramic as a carrier to transport a
traditional Chinese medicine ''Gu-Sui-Bu'' into the bone
cell culture system. Towards this goal, we evaluated the
effect of a Gu-Sui-Bu-immobilized modified calcium
hydrogenphosphate (GI-MCHP) on the bone cells
2. Materials and methods
2.1. Materials used
HMDI was purchased from Aldrich and used in the
experiments without further purification. CHP (CaH-
PO4) powder was prepared by heating CHP dibasic
(CaHPO4?2H2O) at 2001C for about 8h, which has
been proven as pure CHP both by Fourier transforma-
tion infrared (FTIR) and X-ray diffraction (XRD)
spectroscopy. Dimethyl formamide (Aldrich, DMF,
USA) was purified with distillation and stored over
molecular sieves of 4(A. Both hydroquinone and
dibutyltin dilaurate were purchased without further
purification. Gu-Sui-Bu was purchased from a local
Chinese medicine store that would be used as raw
materials to prepare the Gu-Sui-Bu solution. EDAC was
acquired from Sigma (Sigma Co., St. Louis, MO, USA)
without further purification.
2.2. Prepared and characterized surface-modified calcium
hydrogenphosphate (MCHP)
A typical procedure for CHP surface modification
was briefly described as follows. 12.0g of dried CaHPO4
powder with an average grain size of about 0.1mm,
150ml of DMF, dibutyltin dilaurate (0.12ml) and
hydroquinone (200mg) were put into a 250ml flask. In
the system, dibutyltin dilaurate and hydroquinone were
used as catalyst and inhibitor, respectively. The flask
was then stirred for 1h in N2atmosphere with a flow
rate of 100ml/min to make sure the reaction in water-
free condition. Six ml of HMDI was added to the flask
subsequently. The reaction was kept at 501C under N2
protection for 4h to graft HMDI onto the surface of
CHP, which was the so-called surface-modified CHP
(MCHP). The MCHP powder was filtered and washed
with DMF for three times to remove excess HMDI and
HMDI-oligomer. MCHP was then further washed with
acetone for three times to remove the residual DMF.
After CHP reacted with HMDI, the product will be put
into de-ionized water to convert the terminal group of
MCHP into primary amine group. In the study, MCHP
powder would be treated by 10% nitric acid to remove
the ceramic part.
2.3. Preparation of Gu-Sui-Bu solutions
The Chinese medicine: Gu-Sui-Bu [Drynaria fortunei
(kunze) J. Sm] used in this study was supplied in dry
form by the School of Pharmacy, Taipei Medical
University, Taiwan, ROC. Its identification was authen-
ticated by experts in pharmacognosis. The procedures
for extraction of these crude drugs were standardized.
Briefly, 500gm of crude drugs were extracted by 70%
acetone for three times and the solid fragment filtered
J.-S. Sun et al. / Biomaterials 24 (2003) 873–882
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out by using Whatman No. 1 filter paper. After
condensing with reduced pressure at 401C, the solvent
was removed by freeze-drying. The condensate was
stored in freeze as stock extract before use. The stock
extract would be dissolved in water to an appropriate
concentration as Gu-Sui-Bu solution for the later
2.4. The preparation of GI-MCHP
The following methods will be used to prepare GI-
MCHP, the Gu-Sui-Bu was immobilized on the MCHP
powder by EDAC. Ten gm of MCHP powder was
soaked in 100ml of 1366ppm Gu-Sui-Bu solution at a
temperature of 251C. 0.5g of EDAC was then added to
the solution to initiate the reaction of Gu-Sui-Bu and
the primary amine that existed on the surface of MCHP
powder. After 4h, the reaction was terminated by
addition of 0.5m NaOH solution. GI-MCHP powder
would be acquired by filtration of the mixture. The
solution was collected for further analysis. In the first
part of this study, the effects of various sizes of GI-
MCHP powder on bone cell activities were evaluated by
using MTT assay as described below. Six different sizes
of particles (0–37mm, 37–53mm, 53–106mm, 106–
180mm, 180–250mm, 250–297mm) were tested for 1, 3
and 7 days period.
2.5. Osteoblast/osteoclast bone cell culture
The rat alveolar mononuclear cells-calvarias osteo-
blasts co-culture system was the same as previously
described [14]. Newborn Wistar rats (3–5 days old) were
obtained from the laboratory center of the Medical
College, National Taiwan University. Primary osteo-
blastic cells were prepared from newborn rat calvarias.
Osteoblastic cells (1?104cells/well) and alveolar mono-
nuclear cells (7.5?105cells/well) were co-cultured for 6
days in Dulbecco's modified Eagle's medium supple-
mented containing 10% fetal calf serum (Gibco BRL,
Rockville, MD, USA), in the presence of 10nm 1a,
25(OH)2D3 in 6-well plates. Penicillin G sodium
100units/ml and streptomycin 100mg/ml (Gibco BRL,
Rockville, MD, USA) were added. The culture dishes
were incubated at 371C in an atmosphere supplemented
with 5% CO2. After the cells grew to 80% confluence,
1mg/ml of various sized Gusuibu-immobilized MCHP
particles were added.
2.6. Colorimetric MTT (tetrazolium) assay for cell
viability [15]
The mitochondria activity of the bone cells after
exposure to various sizes of Gu-Sui-Bu-immbolized
MCHP was determined by colorimetric assay which
detects the conversion of 3-(4,5-dimethylthiazolyl-2)-
2,5-diphenyltetrazolium bromide (MTT, Sigma catalog
no. M2128, Sigma Co., St. Louis, MO, USA) to
formazan. For the assay, bone cells were incubated in
the presence of 1mg/ml of various sizes of GI-MCHP.
After various time intervals the supernatant was
removed, 100ml per well of MTT solution (1mg/ml in
test medium) was added and the wells were incubated at
371C for 4h to allow the formation of formazan crystal.
Again the supernatant was removed and acid isopropa-
nol (100ml of 0.04n HCl in isopropanol) was added to
all wells and mixed thoroughly to dissolve the dark blue
crystals. After a few minutes, at room temperature, to
ensure that all crystals were dissolved, the plates were
read on Micro Elisa reader (Emax Science Corp.,
Sunnyvale, CA, USA), using a test wavelength of
570nm against a reference wavelength of 690nm. Plates
were normally read within 1h after adding the
isopropanol. According to the result of MTT assay,
we selected the size of 180–250mm GI-MCHP for the
further bone cell activities evaluation.
2.7. Analysis of alkaline phosphatase (ALP), acid
phosphatase (ACP) and lactate dehydrogenase (LDH)
in culture medium
Alkaline phosphatase (ALP) (procedure no. ALP-10,
Sigma Co. USA), acid phosphatase (ACP) (procedure
no. 435, acid phosphatase, leukocyte, Sigma Co., St.
Louis, MO, USA) and lactate dehydrogenase (proce-
dure no. 228-UV, LDL-10, Sigma Co., St. Louis, MO,
USA) activity released from the cells into the medium
was measured with a commercially available assay kit.
The synthesis of ALP, ACP and LDH by bone cells was
measured by multiplying the concentration of each
measurement with the volume of the medium.
2.8. Analysis of cytoplasmic ALP and ACP
At the end of the experimental period, intracellular
ALP and ACP activity was determined following lysis of
the cells with the detergent Triton X-100 (Sigma T 8787,
Louis, MO, USA; 1% in Hanks' balanced salt solution,
30ml at 371C). Cytoplasmic ALP and ACP values were
determined using the methods described for the mea-
surements of culture media.
2.9. Analysis of prostaglandin E2(PGE2) in culture
The production of prostaglandin E2(PGE2) in culture
medium was analyzed by enzyme-linked-immunosor-
bent assay (ELISA) methods. Briefly, 50ml of standard
PGE2 (Cayman Chemical Company, MI, USA) or
sample was added per well. The tested samples were
incubated for 18h at room temperature on the bench-
top. Then 200ml of Ellman's Reagent were added to
J.-S. Sun et al. / Biomaterials 24 (2003) 873–882
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each well and incubated for 1.5h at room temperature.
The reaction was stopped and read by a Microelisa
reader (Emax Science Corp, Sunnyvale, CA, USA) at
2.10. Statistical analysis
The differences between various samples were eval-
uated by an analysis of variances statistical method. The
post hoc test performed was Bonferroni's t-test. The
level of statistical significance was defined as Po0:05:
2.11. Tartrate-resistant acid phosphatase (TRAP) stain
At the end of the experiment, cells were fixed and
stained for tartrate-resistant acid phosphatase (TRAP)
by a commercially available assay kit (procedure no.
386, acid phosphatase, leukocyte, Sigma Co., St. Louis,
MO, USA). Briefly, cells were fixed with citrate/acetone
solution (20ml of 0.038mol/l, pH 5.4, with 30.0ml
acetone) for 30s at room temperature. The fixed cells
were then incubated in the dark with a staining mixture
for 1h at 371C. The cells were washed with deionized
water for 3min, counter-stained with acid hematoxylin,
and then observed and photographed by inverted
microscopy (Olympus, IMT-II, Hatagaya, Shibuya-ku,
Tokyo, Japan).
2.12. Messenger ribonucleic acid (mRNA) isolation and
reverse transcriptase-polymerase chain reaction (RT-
Sequential digestion of newborn Wistar-rat calvaria
was performed by using a modification of the methods
described by Wong and Cohn [16]. For the assay,
1.0?106cells per well were incubated in the presence of
180–250mm GI-MCHP at 1.0mg/ml concentration.
After a predermined time, the RNA was isolated from
osteoblast cells by an acid guanidine method using
RTIzol Reagent (Gibco, USA) and reverse transcribed
to cDNA. For the quantification of mRNA, the method
developed by Nakajima et al. was used [17]. Briefly, 10-
fold titrated cDNA (1mg RNA—1pg RNA) was
amplified by polymerase chain reaction (PCR). The
amplification procedure consisted of 35 cycles (951C for
1min, 581C for 1min, 721C for 10min) with the
oligonucleotide primer sets shown in Table 1. The
cDNA amplified by PCR was fractionated by electro-
phoresis in agarose gel. The relative expression was
evaluated by detection of specific band (Genovese et al.,
1984; Collagen type I: 1208 bp; Lian et al., 1989,
Osteocalcin: 462 bp; Lee et al., 1995; Osteonectin: 890
bp; Oldberg et al., 1986, Osteopontin: 941 bp) [18–21]
visualized by ethidium bromide staining.
3. Results
3.1. Bone cell MTT viability test
zolium bromide] is a pale yellow substrate that produces
a dark blue formazan product when incubated with
living cells. MTT ring is cleaved in active mitochondria,
and the reaction occurs only in living cells [15]. Table 2
shows the size effect of various Gu-Sui-Bu-immobilized
hydrogenphosphates on bone cells viability measured by
MTT assay.
When bone cells are cultured with 1mg/ml concentra-
tions of GI-MCHP for 24h, there is significant increase
in the formation of formazan, while in the size of 180–
250mm GI-MCHP, the increased formation of formazan
attained its maximal effect and this beneficial effect
persists till the end of 7 days' culture (Table 2). In this
study, we selected the size of 180–250mm GI-MCHP for
the further bone cell activities evaluation.
3.2. Alkaline phosphatase (ALP)
Adding 180–250mm sized GI-MCHP (1mg/ml) to
bone cells culture can statistically increase the ALP
secretion into the medium especially during the first 3
days' culture (Po0:05 for the 1st day and Po0:005 for
the 3rd day); however, the intracellular ALP amount did
not show any statistically significant change (Fig. 1).
3.3. Acid phosphatase (ACP)
Adding 180–250mm-sized GI-MCHP (1mg/ml) to
bone cells culture for 7 days can statistically decrease the
intracellular total ACP amount during the experimental
Table 1
Oligonucleotide of 50and 30primers of target genes
Type I
1208 bp
462 bp
890 bp
941 bp
J.-S. Sun et al. / Biomaterials 24 (2003) 873–882
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period (Po0:05 for 1st day, Po0:001 for 3rd and 7th
day); at the end of 7 days' culture, the intracellular ACP
content of tested sample was 75.1 units, while the ACP
content in the control sample was 743.4 units (Fig. 2).
The ACP in the culture media showed statistically
significant increase at the 3rd day's culture (Po0:05)
(Fig. 2).
3.4. Lactate dehydrogenase (LDH) and PGE2in culture
There is no detectable LDH secretion to the medium
noted during the experimental period. The concentra-
tions of PGE2in culture medium of various prepara-
tions were shown in Fig. 3. The PGE2concentration in
the control medium was quite stationary during the 7
days' culture. After adding GI-MCHP, the changes of
PGE2 concentration in culture medium were quite
obvious; the concentrations of PGE2in tested medium
were always significantly higher than that of control
medium (Po0:001) (Fig. 3). The PGE2concentrations
in the tested culture medium reached its peak at the 1st
day after adding GI-CHP particles, then decreased
gradually. At the end of 7 days' culture, the PGE2
concentrations in the medium cultured with 1mg/ml GI-
MCHP was still 4.74 times that of the control medium.
3.5. TRAP stain
After 1 day's GI-MCHP treatment on bone cells, the
size of the osteoclasts detected in the experimental
culture (Fig. 4, Exp: 1D) seems decreased when com-
pared with that of the control sample (Fig. 4, Con: 1D).
After 3 day's GI-MCHP treatment on bone cells, the
osteoclasts in the experimental culture seem lost their
cell integrity and only smaller osteoclasts are still visible
(Fig. 4, Cont: 3D and Exp: 3D). Similar results were
Table 2
Effect of Gu-Sui-Bu [Drynaria fortunei (Kunze) J. Sm.]-immobolized in various-sized modified calcium hydrogenphosphate (GI-MCHP) on bone
cells viability measured by MTT assay (N ¼ 9)
(GI-MCHP: size)1 Day
Mean (SD)
P Value3 Days
Mean (SD)
P Value 7 Days
Mean (SD)
P Value
o 0.001
Fig. 1. Changes in total ALP titer after adding 1mg/ml GI-MCHP
into the bone cells culture (N ¼ 9). ALP amount in culture medium
can be significantly decreased by 1mg/ml GI-MCHP at 3 days' culture
(Po0:05 for the 1st day and Po0:005 for the 3rd day). Intracellular
total ALP synthesis did not show any statistically significant change
(Control-M: control medium, GI-MCHP-M: medium experimental
samples; Control-C: control cell, GI-MCHP-C: cells from experi-
mental samples).
Fig. 2. Changes in total ACP titer after adding 1mg/ml GI-MCHP
into the bone cells culture (N ¼ 9). After adding 1mg/ml of GI-MCH
into bone cell culture, the intracellular ACP titers were all significantly
decreased (Po0:05 for the 1st day, Po0:001 for the 3rd and 7th days);
while the ACP amount in culture medium showed significant increase
at the 3rd day's culture (Po0:05) (Control-M: control medium, GI-
MCHP-M: medium experimental samples; Control-C: control cell, GI-
MCHP-C: cells from experimental samples).
J.-S. Sun et al. / Biomaterials 24 (2003) 873–882
Page 6
observed in the 7 days' culture (Fig. 4, Cont: 7D and
Exp: 7D).
3.6. Reverse transcriptase-polymerase chain reaction
Osteoblast cells cultured with GI-MCHP showed
similar amplification with the detection for osteoblasts
phenotype expression. For the osteocalcin and osteo-
nectin genes, osteoblast cells cultured with GI-MCHP
showed amplification as much as that of control cells.
For that of the collagen and osteopontin genes, slight
decrease in cDNA was detected for the GI-MCHP-
treatment group at the 3rd day of culture for collagen
type I cDNA gene expression and at the 7th day for the
(Fig. 5).
4. Discussion
It has been reported that the use of Chinese medicines
in the treatment of fractures can induce faster and more
uniform bone ingrowths at the fracture site [22]. The
traditional Chinese medicine Gu-Sui-Bu was commonly
used to manage bone disorders and was alleged to have
obtained satisfactory results [7,8]. However, a satisfac-
tory delivery system for Gu-Sui-Bu must be developed
before it can be used in the clinical medicine. This study
sought to establish the relationships between bone cells
and the specific carrier system for Chinese medicine: the
MCHP. The MCHP carrier system for Chinese medicine
(Gu-Sui-Bu) may contribute to the possible justification
for the future clinical application in the treatment of
bone disease.
Calcium phosphate ceramics have been used as drug
carriers and delivery systems for numerous drugs:
bisphosphonates [23], chlorhexidine [24], antibiotics
[25], insulin [26], aspirin [27], hormones [28], coumadin
[29], anticancer drugs [30]. Calcium phosphate ceramics
loaded with bone growth factors are also of interest for
developing biomaterials. In this study, we expect that
the adding of GI-MCHP into the culture medium will
significantly affect the bone cells metabolism. Similar to
that noted in the previous study [8], when bone cells
were cultured with 1mg/ml of Gu-Sui-Bu immobilized
with various-sized MCHP for 24h, there is significant
increase in the formation of formazan with the maximal
effect attained at the size of 180–250mm (Table 2). This
reflected that GI-MCHP could greatly increase prolif-
erative and biosynthetic capacities of bone cells cultures.
As a cytoplasmic enzyme, LDH is present within all
mammalian cells. The plasma membrane is normally
impermeable to LDH and the enzyme is only released
into the extra-cellular fluid when the membrane has been
damaged. The release of LDH is therefore a sensitive
and accurate marker for cytotoxicity. Assays for LDH
have been used in immunology for detecting cellular
cytotoxicity [31], and in biocompatibility studies for
measuring the toxicity of biomaterials in vitro. In this
study, there is no detectable amount of LDH release
noted during the experimental period. This result means
that GI-MCHP had little or no cytotoxic effect.
The addition of Gu-Sui-Bu into bone cells culture has
a potential beneficial effect on the osteoblasts as
evidenced by the increase in both the intracellular total
ALP amount and the ALP secreted in the medium [8]. In
this study, the Gu-Sui-Bu is chemically immobilized
onto the surface of MCHP. The results showed that
when Gu-Sui-Bu was immobilized by MCHP, there was
no significant change in the intracellular ALP content
but the ALP secretion into medium still significantly
increased during the first 3 days (Fig. 1).
It was demonstrated that there was a slow release of
Gu-Sui-Bu from the GI-MCHP [32]. In the initial 12-h
soaking in the distilled water, only 19.2% of Gu-Sui-Bu
releases from the GI-MCHP. After 2-months soaking,
about 70% of Gu-Sui-Bu is still left on the surface of
GI-MCHP that is due to the covalent bond formed
between MCHP and Gu-Sui-Bu through the treatment
of EDAC. The sequential release of Gu-Sui-Bu is the
effective element in the stage of bone healing. The
released Gu-Sui-Bu can attract bone-healing factors to
the wound site and can be a long-term effective factor to
stimulate proliferation of osteoblasts, osteoprogenitors,
and bone mineralization, which will be helpful for bone
Fig. 3. Changes in PGE2titer after adding 1mg/ml GI-MCHP into the
bone cells culture (N ¼ 9). In the control medium, the PGE2
concentration was quite stationary during the 7 days' culture. The
concentrations of PGE2 in tested medium are always significantly
higher than that of control medium (Po0:001). The PGE2concentra-
tions in the tested culture medium reached its peak at the 1st day after
testing, then decreased gradually. At the end of 7 days' culture, the
PGE2concentrations in the tested sample were still 4.74 times that of
the control sample (Control-M: control medium, GI-MCHP-M:
medium experimental samples; Control-C: control cell, GI-MCHP-C:
cells from experimental samples).
J.-S. Sun et al. / Biomaterials 24 (2003) 873–882
Page 7
regeneration [32]. This reflected the fact that the
immobilization of Gu-Sui-Bu will slow down the
releasing rate of Gu-Sui-Bu and therefore the beneficial
effect on the osteoblasts proliferation is evident in the
early stage of the culture period and will be possibly
persisted for a longer duration (Fig. 1).
It is well established that osteoclasts, specialized
multinucleated giant cells responsible for bone resorp-
tion, i.e. osteoclasts, were derived from activated
macrophages and play a role in the direct lysis of bone
and release of the lysosomal enzyme acid phosphatase at
the bone–cell interface [33]. After adding 1mg/ml GI-
MCHP to bone cells culture, the intracellular ACP
amount decreased significantly, while the ACP in culture
medium was significantly increased during the experi-
mental period (Fig. 2). The decrease in the intracellular
ACP suggested that the osteoclasts cell population
decreased during the experimental period. Histomor-
phological study of the bone cells culture with
GI-MCHP treatment revealed that the size of the
Fig. 4. TRAP stain for bone cells culture after GI-MCHP treatment. In the control samples, osteoclasts were well-differentiated and progressively
increase in their size (Fig. Cont: 1D, 3D and 7D), while the size of the osteoclasts detected in the experimental culture seems decreased when
compared with that of control sample at 1 day's culture (Fig. Exp: 1D). After 3 day's GI-MCHP treatment on bone cells, the osteoclasts in the
experimental culture seem to have lost their cell integrity and only smaller osteoclasts are still visible (Fig. Exp: 3D). Similar results were observed in
the 7 days' culture (Fig. Exp: 7D) (Bar=200mm).
J.-S. Sun et al. / Biomaterials 24 (2003) 873–882
Page 8
osteoclasts detected in the experimental culture seems
decreased accompanied with their loss in cell integrity
when compared with that of the control sample (Fig. 4).
As noted in our previous work, adding Gu-Sui-Bu into
bone cell culture will induce the apoptosis of the
osteoclasts [34], the transient increase in ACP content
of the medium may manifest the release of intracellular
ACP into extracellular environment during the apoptic
process. This result suggested that the immobilized Gu-
Sui-Bu still preserved its original property such as
beneficial effect on the osteoblasts and inhibitory effect
on the osteoclasts [8].
The increased synthesis and release of PGE2may act
as an autocrine or paracrine factor. Also, the adminis-
tration of PGE2in various in vitro and in vivo models
has led to increased bone remodeling of turnover
[35–37], attributable to increased bone resorption
[38,39] or bone formation [40,41], or both. Prostaglandins
are important local regulators of bone cell function and
have been shown to have multiple effects on osteoclasts.
Using a human bone marrow culture system, Chenu
et al. have shown that PGE2 is a potent inhibitor of
multinucleate cell formation in human marrow cultures
[42]. In this study, there is a significant elevation in
PGE2secretion into the medium (Fig. 3) accompanied
with significant decrease in intracellular ACP synthesis
(Fig. 2). This is inconsistent with that of Chenu et al.,
i.e., the GI-MCHP is a potent inhibitor of osteoclasts
RT-PCR analysis was adopted for the study of cell–
biomaterials interactions at the genetic level of mRNA
expression. For the evaluation of RNA expression, the
northern blotting analysis is used as usual, but this
requires more than 1mg mRNA for one sample; i.e.,
over 107cells are needed to obtain 1mg mRNA, so it is
difficult to use northern blotting for studying cell–
biomaterials interactions [43]. In fact, RT-PCR analysis
seemed to fit our purpose, as it needs only a small
amount (pg) of mRNA for detection. In this study,
collagen type I, osteocalcin, osteonectin and osteopontin
production were measured to assess the influence of
surface composition on bone cell specific markers.
Collagen type I expression is considered an early
differentiation marker of the osteoblast phenotype
[44,45]. Osteocalcin is one of the most abundant non-
collagenous proteins in bone. The biologic function of
osteocalcin has been recently defined to act in combina-
tion with other hydroxyapatite-binding proteins as a
signal for bone remodeling [46]. Osteopontin is sup-
posed to promote or regulate the adhesion and
attachment [47]. Another protein abundant in bone
with potential roles in regulating cellular activities,
albeit not in binding the cells, is osteonectin [48]. Studies
in vitro have shown roles of osteonectin in modulating
cell division and cell migration [48] and perhaps
initiating active mineralization in the normal skeletal
tissue [49].
In the current study, cells cultured with GI-MCHP
showed similar amplification with the detection for
osteoblasts, a phenotype expression. For the osteocalcin
and osteonectin genes, osteoblast cells cultured with GI-
MCHP showed amplification as much as that of control
cells, whereas for the collagen and osteopontin genes,
only a slightly decrease in cDNA was detected by the
GI-MCHP-treatment (Fig. 5). Again, this result sug-
gested that the immobilized Gu-Sui-Bu still preserved its
original property such as the beneficial effect on the
osteoblasts [8].
Chinese biomedical scientists are now developing a
new approach to medicine by combining traditional
Chinese medicine and Western biomedical science.
Despite encouraging preliminary clinical reports about
clinical improvement with the use of the traditional
Chinese medicine Gu-Sui-Bu, basic science and con-
trolled studies reported to date have not been estab-
lished. In this study, we elucidate that modified calcium
hydrogenphosphate is a potential carrier system for the
Gu-Sui-Bu [Drynaria fortunei (Kunze) J. Sm.] which will
preserve its potential beneficial effects on the bone cells.
Although our study had limitations and our findings are
preliminary, we believe that continued and advanced
study will provide a basis for understanding the
observed bone cells responses to various traditional
Chinese medicines and develop its possible clinical
Fig. 5. Phenotype gene expression of osteoblasts after GI-MCHP
treatment. For the osteocalcin and osteonectin genes, cells cultured on
GI-MCHP showed amplification as much as that of control cells,
whereas for the collagen and osteopontin genes, a slight decrease in
cDNA was detectable for the GI-MCHP-treatment group at the 3rd
day of culture for collagen type I cDNA gene expression and at the 7th
day for the osteopontin cDNA gene expression, respectively.
J.-S. Sun et al. / Biomaterials 24 (2003) 873–882
Page 9
The authors sincerely thank the National Health
Research Institutes (ROC) for their financial support of
this research.
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[18] Genovese C, Rowe D, Kream B. Construction of DNA sequences
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[19] Lian J, Stewart C, Puchacz E, Mackowiak S, Shalhoub V, Collart
D, Zambetti G, Stein G. Structure of the rat osteocalcin gene and
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Sci USA 1989;86:1143–7.
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[21] Oldberg A, Franzen A, Heinegard D. Cloning and sequence
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[22] Huang HF, You JS. The use of Chinese herbal medicine on
experimental fracture healing. Am J Chin Med 1997;25:351–6.
[23] Denissen H, van Beek E, Lo wik C, Papapoulos S, van den Hooff
A. Ceramic hydroxyapatite implants for the release of bispho-
sphonate. Bone Miner 1994;25:123–34.
[24] Sodhi RNS, Grad HA, Smith DC. Examination by X-ray
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on hydroxyapatite. J Dent Res 1992;71:1493–7.
[25] Tung IC. In vitro drug release of antibiotic-loaded porous
hydroxyapatite cement. Artif Cells Blood Substit Immobil
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bovine insulin and bovine albumin. J Pharm Sci 1994;83:255–8.
[27] Otsuka M, Matsuda Y, Suwa Y, Fox JL, Higuchi WI. A novel
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for the quantitation of lactate dehydrogenase release in measure-
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of Chinese medicine on bone cell activities. Am J Chin Med,
accepted for publication.
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of subcutaneously administered prostaglandin E sub 2 on
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Res 1993;11:110–21.
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[39] Saffar JL, Leroux P. Role of prostaglandins in bone resorption in
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Recent Prog Horm Res 1988;44:223–62.
[41] Rasmussen H. The calcium messenger system (part 2). New
England J Med 1986;314:1164–70.
[42] Chenu C, Kurihara N, Mundy GR, Roodman GD. Prostaglandin
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[44] Lynch MP, Stein JL, Stein GS, et al. The influence of type I
collagen on the development and maintenance of the osteoblast
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[45] Stein GS, Lian JB. Molecular mechanisms mediating proli-
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development of the osteoblast phenotype. Endocr Rev 1993;1:
[46] Gunberg CM, Nishimoto SK. Vitamin K-dependent proteins of
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during progressive
J.-S. Sun et al. / Biomaterials 24 (2003) 873–882


text:
Membrane Domain
- Save the Monkey Puzzle TreeWildfires in Chile are threatening the Monkey Puzzle trees (Araucaria araucana) some of which are over a thousand years old. Chile has been in an 8 year drought that is largely blamed on global warming. "Faced with this critical situation, there is no choice but to assume that the lack of water resources is a […]
- China And Climate ChangeIn a unique statement, the ranking weather official for China has come out publicly about global warming. The BBC reports: Climate change could have a "huge impact" on China, reducing crop yields and harming the environment, the country's top weather scientist has warned, in a rare official admission. Zheng Guogang told Xinhua news agency that […]
- California Running DryCALIFORNIA — Jay Famiglietti, a senior water scientist at the NASA Jet Propulsion Laboratory in California said, "As our 'wet' season draws to a close, it is clear that the paltry rain and snowfall have done almost nothing to alleviate epic drought conditions. January was the driest in California since record-keeping began in 1895. Groundwater […]
- Save the Monkey Puzzle Tree
Global Warming
- México anuncia ambiciosa meta de reducción de emisiones post 2020March 27, 2015
- Harry Reid "Sees a Future with Both a Healthy Climate and a Healthy Economy"March 27, 2015
- Vote-a-Rama Reveals Senators' Environmental AgendaBy Jeremy SymonsIt's been a big news day in the U.S. Senate, with Senate Democratic Leader Harry Reid announcing he won't run for another term. But that's not the only news. We have had our eyes on the Senate's marathon "Vote-a-Rama" budget process that wrapped up around three-thirty this morning. A number of environmental and […]
- México anuncia ambiciosa meta de reducción de emisiones post 2020
State Of The Climate
- February 2015 National OverviewPlease see February 2015 National Overview report for more information.
- February 2015 Global AnalysisPlease see February 2015 Global Analysis report for more information.
- February 2015 DroughtPlease note that the values presented in this report are based on preliminary data. They will change when the final data are processed, but will not be replaced on these pages.
- February 2015 National Overview
Global Climate Change – Vital Signs of the Planet – News RSS Feed
- It's 'full spin ahead' for soil moisture mapperThe 20-foot (6-meter) "golden lasso" reflector antenna atop NASA's new Soil Moisture Active Passive (SMAP) observatory is now ready to wrangle up high-resolution global soil moisture data, following the successful completion of a two-part procedure to spin it up to full speed. Mission controllers at NASA's Jet Propulsion Laboratory in Pasadena, California, on Thursday, March […]
- It's 'full spin ahead' for soil moisture mapper
Natural Hazards
- In the Aftermath of Cyclone PamTwo of the hardest hit islands in Vanuatu saw vegetation go from green to brown because of the storm's fury.
- In the Aftermath of Cyclone Pam
Tag Archives: forest fires
Australia Fires
By admin | Published: November 18, 2011
Photograph of the fires taken from the sky by NASA Fire season in northern Australia usually wraps up by October, but 2011 has been an exceptional year. In mid-November, dry grasses and low humidity combined to create extremely dangerous fire conditions, prompting the Queensland Fire and Rescue service to issue a warning. Because of the […]
Posted in Uncategorized | Also tagged Australia, burn scar, climate change, fire, fires, global warming, human induced, wild fires | Comments closed

subset: commoncrawl


text:
Windows Help
It's free to register, to post a question or to start / join a discussion
Ram Problem
Likes # 0
Posted November 8, 2009 at 10:58AM
I had to install windows vista ultimate x64
i had 4 GB ram supported
Now I have Windows 7 ultimate x64 and it's
only using 3.25 GB ram
anybody knows why or what can i do to use all of the 4 GB ram ?
Thank You
Likes # 0
Posted November 8, 2009 at 7:23PM
it's showing
4.00 GB Ram (3.25 Usable)
Likes # 0
Posted November 8, 2009 at 11:55AM
if you click on the start orb and right click computer is it showing as 3.25gb there?if so try rerunning the windows experience index.
Likes # 0
Posted November 9, 2009 at 2:11AM
Click the start orb and type in "msconfig" and go to the tab labeled "boot". Go to "Advanced options" and make sure the "Maximum memory" checkbox is unchecked.
Likes # 0
Posted November 8, 2009 at 7:40PM
if you have onboard graphics it is probably using it
Reply to this topic
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Sign in to digital edition
Saturday 28 Mar 2015
By Julia Halperin. Web onlyPublished online: 01 May 2013
No one could accuse Michael Govan of being unambitious. The director of the Los Angeles County Museum of Art (Lacma) is launching a $650m capital campaign to fund the construction of an expansive new home for the institution along Wilshire Boulevard. The new museum, designed by the Pritzker Prize-winning Swiss architect Peter Zumthor, would require the demolition of much of Lacma's main campus, including three 1965 buildings by William L. Pereira—the Ahmanson, Hammer and Bing wings—as well as the 1986 Art of the Americas addition by Hardy Holzman Pfeiffer Associates of New York, according to the Los Angeles Times.
First reported in the Wall Street Journal, Zumthor's proposal would replace the angular existing galleries with sinuous glass structures that allow passersby to peek at exhibitions. Before it moves forward, the plan must be approved by Lacma's board of trustees and the Los Angeles County Board of Supervisors. In 2001, the board approved an expansion by Rem Koolhaas, but the museum aborted the project less than two years later after struggling to raise funds. Lacma instead opened the Broad Contemporary Art Museum in 2008 and the Lynda and Stewart Resnick Exhibition Pavilion in 2010, both designed by Renzo Piano. LA denizens will be able to preview the proposed building next month at "The Presence of the Past: Peter Zumthor Reconsiders Lacma" (9 June-13 September). The exhibition, part of the Getty-sponsored initiative "Pacific Standard Time Presents: Modern Architecture in LA" and co-curated by Govan, may aim to steer public opinion about the project. According to the press release, "For the first time in an exhibition, Lacma will analyse the development of its campus and explain how financial restrictions, political compromises, and unrealised plans have impacted the museum's architectural aesthetic and art-viewing experience."
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NewsAlert: German prosecutors say no suicide note or claim of...
Uncertainty awaits Sharks after drama-filled season goes off the rails
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AJAX -- PFLAG Durham Region recently brought a gender-neutral restroom challenge to Ajax council to help raise awareness about gender-neutral washrooms.
Members from PFLAG Durham Region challenged councillors to use only gender-neutral restrooms during November, to get a small sense of what it feels like for trans-gendered and gender-variant people who have to seek out such restrooms. PFLAG was hoping to inspire councillors and residents to make the washrooms available in public places.
The PFLAG campaign notes that using gendered restrooms can lead to unsafe situations for transgendered and gender-variant people, who risk being verbally abused or physically assaulted. Gendered restrooms are those designated for men or women only.
"As it stands right now, to go into that washroom is to encounter discrimination and harassment and possibly feel unsafe in that environment," said Kim Dowds from PFLAG Durham Region. "Unfortunately for many transgendered people ... they have to literally hold it."
Family restrooms are considered gender-neutral, but most do not have signage designating them as such, which Ms. Dowds said can lead to discrimination of individuals using them. She also asked Ajax council to consider adding gender-neutral signs to the Town's family washrooms.
The PFLAG challenge was presented to councils throughout Durham.
"Ajax council has asked the Town's diversity and community engagement citizen advisory committee to review the PFLAG request," noted Christie McLardie, Ajax manager of communications. "In addition, we are pleased to share that PFLAG was invited to meet with our diversity and community engagement staff committee to discuss strategies to ensure the Town continues to be supportive and inclusive to our LGBTQ community. The Town of Ajax is committed to continuing to work pro-actively to support inclusive communities through mechanisms such as our diversity and community engagement plan."
Ajax councillor Marilyn Crawford accepted the restroom challenge, and recommended others try it too.
"It was not something that I had ever thought of, so I thought, well, I'm going to see what it's like and to kind of put your feet in somebody else's shoes for a change," Coun. Crawford said. "(Family-style washrooms) serve a multi purpose, certainly for the gender-neutral and also for the families, so I think that it's essential that that is open and an option in any public facility. I think it's important, we live in a diverse world and we have to be able to be sensitive to that and to everyone's needs."
The campaign notes there are people who may require the privacy of gender-neutral, single-stalled restrooms for several reasons, including shyness and medical needs.
PFLAG Durham Region is an independently-operated, not-for-profit chapter of PFLAG Canada. The organization provides support, education and resources related to sexual orientation and gender identity and expression. For more information, visit www.PFLAGDurhamRegion.com, or contact email@example.com or 905-231-0533.
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text:
Several years ago the City of Lakewood created a list of shared organizational values that would serve as a code of conduct for City Staff. This resulted in four "Guiding Principles":
- Service
We provide prompt, courteous, and effective service, emphasizing problem solving and creativity.
- Teamwork
We communicate and work cooperatively with others, recognizing and appreciating the contributions of all.
- Integrity
We are honest, accountable and honor our commitments.
- Respect
We embrace differences and treat each other with dignity.
The Guiding Principles have stood the test of time and exist in their original form today. New employees learn about the Guiding Principles from the City Manager after they are hired; and existing employees nominate and award employees quarterly who embody the Guiding Principles. City leadership remains committed to instilling and maintaining the Guiding Principles as part of City of Lakewood's customer service oriented culture.


text:
1946 Indian Chief, black and red, recently restored, engine and body, less than 500 miles since resto. could use tires, good tread but old. on... More
2002 Indian Chief Roadmaster Ausstattung The Indians were one of the first bikes ever built. They have some experience in building bikes and therefore... More
1928 Indian 101 ScoutTo be OFFERED AT AUCTION at Auctions Americaâs Auburn Spring event, May 7-9, 2015. Click here to view this listing on... More
Offered here is an original 1948 Indian Chief Motorcycle. I am advised that only 3000 Chiefs were manufactured by the company in 1948 making... More
This Indian Scout combines exhilarating power, precise handling and rock-solid stability with laid-back comfort that riders of all sizes and levels of experience can... More
1940 Indian Four, older restoration about 15 years ago and only about 500 miles on it since restored. It's been garage kept since it... More
INDIAN: 1947 service cycle, tow bar & strap, red, single cylinder, rare (only two known), original paint, seat, $13,500. 215-536-0598, PA.... More
INDIAN motorcycles: purchase one of our fine restored motorcycles or have Maciag restore one for you. Maciag Motorcycle, 351 Park St, New Britain, CT... More


text:
The Art of Emma Johnson
5 CD / ASV / 2004-11-23Works: Wolfgang Amadeus Mozart: Concerto for Clarinet in A major, K 622; Quintet for Clarinet and Strings in A major, K 581; Bernhard Henrik Crusell: Concerto for clarinet & orchestra no 1 in E flat major, ...
Performers: Back, Gordon; Boulton, Timothy; Hirsch, Rebecca; Johnson, Emma [Clarinet]; Martineau, Malcolm; Shulman, Andrew; Takacs-Nagy, Gabor
Conductors: Leppard, Raymond; Herbig, Günther; Groves, Charles, Sir; Tortelier, Yan Pascal; Schwarz, Gerard; Bolton, Ivor
Ensemble: ; English Chamber Orchestra; Royal Philharmonic Orchestra
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Arnold, Britten, Maconchy: Clarinet Concertos / Thea King
1 CD / Hyperion / 2001-11-01Works: Malcolm Arnold: Concerto for Clarinet no 1, for clarinet & strings, Op. 20; Concerto for Clarinet no 2, Op. 115; You Know What Sailors Are, film score; Elizabeth Maconchy: Concertino for clarinet & o...
Performers: King, Thea
Conductors: Wordsworth, Barry
Ensemble: English Chamber Orchestra
"An exceptionally attractive disc, beautifully recorded and superbly performed." -Gramophone...continue...
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Come celebrate your birthday or special occasion at the best party place in town.
Our state of the art sound and lights bring Whiskey River to life. Come dance on CorpusChristi's Texas size dance floor.
DJ Twinky is our resident DJ who will be playing all your favorite Country and hip hop Videos.
Whiskey River Nightclub
Corpus Christi, Texas
361-992-4445 Club or 361-331-5066 Mobile
Cash Balloon Drop * Texas Size Food Buffet * Party Favors* Complimentary Champagne Toast at Midnight
$10 Pre Sale New Years Admission Ticket $15 Table Reservation 4 person purchase at door
$10 Table Reservation 2 person Sold Out
No refunds. The seating around the dancefloor will be first come first serve. An admission ticket is required for entry. Table reservations are optional. All food is complimentary with admission. Tickets and reservations are also available in the club during business hours.


text:
Cleaning Countertop Seams?
I have light quartzite countertops that have begun to show dark "crud" for lack of a better term in the seams. I've tried the normal warm soapy water, the 50/50 alcohol mix and even the thumbnail action, but no luck. Ha anyone found a way to clean the seam?
The best way to keep your granite counter tops looking good is to perform your granite counter top cleaning after each use. Wipe down the counters with a non-abrasive sponge or damp rag after each use.If the counter tops are sticky, spray the granite with a neutral PH granite cleaner after the initial wipe down. (NOTE: Always test your granite cleaner in a small inconspicuous area prior to use on the entire surface to determine if the product is suitable. After cleaning the granite counter tops, dry with a soft, clean rag.


text:
This standup features Captain Jack at his best sword in hand and hat in place. The standup requires minimal assembly and is a high quality cardboard with vibrant lasting color. It can be hung on the wall or left free standing.
" Thank you so much. I really love your store and think its great i can cruise around online. I really appreciate what you've done, I feel great about... "


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Prep Wrestling: ACGC picks up 2 more routs at Paynesville Tri
PAYNESVILLE — Atwater-Cosmos-Grove City beat Paynesville 58-9 and Minnewaska 44-10, and Paynesville topped Minnewaska 52-16 on Thursday at the Paynesville Triangular.
Larry Bomstad (152), Jeremy Nelson (138), Derek Dengerud (145) and Maverick Whitcomb (195) won both of their matches for ACGC.
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Google AdSense Removes Invalid Activity From Reporting
Starting this month, publishers using Google AdSense will no longer see invalid click and impression activity included in their AdSense reporting. This also means earnings deducted from that invalid activity won't be included in the reports.
This change does not impact how earnings are calculated, how publishers get paid, or how Google monitors and filters invalid activity. It just means reports will only include the activity for which publishers actually earn revenue. Google states,
In most cases, removing the invalid activity from your reports means you can expect to see a slight decrease in clicks and impressions, causing a slight increase in CPC (cost-per-click) and RPM (revenue per thousand impressions).
Essentially, you'll see a clearer representation of valid activity performance, including click-through rates, without having to deduct the invalid impressions and clicks.
Attend MarTech and hear first-hand how brands like Coca-Cola, Aetna, Dell, EMC and Netflix are harnessing the power of technology to produce exceptional customer experiences that deliver business results. Visit with over 60 companies in expo hall. Don't miss the only US-based MarTech conference this year. Register today!
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Top news and emerging ideas in display advertising, delivered every Monday.


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Register NOW for your chance to WIN 4 Tickets to expierence the bone-jarring, high-flying spectacle that is
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Promotion Description. This promotion is sponsored by WXIX-TV, 635 West Seventh Street, Cincinnati, OH 45203, and Feld Motor Sports, Inc. ("Sponsor(s)"). The promotion begins at 12:00 PM (ET), March 27, 2015 and ends at 11:59 PM (ET), April 5, 2015. Entries must be received by 11:59 PM (ET), April 5, 2015.
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List of Winners. For a list of winners, send a self-addressed stamped envelope to WXIX-TV – 635 West Seventh Street – Cincinnati, OH 45203 – attention MONSTER X Ticket Giveaway.


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Probably won't be many more of these this year
"Like". :)
Great shot!
Yes, nice!
Thank You
Nice,..these full 180 degree are hard to find!
Got one in August.
Well that rainbow sure took it's time coming out my way. Looks like a dome over all those houses. Neat.
Under the dome? Good luck to you.
Nice Pic.
WOW! Nice to see a rainbow in Oct for sure!!!!!! :-) Thanks.


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Get outside and explore this Easter! Fresh ideas for children from the Woodland Trust
Join the naturedetectives CLUB
Lots of ideas for fantasticwildlife adventuresin the woods. Great gift! find out more
Flash player is freely available at the following location:
Flash Player Download Center
Mapping system, recording system and live tracking maps by Internova.
spring fun things to do
birds Look out for our feathery friends
sticks 20 activities
spotter sheets from bugs to poo!
find a wood for winter play
grown-ups get ideas in your newsfeed
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Forum - View topicNEWS: Aniplex USA Adds Sword Art Online, Blast of Tempest, Magi Anime
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Posts: 138
Location: Bay Area
I have to lol at your friend for flipping out. The Beatles are one of the most seminal bands in the 20th century who helped chart the course of pop music's history. As a band who had a massive imprint on society, culture and music, OF COURSE they will be copied, parodied and influenced both in looks, style and music. DRRR was not the first nor will be the last to use such image in the promotion or use in their product.
And the focus on whether PMMM is great or not in Vata Raven's eyes detracts from the main point s/he was making, that AoA will put out product and price as they see fit and each individual consumer will use their own internal logic and decision making on whether to buy or not buy. Why someone chooses to get something should not bother or affect you in any way.
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Posts: 180
Location: Toronto, Canada
It's not the fact that he's buying the stuff that's bothering me in anyway, I couldn't care less whether he buys a release or not. For example of he/she said "I'm not buying this series". Well I wouldn't give it a second thought.
What does bother me if someone disagrees with my opinion without giving me a reason because then I'm just left wondering why he hates something so much. Their argument doesn't make sense. Now you could argue that it's none of my business but once someone disagrees with my opinion it kind of IS.
It's like as if someone just denounced something you feel really passionate about to your face then left without giving a single reason for what he/she did. I'm sure that would be upset and wondering what the heck is his logic?
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Vata Raven
Posts: 711
Location: TN
Why the bloody hell does it bloody matter if I go into bloody detail in why I hate something? Oh, that's right, because the fans can't expect that someone might hate a "great" show.
Refusing to watch a show because of a picture is a dumbass reason. At least I watched an episode of PMMM before putting it into the unwanted list. And so what if I had to look up the Beatles pictures, I could care less about some French band. I don't keep up with bands, I don't keep up with pop cutler, I never understand it when Funimation writes in American pop cutler.
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Posts: 138
Location: Bay Area
@manicli: I totally understand your feeling. I always want to know the mindset of someone who dislikes something I like or takes a contrary position to an issue near to me. But I've learned, you just sometimes will not get that reason and even if you do, sometimes its not one that will make a lot of sense to you anyways. So I learn to just put my hands up and and walk away. Its just going to be something that unnecessarily upsets you.
@Vata: The Beatles are English not French. And considering how old the Beatles catalog is (40+ years) it doesn't suprise me less people know about them since you only hear their songs on oldies or soft rock stations at this point. And I don't follow pop cutler either, but it sounds good to eat. In fact, its dinner time right now so I'm off for food.
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Posts: 13158
Location: NZL
One episode, huh? Then you haven't seen anything yet.
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Posts: 8451
Surely you don't mean to suggest she doesn't have anime clairvoyance?
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Posts: 13158
Location: NZL
LOL, nice one.
I've dropped plenty of shows after the first episode, usually because it is very obvious from the first episode what the rest of the show will be like. But PMMM's first episode is famous - even infamous - for not being entirely truthful. Each and every prospective viewer should give the show at least three episodes.
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Posts: 180
Location: Toronto, Canada
Because in the real world you have to justify what you say. So I thought you'd be mature enough to understand that. Do you think that if you go against someone at work without explaining your reason, its going to fly? Ofcourse not, you'll probably be looked down upon. I always have to justify what I say every week when I write a lab report(s). Heck I even have to justify myself when doing regular assignments.
Do you think I can just walk into my lab and say to my TA I think this will happen? Of course not, I'd probably fail my lab if I didn't justify myself. Do you think anyone will take me seriously if suddenly came up with a hypothesis with no evidence? No I'd probably be the laughing stock of my entire class not to mention I'd fail the assignment/lab report.
I guess logical argument is beyond you which is quite a shame because you seem to be quite opinionated but none the less, opinion without reason or evidence is just worthless. I guess humanity has declined. (I've yet to watch the anime)
As for the picture thing, I've watched the first 5 episodes of Durarara and concluded that this show has nothing to offer me in terms of entertainment or knowledge. I would be neutral but the show just had to copy something famous. And The Beatles aren't french!
So now I will conclude that not only do you fail to have cognitive abilities as you refused to understand my reasoning but your lack of research ability shows that you clearly didn't have a good education and either aren't very good at researching or you just can't read. Which ever the case may be, the appropriate plebeian term for you would be a dumbass. Either way it seems language and the ability to comprehend is beyond you. I recommend going to see a psychiatrist as this could be masking a more serious brain disease.
I suppose you're right to some extent. Though it still amazes me some people can say what ever without a reason and believe they are right or that other people will show them any respect. I guess I'm just astonished at how ignorant people have become.
But back on to topic here, it's going to be expensive but I think I'll start saving up for it so when it comes out I'll be able to afford it.
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Get off my lawn!
Posts: 2345
Location: Bellevue, WA
Aniplex, eh? Well... I guess that means these titles will be out of my price range for the foreseeable future.
I'm a sad panda. :/
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Posts: 2017
I thought the regular/bare bones edition of Madoka Magica was too expensive, not for the series but for just a DVD/BR.
However I bought it anyway so I guess my head saying it was too expensive and what I wanted conflicted and what I wanted won out.
I did get it signed by Gen as well so I guess that also helped off set the cost.
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Posts: 577
Location: Minnesota, USA
If SAO ends up being $150 for a BD box (Though who knows if that's the release format it'll get, AoA does not have an established release pattern yet), I still won't find it overpriced despite the fact I'm beginning to hate the series.
Some people need to realize "Not worth the money=/=overpriced".
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It...it's not like I post for you or anything!
Posts: 15667
Location: Toronto, Canada
|Whether something is overpriced is a subjective opinion. However, with AoA, it's possible to look at what they put out and compare it to similar releases by other R1 distribs. Many of their releases are considerably more expensive than similar offerings by other companies. Hell, many of AoA's LEs are still more expensive at their discounted price than Funi's LEs are at their full MSRP. So, in that context, it is fair to say that in comparison to other companies, many of AoA's products are overpriced given what the R1 market normally sets as a baseline price.
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Posts: 577
Location: Minnesota, USA
There is no such thing as objectively good, and objectively bad, especially when it's coming out of the mouth of a human. In order to be fully objective, you first need to get rid of all biases, which is impossible. There is no such thing as a person with no bias.
And really how is it objectively good? I've seen people like the guy who wrote this review (Actually an in-depth analysis that basically dissected the entire show) argue it's objectively bad a bit.
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Posts: 13158
Location: NZL
[If you think he's wrong then make your case. "You're wrong" is not adding to the discussion.]
This again. I'm really sick and tired of people using that moron's "review" (read: rant) as 'proof' that the show isn't all that good.
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Posts: 5086
Yeah, you realize that just because someone bloviates for 3000 words does not mean their review is an "in depth analysis", right?
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The Set List - Top 10 OPs of 2004
It's time to fly back an entire decade to the anime theme songs of yesteryear! What were the coolest openings of 2004? ― George Bush Jr. was re-elected, World of Warcraft was launched, but most importantly, a little show called Beet the Vandel Buster stole the hearts of otaku everywhere. The year was 2004, and I'm sure I remember it perfectly. Wait, maybe Beet wasn't the big anime hit of that year. ...
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