text:
I just finished my second batch of brew and, for the second time, my inital gravity was less than the expected by about 0.06 or so. The previous batch was off just about the same amount.
I am wondering if my hydrometer is a bit off.
I am currently an extract brewer. I steeped the cracked barely in the brew pot as the water temperature came up to 170 degees. I then added then added and boiled all of the extracts. After the 60 min of boiling, I lost about .5 gal to steam. After cooling and straining the work, I toppd off the primary fermenter to 5 gal. I am just eyeballing the 5 gal mark, but, based on the graduations marked on the ale pale, I am certain that I am reasonably close to the proper water level.
Should I agitate the barley while it is steeping? Perhaps I am losing a bit during the straininig process?
Any thoughts?
What was your OG?
And what grains/extracts did you use?
And how much of each?
Beer. It's what's for dinner.
I'm assuming you dropped a "0" and the reading was off 0.006. I would suspect your hydrometer is off or you aren't mixing the primary enough. The steeping grains add very little to the OG, they are just there for flavor. Check the hydrometer in water at 60F, it should be exactly 1.000. Sometimes the paper scale slides.
Remember one unassailable statistic, as explained by the late, great George Carlin: "Just think of how stupid the average person is, and then realize half of them are even stupider!"
"I would like to die on Mars, just not on impact." Elon Musk
Well, I tested my hydrometer. It was a bit of a task trying to find a glass tall enough to work. I was not willing to fill up my bottling bucket for this test. Anyway, I found an empty vodka bottle that worked and the hydrometer checked out.
I was wondering, how much of an affect would testing highly oxygenated beer have on the reading. I tested my initial gravity right after topping off the water level to 5 gal. The wort was so oxygenated that it about 3 - 4 inches of foam on the surface. I did remember to spin the hydrometer to free any clinging bubbles from the surface of the glass. I would gues that the oxygen would decrease the density of the wort, but could it possibly account for an error of 0.006?
I've been wondring the same thing about my hydrometer: I'm consistently about 4 points below the calculated gravity. Last night, I did a hefeweizen with no grains to steep--all extract. (Extracting sugars from steeping is always a bit of a crap shoot.)
5.5 gallons, 6.6 lbs wheat LME, .5 lbs wheat DME
Should be an OG of 1.048. MEasures OG (at 78 degrees, temp corrected) was 1.043.
This is how my batches always go. I measure very carefully and go to great lengths to make sure I'm getting every last drop of LME out of the can, etc.
My hydrometer checks out at 60 degrees in plain water. I'm thinking of doing a test with .25 pounds of table sugar dissolved in .25 gallons of boiling water and chilling to 60 degrees. Table sugar is a highly refined product and should always be pretty close to the target gravity of 1.046 (per pound/gallon), I would think.
I eyeball the volume markings on my primary fermenter, but the yields I get at bottling tell me that I'm not putting too much top-off water in.
[/I] Up Next - HobgoblinAfter That - Czech PilsnerPrimary - Humboldt Hop Rod (4/24)Primary - NOT Wheat AG SNCA (5/5)Secondary - Conditioning - SNCA Clone (3/3),


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Constructed on the site of a much earlier mill, the current Grade II listed Felin Cochwillan building dates back to the late 18th century. A wooden-spoked, breast-shot iron waterwheel, driven by water from the Afon Ogwen, provided the power for what was originally a 'pandy', or fulling mill. The fulling process cleanses, thickens and strengthens woven cloth, and involves washing to remove... [more...]
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N or M?
A Tommy and Tuppence Mystery
"N or M Song Susie" were the last words uttered by a government agent. Now Tommy Beresford is asked by the British Intelligence Service to try to smoke out an enemy agent who is believed to be living at the Sans Souci Hotel in the seaside town of Leahampton. Refusing to be left behind, Tuppence joins him.
Once inside the Sans Souci, the intrepid Beresfords are greeted by hostile guests, a mysterious hotelier, ransacked rooms, and a disappearance. And when Tommy himself disappears, Tuppence realizes that the stakes couldn't possibly be higher.
It is World War II, and while the RAF struggles to keep the Luftwaffe at bay, Tommy and Tuppence face an extremely sinister threat from "the enemy within."
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A TEENAGER from South Lakeland has outperformed almost 9,000 others to sing her way to the semi-final of a national singing competition.
Grace Heap, 18, of Grange-over-Sands, has made it through two gruelling heats after impressing TeenStar judges - and just one more round now stands between her and the grand final, at the O2 Arena in London.
"It's quite overwhelming," said the former Cartmel Priory pupil, who is a member of Grange Opera.
"I wasn't really expecting to get through this round but I'm very excited about the next bit and hope I can do well in that too."
Grace, who attends sixth form at Ripley St Thomas in Lancaster, got through an initial first round of heats, which around 9,000 people entered.
And after performing 'Bound To You' by Christina Aguilera at the regional final, she became one of just 12 'late teens' chosen to progress to the area final, which will take place in May with entrants from the whole of the North West and Scotland.
- A golden day as end-of-life-care nursing staff at Royal Lancaster Infirmary receive top award
- Revamp for people's park is 'over the top' claim
- Police seek man over approach to teenage girl in Furness
- Left in the dark - ill-equipped hikers call rescuers after being overtaken by nightfall
"I'm so proud of her," said mum Ann Heap.
"She's done so well and worked so hard and now she fully deserves to get through to the final."
The winner of the grand final will take away time in a recording studio, 'music development' classes and £5,000 to spend on the development of their act.
The competition was won last year by Luke Friend, who went on to X Factor.


text:
Page 1
Activation of multidrug efflux transporter activity at fertilization in
sea urchin embryos (Strongylocentrotus purpuratus)
Amro M. Hamdouna,*, Gary N. Cherrb,c, Troy A. Roepkec, David Epela
aHopkins Marine Station, Stanford University, Pacific Grove, CA 93950, United States
bDepartments of Environmental Toxicology and Nutrition, University of California, Davis, CA 95616, United States
cBodega Marine Laboratory, UC Davis, Bodega Bay, CA 94923, United States
Received for publication 8 August 2004, revised 8 September 2004, accepted 9 September 2004
Available online 14 October 2004
This study presents functional and molecular evidence for acquisition of multidrug transporter-mediated efflux activity as a consequence
of fertilization in the sea urchin. Sea urchin eggs and embryos express low levels of efflux transporter genes with homology to the multidrug
resistance associated protein (mrp) and permeability glycoprotein (p-gp) families of ABC transporters. The corresponding efflux activity is
low in unfertilized eggs but is dramatically upregulated within 25 min of fertilization; the expression of this activity does not involve de novo
gene expression and is insensitive to inhibitors of transcription and translation indicating activation of pre-existing transporter protein. Our
study, using specific inhibitors of efflux transporters, indicates that the major activity is from one or more mrp-like transporters. The
expression of activity at fertilization requires microfilaments, suggesting that the transporters are in vesicles and moved to the surface after
fertilization. Pharmacological inhibition of mrp-mediated efflux activity with MK571 sensitizes embryos to the toxic compound vinblastine,
confirming that one role for the efflux transport activity is embryo protection from xenobiotics. In addition, inhibition of mrp activity with
MK571 alone retards mitosis indicating that mrp-like activity may also be required for early cell divisions.
D 2004 Elsevier Inc. All rights reserved.
Keywords: Multidrug efflux; Fertilization; Embryo; mrp; p-gp; Activation; psc833; mk571; Calcein-am; SpABCB1; SpABCC1; SpABCC2; Mitosis
Fertilization results in physiological and structural
modification of the egg. In sea urchins, the changes in
egg activity result from a transient rise in calcium and
permanent rise in intracellular pH; these initiate structural
and enzymatic changes that ultimately lead to initiation of
DNA synthesis and rapid cell divisions (Epel, 1990; Jaffe et
al., 2001; Runft et al., 2002). The structural changes center
on reorganization of embryonic surfaces, initially arising
from cortical granule exocytosis and formation of the
fertilization envelope. The exocytosis is followed by
endocytosis and later sequential movements of vesicles to
the cortex (Matese et al., 1997; Connor and Wessel, 1998;
Smith et al., 2000). Some of these vesicles contain
constituents of the extracellular matrix and their movement
is sensitive to cytochalasin and latrunculin suggesting that
the trafficking occurs on microfilaments (Ikebuchi et al.,
2001; Wessel et al., 2002). It is also hypothesized that some
of these vesicles contain amino acid transporters that are
similarly inserted into the plasma membrane and then used
to bring in amino acids from the surrounding seawater
(Epel, 1972; Schneider, 1985; Wright and Manahan, 1989).
Here, we show that the fertilization response involves
upregulation of multidrug efflux transport activity, as part of
the re-organization of the cytoplasm and plasma membrane.
This activity protects the embryo from exogenous toxicants
and inhibition of the efflux activity affects cell division,
suggesting that the transporter activity also has some role in
passage through mitosis.
These multidrug (mdr) efflux transporters are members of
the ATP Binding Cassette (ABC) super-family and actively
0012-1606/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
* Corresponding author. Hopkins Marine Station, Stanford University,
120 Oceanview Boulevard, Pacific Grove, CA 93950.
E-mail address: email@example.com (A.M. Hamdoun).
Developmental Biology 276 (2004) 452–462
Page 2
export or sequester endogenous and exogenous compounds
(Borst et al., 2000; Dean et al., 2001; Litman et al., 2001)
including various xenobiotics (Leslie et al., 2001). The
xenobiotic transporters include permeability glycoproteins
(p-gp; Ambudkar et al., 1999), multidrug resistance associ-
mitoxantrone (mxr) resistance proteins (Litman et al., 2000).
In addition to providing protection by xenobiotic transport,
these proteins regulate cellular and developmental physiol-
ogy by efflux of endogenous signaling molecules. In
Dictyostelium, the ABC transporter RhT regulates terminal
stalk cell differentiation by efflux transport of DIF-1, an
endogenously produced differentiation factor (Good and
Kuspa, 2000). Efflux transporters are also involved in
establishing the auxin gradients required for plant develop-
ment (Friml et al., 2003; Gaedeke et al., 2001).
Most attention has focused on the protective role of these
transporters in somatic cells but they are also important in
embryo protection. For example both, embryos and adults of
the marine echiuroid worm Urechis caupo express a p-gp
transporter which protects them from naturally occurring
microbial toxins (Hamdoun et al., 2002; Toomey and Epel,
1993; Toomey et al., 1996). Embryos of Caenorhabditis
elegans express p-gp and mrp genes that are associated with
tolerance to heavy metals (Broeks et al., 1996). Similarly
mouse embryos have an efflux transport phenotype thought
to be mediated by a p-gp transporter and which protects the
embryo from xenotoxins (Elbling et al., 1993).
Previous work had suggested that early sea urchin
embryos and larvae do not have p-gp-mediated toxicant
defenses (Hamdoun et al., 2002; Toomey and Epel, 1993),
although the adults can live in polluted environments. Here,
we show that several mdr transporter-like genes are
expressed in the embryo, including at least one with
homology to p-gp and two with homology to mrps. The
major effluxactivity in the sea urchinegg is mrp-like and this
activity is dramatically upregulated within 25 min of
fertilization. This increase does not involve de novo tran-
scription or translation, but rather the transporter proteins are
present in eggs and their activity increases by a microfila-
ment-dependent process, most likely involving movement of
transportervesiclestotheeggsurfaceand insertion. Weshow
that this transport activity protects the embryo from toxic
xenobiotic compounds such as vinblastine. Finally, inhib-
ition of mrp-like transport activity slows progression through
mitosis with a major effect on progression through anaphase.
This suggests that mrp-mediated transport of some endog-
enous substance may also be required for early cell divisions.
Materials and methods
Purple sea urchins, Strongylocentrotus purpuratus, were
kept in flow through sea water tanks (approximately 138C)
and fed kelp. Gametes were collected by intracoelomic
injection with 0.5M KCl. Eggs were washed several times in
1.0 Am filtered seawater (FSW) prior to use in experiments.
For individual embryo transport measurements, embryos
were prepared by mixing eggs from 2 to 4 females, with
diluted sperm from one male. Only batches of embryos with
N90% successful development (assessed at the two-cell
stage) were used for experiments. All experiments were
conducted at 148C.
Fluorophores and inhibitors
MK571 was obtained from Cayman Chemicals. PSC833
was a gift from Novartis (Basel, Switzerland). Reversin
(R205) was initially obtained by gift of Dr. Balzas Sarkadi
and later purchased from Sigma (St. Lois, MO). Actino-
nmycin-D, emetine and verapamil were also purchased from
Sigma. Calcein-acetoxymethylester (c-am), cytochalasin-D
and latrunculin-A were obtained from Calbiochem. Sytox-
green was from Molecular Probes (Eugene, OR). DMSO was
used as a solvent for all stocks. Chemical stocks were
prepared at approximately 1000? the final desired concen-
tration such that highest concentration of DMSO did not
exceed 0.5% of the experimental volume; this solvent
concentration has no effect on transport activity or activation
of transport.
Degenerate PCR and cDNA library screening
Packed eggs or embryos were flash frozen in liquid
nitrogen, homogenized in a liquid nitrogen cooled ceramic
mortar, and then thawed into 10 volumes of homogenization
buffer (Kalinowski et al., 2003) containing proteinase K (200
Ag/ml). Homogenates were incubated for 45 min at 378C
before phenol-chloroform extraction and ethanol precipita-
tion. RNA was further purified by 8 M lithium chloride
precipitation (Sambrook and Russell, 2001). cDNA was
synthesized with an oligo-dT 12–18 primer and Superscript
Reverse Transcriptase (Invitrogen, Carlsbad, CA) according
to manufacturer's instructions. Degenerate primers (Allik-
mets and Dean, 1998; Lee et al., 1998) were used to amplify
conserved fragments of the corresponding transporter
cDNAs by RT-PCR and clone a heterologous cDNA
fragment containing a conserved 230-bp region of sea star
(Asterina miniata) transporter cDNA; 5VTCGGGATC-
TCTATCTCCTTGACGACGAATTCCG3V. This fragment
was random primed and used to screen cDNA macroarray
filter libraries of S. purpuratus (Cameron et al., 2000; Clark
et al., 1999) using standard filter hybridization techniques
(Sambrook and Russell, 2001).
A.M. Hamdoun et al. / Developmental Biology 276 (2004) 452–462
Page 3
cDNA characterization
Clones corresponding to positive coordinates on the
cDNA library screen were obtained from cDNA libraries
maintained at Caltech as part of the Sea Urchin Genome
Project (SUGP). Clones were grown on selective plates and
individual colonies were selected for sequencing. cDNA
inserts (pSport vector; Invitrogen) were analyzed for size on
agarose gels and SP6 and T7 ends were sequenced (Davis
Sequencing, Davis CA) to confirm the identity of each
clone. All of the positive clones were completely sequenced
using a transposon-based shotgun sequencing approach
(GPS, New England Biolabs; Beverly MA) on an ABI
3100 sequencer using standard cycle sequencing protocols.
Contiguous partial cDNA sequences were edited and
assembled using Sequencher. Sequencing coverage was at
least 2? for all sequences. The reading frame, homology
and sequence analyses of the putative proteins encoded by
these partial cDNAs were determined using tools at the
Expasy Molecular Biology Server (http://us.expasy.org/)
and at NCBI.
Real-time PCR
Real-time PCR was performed at the Lucy Whittier
molecular core facility, UC Davis according to the following
protocol. Fluorescent labeled TaqMan probe (5Vend, reporter
dye FAM (6-carboxyflourescein), 3V end, quencher dye
TAMRA (6-carboxytetramethylrhodamine)) were designed
using Primer Express software (Applied Biosystems, Foster
City, CA). SpABCB1 primer sequences were forward
GGAGGATGAAACCACACTCTG3V with the probe
sequence 5VTGATGCGAGTCTGGAAGCTCAACACC3V.
SpABCC1 forward primer was 5VGATGGCGATGTAAC-
GGACAGT3V and reverse 5VTGATGGTCACCGAAA-
GATCGT3V with the probe 5VCCGAGAGCGTGGGTAA-
TTCACACGA3V. TaqMan PCR systems were evaluated
according to Leutenegger et al. (1999).
For q-pcr experiments, three separate batches of sea
urchin eggs and embryos were prepared for sample
collection. Samples were taken immediately after termi-
nation of the experiments and lysed in 1? ABI lysis
buffer (Applied Biosystems). Before RNA extraction, the
lysates were transferred into 96 deep well plates and
digested with Proteinase K (Invitrogen). The total RNA
was extracted from the lysates using a 6700 automated
nucleic acid (ANA) workstation (Applied Biosystems)
according to manufacturer's instructions. Total RNA was
eluted in 100 Al of elution buffer. Complementary DNA
(cDNA) was synthesized using 100 units of SuperScript
III (Invitrogen), 300 ng random hexadeoxyribonucleotide
(pd(N)6) primers (random hexamer primer) 10 U RNase-
Out (RNase inhibitor), and 1 mM dNTPs in a final
volume of 40 Al. Each PCR reaction contained 400 nM
of each primer, 80 nM of the TaqMan probe and
commercially available PCR mastermix (TaqMan Univer-
sal PCR Mastermix, Applied Biosystems) containing 10
mM Tris–HCl (pH 8.3), 50 mM KCl, 5 mM MgCl2, 2.5
mM deoxynucleotide triphosphates, 0.625 U AmpliTaq
Gold DNA polymerase per reaction, 0.25 U AmpErase
UNG per reaction and 5 Al of the diluted cDNA sample
in a final volume of 25 Al. The samples were placed in
96 well plates and amplified in an automated fluorometer
(ABI PRISM 7700 Sequence Detection System, Applied
Biosystems). Amplification conditions were 2 min at
508C, 10 min at 958C, 40 cycles of 15 s at 958C and 60
s at 608C.
Final relative quantitation was done using the compara-
tive cycles to threshold (CT) method (User Bulletin #2,
Applied Biosystems) and is reported as relative transcription
or the n-fold difference relative to eggs. The endogenous
control for the sea urchin samples 18S rRNA was used to
normalize the mrp TaqMan signals. The DCTvalues were
calibrated against the control DCTvalues for each target
gene. The relative linear amount of target molecules relative
to the calibrator, was calculated by 2?DDCT.
Fluorescence microscopy
Accumulation of calcein was used to measure mrp trans-
porter activity in eggs and embryos. Calcein-AM (c-am) is a
non-fluorescent mrp and p-gp substrate. Eggs and embryos
is rapidly hydrolyzed by esterases to form fluorescent,
membrane-impermeant calcein, which is detected as intra-
cellular fluorescence within minutes of extracellular c-am
results in high calcein accumulation and high fluorescence
while increased transporter activity results in reduced intra-
cellular accumulation of calcein and reduced fluorescence.
Visualization of DNA for cell division experiments was
performed by fixing embryos overnight in 2% formaldehyde
and 0.1% Tween 20. Fixative was washed out 2? in Tris-
buffered saline with 0.1% Tween (TBST). Embryos were
then resuspended in 10 nM Sytox green (Molecular Probes)
in TBST. DNA was visualized with FITC wavelength filters
(as described below).
Fluorescence imaging was performed using a fixed stage
upright microscope (Olympus BX50-WI) with xenon
illumination and a cooled (148C) stage, or an inverted
microscope (Zeiss Axiovert S-100; Germany) equipped with
mercury illumination. Excitation was set to 488 Dm using a
Lambda-10 automated filter wheel (Sutter Instruments,
Novato, CA). Images were captured using a Photometrics
Coolsnap FX cooled charge coupled device (CCD) camera
(Olympus BX50-WI; Tokyo, Japan) or a Princeton Instru-
ments cooled CCD camera (Zeiss S100). A 10? objective
was used for all physiology experiments except the time-
lapse recordings of eggs at fertilization which were collected
using a 20? objective. Pseudo color micrographs and the
visualization of DNAwere performed with a 40? objective.
A.M. Hamdoun et al. / Developmental Biology 276 (2004) 452–462
Page 4
Data analysis and presentation
Fluorescence intensity of individual eggs and embryos
was measured using image analysis macros provided with
MetaMorph (Universal Imaging Corp., Downingtown, PA)
software. All comparisons of changes in fluorescence
intensity between treatments were made from data corrected
for background (70 units). P values were computed from
two sample t tests of means.
Identification of transporter genes in the egg
We confirmed that several efflux transporter mRNAs
are expressed in the sea urchin egg and we characterized
partial cDNAs encoding large fragments of three unique
mRNAs; SpABCB1 (1289AA; Genbank accession number
CK995664), encoding a p-gp, and large fragments of two
distinct mrp-like cDNAs, SpABCC1 (765AA; CK995665)
and SpABCC2 (709AA; CK995667). BlastP comparison of
the translated cDNAs on Genbank (NCBI) indicate highly
significant homology (E b 1 ? 10?50) to mammalian p-gp1,
mrp2, mrp5, and sulfonylureas (ABCC8/9). Each partial
mrp cDNA encoded about half of the expected protein with
at least one ABC transporter ATPase domain and one ABC
transporter type-1 integral transmembrane domain (Prosite).
The p-gp cDNA was nearly full-length, as expected by
homology to other p-gps. Multiple alignment of the protein
sequences by ClustalW revealed that observed urchin
sequences align with other vertebrate and invertebrate
sequences consistent with their predicted BlastP identities
(Fig. 1).
Basis of efflux transport assay
We adapted the methods of Homolya et al. (1996) and
Essodaigui et al. (1998) which employ calcein-acetoxyme-
thylester (c-am) to measure mdr transporter mediated
transport activity (Essodaigui et al., 1998; Hollo et al.,
1994, 1996, 1998; Homolya et al., 1996). Calcein-AM is a
non-fluorescent compound that is a substrate of both mrp
and p-gp. If the cell has low p-gp or mrp activity, or no
activity, c-am accumulates in the cells and is rapidly
hydrolyzed by non-specific esterases to form green,
fluorescent calcein. Unlike c-am, free calcein is relatively
membrane impermeant and therefore remains in the cell
(Essodaigui, 1998). If a cell has high levels of p-gp or mrp
transport activity, c-am is prevented from accumulating in
the cell by efflux transport and fluorescent calcein accumu-
lates to a lesser extent.
A specific test for multidrug transporter-mediated efflux
activity is to measure intracellular calcein accumulation in
the presence or absence of mdr inhibitors. If there is no or
little transport activity, incubation in a transport inhibitor has
little or no effect and there is no or little increased
fluorescence in the cell. In contrast, if there is transport
activity, incubation in an inhibitor results in increased
calcein accumulation and fluorescence as compared to the
fluorescence in the cell when no inhibitor is present.
Initiation of transport at fertilization
Fig. 2 compares the kinetics of calcein accumulation in
the presence of 250 nM extracellular c-am in eggs and in
2–4 cell embryos. Calcein accumulates in unfertilized eggs
in a linear fashion over time whereas in 2–4 cell embryos
intracellular calcein fluorescence is barely detectable even
after 2 h of incubation in c-am. The reduced calcein
accumulation in embryos relative to eggs could be a
consequence of reduction in calcein-AM permeability, a
reduction in intracellular esterase activity, or result from
increased expression of a calcein efflux transporter activity.
Fig. 3 shows that embryos incubated in the presence of the
mdr inhibitors cyclosporin-A, reversin-205, verapamil,
PSC833, or MK571 accumulate increased levels of calcein.
This indicates that the reduced accumulation in the
embryos largely results from increased efflux transport,
since the addition of the various mdr inhibitors results in
calcein accumulation.
We found that differences in passive c-am permeability
and esterase activity do not account for the changes in c-am
accumulation at fertilization since the absolute fluorescence
Fig. 1. Multiple sequence alignment (ClustalW) shows that the sea urchin multidrug transporter proteins and partial cDNAs segregate into three lineages
relative to human and invertebrate mdr proteins. SpABCB1 is a p-gp that aligns more closely with p-gp and mxr (a functional half transporter) than any of the
other proteins. The partial cDNAs SpABCC1 and SpABCC2 are both mrp-like and the former most similar to human mrp5 and 8, whereas the latter is closer to
the other invertebrate (C. elegans) mrps and human mrp2.
A.M. Hamdoun et al. / Developmental Biology 276 (2004) 452–462
Page 5
levels of embryos treated with 10 AM MK571 or PSC833
were not significantly different (P = 0.34 and 0.4,
respectively) from those of eggs treated with the same
inhibitors (Figs. 4A, B). This indicates that the levels of c-
am hydrolysis by esterases and passive permeability are
equivalent in eggs and embryos; rather, fertilization results
in a change in transport-inhibitor sensitive activity. Thus, the
change in c-am accumulation that occurs post-fertilization is
related to upregulation of transport activity. The results also
demonstrate that there is a low level of MK571-sensitive
transport in eggs prior to fertilization.
Fig. 5 shows continuous, time-lapse recording of calcein
accumulation in seven sea urchin eggs at fertilization. In this
experiment eggs were attached to a poly-lysine coated dish
and recordings were started immediately after addition of c-
am. Fluorescence was measured every 2 min and points
represent the mean fluorescence intensity in seven embryos
(Fone standard deviation). As seen, fluorescence increases
in a linear fashion prior to fertilization, indicating low efflux
activity. Within 20–30 min after fertilization, a leveling of
intracellular fluorescence accumulation occurs. This indi-
cates that a distinct increase in efflux activity begins at 20–30
min after fertilization, and a corresponding reduction in the
accumulation of intracellular calcein is observed. The
addition of the mrp-specific inhibitor MK571 (10 AM)
post-fertilization restores the unfertilized rate of calcein
Mrp- and p-gp-specific embryo transport
The results in Figs. 3 and 4 show that both mrp and p-gp-
like transport activities are upregulated in embryos but
suggest that mrp is the major transporter since the increase
in fluorescence is greater with the mrp-specific inhibitor
MK571 than with the p-gp inhibitors cyclosporin-A,
reversin 205, verapamil, and PSC833.
The results in Fig. 6 examine in greater detail the relative
contribution of p-gp and mrp transporters to c-am efflux in
embryos using the mrp- and p-gp-specific inhibitors,
MK571 and PSC833. PSC833 is effective at lower
concentrations but the effect saturates at about 3 AM and
increased levels of drug do not result in any additional
Fig. 2. Typical plot of calcein accumulation in unfertilized eggs and
embryos (250 nM extracellular c-am). Calcein-am is excluded from
embryos but in eggs it enters the cell, is hydrolyzed to fluorescent calcein,
and accumulates in a linear fashion over time (points represent mean F 1
SD of 20–100 individual embryos from four females).
Fig. 3. Change in intracellular fluorescence in eggs and 2-cell embryos treated with mdr inhibitors for 90 min, as compared to untreated controls of the same
developmental stage (bars represent means F 1 SD of 3 batches of eggs or embryos). There is a small increase in fluorescence intensity in eggs treated with the
mrp-specific inhibitor MK571 as compared to controls, but in embryos the fold change is much greater due to the significant decrease in calcein accumulation
that occurs at fertilization. MK571, 10 AM, is more potent than any of the other p-gp-specific or general mdr inhibitors at inhibiting transport in the embryos.
A.M. Hamdoun et al. / Developmental Biology 276 (2004) 452–462
Page 6
accumulation. The effect of MK571 also saturates, but at a
higher concentration. Importantly, the amount of intra-
cellular fluorescence in saturating doses of MK571 is four
to five times higher than when transport is maximally
inhibited by PSC833, indicating a greater contribution of
efflux transport activity from mrp transporters. These results
indicate that both p-gp and mrp transport activity are present
in these embryos, but that the p-gp contribution is minor
compared to the mrp contribution.
Mechanism of transport initiation at fertilization
Translation and transcription
To determine the significance of transcription and trans-
lation in the upregulation of efflux transport, eggs were
fertilized and appropriate inhibitors added after confirmation
of fertilization envelope elevation (5 min post-fertilization
(PF)). At 60 min PF the inhibitors were washed out and c-
am was added. Embryos were incubated for 90 additional
min and fluorescence intensities were measured at 150 min
PF (2-cell stage).
Initiation of transport is not affected by inhibitors of
transcription or translation, actinomycin and emetine,
respectively (Fig. 7). As demonstrated previously (Kiefer
et al., 1969; Wagenaar, 1983), embryos progress through
first cleavage normally when treated with actinomycin but
arrest prior to first cleavage with emetine treatment. The
lack of effect of actinomycin and emetine on transport
activity suggests that transcription and translation do not
mediate post-fertilization changes of transporter activity. In
addition, we directly measured the levels of transcripts of
SpABCC1 and SpABCB1 in eggs and early embryos by
real-time PCR (Table 1). These results showed an insignifi-
cant decrease in transcript abundance at fertilization
followed by a later increase in transcript abundance 90–
120 min PF. The increase in transcript abundance occurs
later than the increase in transporter activity, confirming that
the large induction in transporter activity occurs prior to de
novo transcription of transporters. It is also important to
note that the levels of these transcripts appear to be quite
low (b1 copy/egg) as determined by comparison of CT
values in eggs to those from plasmid standards (data not
Role of microfilaments in upregulation of transport
Earlier work has shown that transport systems for amino
acids and nucleosides are also activated after fertilization
and that this process is prevented by microfilament
inhibitors (Epel, 1972; Schneider, 1985; Swezey et al.,
1987). The stimulus-induced delivery of vesicles to the
plasma membrane is typically a microfilament-mediated
process requiring actin polymerization (Ikebuchi et al.,
2001; Kato et al., 2004; Wessel et al., 2002). We examined
the role of microfilaments in upregulation of transport using
the actin polymerization inhibitors cytochalasin-D (CD) and
latrunculin-A (Fenteany and Zhu, 2003; Schatten et al.,
1986; Wessel et al., 2002) and found that both compounds
prevented the upregulation of efflux transport activity since
significantly more calcein accumulated in the embryos
fertilized in these inhibitors than controls (Fig. 7). Because
Fig. 4. (A) Absolute fluorescence levels in eggs and embryos before and
after treatment with mrp- and p-gp-specific inhibitors (MK571 and
PSC833; bars represent means F 1 SD of 3 batches of eggs and embryos).
The addition of inhibitors to embryos restores fluorescence to similar
levels to eggs suggesting that bpassiveQ permeability of eggs and embryos
is similar but that MK571 and PSC833 sensitive transport increase at
fertilization. (B) Representative pseudo-color micrographs of eggs
embryos treated with mrp and p-gp inhibitors. Color bar (left) indicates
relative fluorescence intensity (white is brightest fluorescence, violet
Fig. 5. Time-based measurement of calcein accumulation in eggs (n = 7)
before, during and after fertilization. Calcein accumulates in a linear fashion
in unfertilized eggs (A) but calcein accumulation stops within 25 min of
fertilization (B) and remains at a steady state (C) until the mrp-specific
inhibitor MK571(10 AM) is added (D). These results indicate that c-am
transport is activated at fertilization.
A.M. Hamdoun et al. / Developmental Biology 276 (2004) 452–462
Page 7
these inhibitors were added 5–10 min after fertilization and
washed out prior to c-am addition this effect was not due to
their competitive inhibition of transport activity.
Latrunculin caused a nearly 30-fold increase in calcein
accumulation and its efficacy as an irreversible microfila-
ment inhibitor was also evidenced by the fact that it blocked
cell division, even though it was washed out (to prevent any
possible interaction with the transporters as a competitive
substrate for efflux) at 60 min post-fertilization. In contrast,
with 2 AM cytochalasin-D, half of the embryos completed
the first cell division consistent with reversible inhibition of
actin polymerization in these embryos (Wessel et al., 2002).
In cleavage-arrested, CD-treated embryos, there was a 7-
fold increase in fluorescence relative to controls, indicating
inhibition of the transporter. However, in CD-treated
embryos that divided, fluorescence levels were similar to
controls and we observed this effect even with 4 AM CD
(not shown) suggesting recovery from CD treatment, rather
than threshold dosage effects (Wessel et al., 2002). These
results show that upregulation of transporter activity at
fertilization is microfilament dependent.
Role of efflux transport in the embryo
Protection from xenobiotics
We tested the hypothesis that the major, mrp-
mediated, transport activity protects embryos from
xenotoxins by measuring the toxicity of the mrp
substrate vinblastine to embryos during the first cell
division in the presence or absence of 5 AM MK571. As
seen in Fig. 8, MK571 increases sensitivity to vinblastine
by at least one order of magnitude confirming that mrp
activity is protective.
Progression through the cell cycle
We also observed that treatment of embryos with 10 AM
or higher levels of MK571 alone, retards the first cell
division. To analyze this in depth, we fixed embryos at
various intervals following fertilization and chromosomal
events during the cell cycle were visualized using the
fluorescent DNA probe Sytox green. We found that
embryos treated with 10 AM MK571 take a longer time to
complete anaphase than do controls, though progression
through earlier stages was not affected (Fig. 9A). Although
the 10 AM MK571-treated embryos eventually complete cell
division and continue to divide, many develop abnormally
and have unevenly sized blastomeres (Fig. 9B) As opposed
to the 10 AM concentration of MK571, concentrations of 15
AM or higher slowed progression through all stages of the
cell cycle, and concentrations in excess of 20 AM stop cell
division all together (not shown).
This study shows a post-fertilization upregulation of
efflux transporter activity that begins 25 min post-fertiliza-
tion, coincident with the later responses to fertilization that
include delivery of other stored maternal proteins to
embryonic surfaces (Matese et al., 1997), pronuclear fusion,
and DNA synthesis (Epel, 1990). The confluence of
changes at this time may indicate a second phase of the
post-fertilization events that is linked to resumption of the
cell cycle (Jaffe et al., 2001).
This efflux activity is sensitive to inhibitors of mdr
transporters, including specific inhibitors of p-gp and mrp,
PSC833 and MK571, respectively (Gekeler et al., 1995; Liu
et al., 2001; Mayer et al., 1997; Miller et al., 2002a), and is
most effectively blocked by the mrp-specific inhibitor
MK571. The data indicate that mrp-like activity is the
major high-capacity transport activity in the sea urchin egg
and embryo but the results also reveal a lower capacity, p-
gp-like transport. The mrp activity appears to have several
Fig. 6. Representative dose–response curves of the p-gp- and mrp-specific
inhibitors (PSC833 and MK571, respectively) on calcein-am transport in 2
cell embryos. PSC833 inhibits transport at low doses, but MK571 is much
more effective at high doses.
Fig. 7. Transcription and translation (actinomycin-D and emetine) inhibitors
do not block the initiation of transport at fertilization, but cytoskeleton
disrupting compounds cytochalasin-D and latrunculin-A do. This suggests
that the initiation of transport occurs in some microfilament-dependent
process such as movement of vesicles to the plasma membrane.
A.M. Hamdoun et al. / Developmental Biology 276 (2004) 452–462
Page 8
roles. Inhibition of activity enhances toxicity of vinblastine,
indicating that this efflux transport can protect against
xenobiotics, as seen in other organisms. Inhibition of the
transporter also retards progression through the cell cycle,
with major effects on anaphase. This effect on mitosis and
the timing of upregulation of activity, point to a role for
efflux transport in promoting entry or progression through
the first cell division.
Our molecular data demonstrate that several mdr trans-
porter mRNAs are present in the egg including SpABCB1,
SpABCC1, and SpABCC2, and that they encode gene
products that could play roles in the observed xenobiotic
efflux. SpABCB1 encodes an efflux transporter similar to p-
gp, an extensively studied xenobiotic transporter that
transports c-am and various xenobiotics in mammalian cells
(Ambudkar et al., 1999). SpABCC1 encodes a protein with
homology to mammalian mrp5 which is an efflux trans-
porter with broad substrate specificity and the capacity to
transport characteristic mrp substrates including cyclic
nucleotides, anti-viral drugs and anti-cancer drugs (Kruh
and Belinsky, 2003). SpABCC1 was highly represented in
the sea urchin egg cDNA library and an analysis of the
translated partial cDNA shows that the protein it encodes
shares 65% amino acid identity to murine mrp5 in the
conserved ABC transporter ATPase domain and an overall
identity of about 22% to the same murine protein. In plants
mrp5 homologs establish auxin gradients that regulate root
development (Friml et al., 2003; Gaedeke et al., 2001).
SpABCC2 encodes a protein with homology to mammalian
sulfonylurea receptors which regulate K+ conductance in
insulin-sensitive cells and with transport properties similar
to those of mrp5 (Bryan and Aguilar-Bryan, 1997; Kruh and
Belinsky, 2003).
In addition to molecular approaches, different fluoro-
phores are used to identify the transporters responsible for
particular transport phenotypes (see Litman et al., 2001 for
review). For example, while c-am is extruded by both p-gp
and mrp proteins, rhodamine is only transported effectively
by cells that express p-gps. Consistent with this observation,
Urechis embryos and larvae express p-gp, and are capable
of high-capacity rhodamine transport, while sea urchin eggs,
embryos and larvae are not (Hamdoun et al., 2002; Toomey
and Epel, 1993). It is probable that these same studies did
not identify drug-sensitive p-gp transport in sea urchin eggs
and embryos because rhodamine was used at concentrations
(1 AM) that saturate the low-capacity p-gp-like transport we
identified here.
Similarly, our previous studies did not detect p-gp
protein (Hamdoun et al., 2002; Toomey and Epel, 1993)
on western blots of sea urchin egg proteins using the
monoclonal antibody C219 which reacts strongly with the
conserved the epitope VQEALD (Georges et al., 1990). The
corresponding epitope in sea urchin p-gp has a single amino
acid substitution of the glutamate with aspartate, which
could account for the lack of immunoreactivity of C219
with sea urchin p-gp. Given the molecular evidence and
PSC833 sensitivity, it is clear that sea urchin embryos have
some p-gp-mediated efflux activity.
Analysis by real-time-PCR (RT-PCR) shows that
SpABCC1 and SpABCB1 are rare transcripts and that
their level does not significantly increase until 90–120 min
post-fertilization. In contrast, transport activity increases
within 25 min of fertilization and the increase in efflux
activity occurs in the presence of protein and RNA
synthesis inhibitors. These data then all indicate that the
Table 1
Results of real-time q-pcr analysis of mrp (SpABCC1) and p-gp (SpABCB1) transporter mRNA abundance before and after fertilization are shown
Time PF
CT18s rRNA
CTSpABCB1Fold change SpABCC1
(relative to egg)
Fold change SpABCB1
(relative to egg)
The cycles to threshold (CT) values for each transcript measured are shown. The high CTvalues for mrp and p-gp suggest that these are low abundance
transcripts. A standard curve relative to a recombinant standard suggests they are both at b1 copy/egg (not shown). The fold change in mrp and p-gp is
expressed relative to the value of the internal standard (ribosomal RNA) for each sample. Given the low abundance of these transcripts, the changes in
transcript abundance are not likely to be significant. Values represent the means (Fone standard deviation) for three batches of eggs and embryos.
Fig. 8. Competitive inhibition of mrp activity with 5 AM MK571 sensitizes
the embryo to vinblastine measured as failure to enter and complete first
cell division. Vinblastine was added 45 min PF (after efflux transport is
initiated) either in the presence or absence of MK571 (n = 100 embryos per
point). At 145 min PF embryos were fixed and scored for completion of
first cell division.
A.M. Hamdoun et al. / Developmental Biology 276 (2004) 452–462
Page 9
upregulation of mdr efflux activity at fertilization is
mediated post-translationally.
The upregulation of mrp following fertilization may
share a common mechanism with the previously described
increase in activity of amino acid transporters after
fertilization. Activation of mrp and amino acid transport
activity are both insensitive to pharmacological inhibition of
transcription or translation, sensitive to microfilament
polymerization inhibitors and activity becomes apparent
after cortical exocytosis (Epel, 1972; Schneider, 1985). This
similar behavior among transporters suggests that they are
translocated to the plasma membrane in vesicles after
fertilization. Indeed, this may be part of a cascade of vesicle
movement events after fertilization which also includes
movement and exocytotic deposition of vesicle contents
(Matese et al., 1997) and, in at least some cases, this
movement is similarly carried out on microfilaments (Kato
et al., 2004).
This phenomenon is analogous to numerous other types
of signal-induced changes in cellular transport physiology
that are mediated by delivery of transporters to the plasma
membrane (see Bryant et al., 2002 for review), including the
well-studied example of insulin-induced translocation of the
glucose transporter GLUT4 to the membrane (Watson and
Pessin, 2001). The microfilament-mediated increase in mrp
activity in the sea urchin embryo at fertilization may also be
accompanied by other post-translational events such as
protein kinase-mediated upregulation of pre-existent and/or
newly inserted membrane transporter activity (Miller et al.,
One role of these transporters at fertilization is protec-
tion, as is demonstrated by the sensitization of embryos to
vinblastine by competitive inhibition of mrp activity with
MK571. This role is similar to the protective functions
ascribed to these transporters in somatic cells and consistent
with their expression in the adult blood–brain barrier (Miller
et al., 2002a), blood–testis barrier (Melaine et al., 2002), and
in intestinal epithelia (Miller et al., 2002b). Similarly, this is
also consistent with a role for these transporters in
protection of embryos and fits with their expression both
in embryos (Broeks et al., 1996; Elbling et al., 1993;
Toomey and Epel, 1993) and the placenta (Leazer and
Lassen, 2003; Pascolo et al., 2003).
An unexpected finding was that embryos treated with
MK571 alone exhibit a mitotic delay seen as a retardation
of progression through anaphase at 10 AM MK571 and
overall retardation at higher levels of the inhibitor. One
possibility is that the effect is a non-specific inhibition of
one of the enzymes involved in mitotic progression, such
as polo or aurora kinase. A second possibility is that the
transporter is pumping out some generally toxic substance
produced by metabolic activity and when this is not
removed cell activity is retarded with anaphase being
particularly sensitive. A reasonable candidate in this
regard is 4-hydroxynonenal which is a toxic lipid
peroxidation product associated with free radical produc-
tion and which is a known mrp substrate (Renes et al.,
2000). A third possibility is that there is some regulatory
substance that needs to be removed in order for the cell to
enter anaphase. Regulatory proteins are released by the
kinetochore and most emphasis has focused on inductive
roles of these agents; perhaps, there is also a negative
Fig. 9. (A) Graph shows the progression of control and 10 AM MK571-
treated (5 min PF) embryos through first cell division (n = 50–100
embryos per point). Progression through metaphase is similar in controls
and MK571-treated embryos. In contrast at 90 min post fertilization N60%
of control embryos are past anaphase, while virtually none of the MK571-
treated embryos have completed this stage. These MK571-treated
embryos later complete anaphase demonstrating MK571-induced devel-
opmental delay rather than cell death. (B) Micrograph. (i) Control
embryos enter prophase of the second cell division by 120 min following
fertilization. In contrast, cell division is retarded in MK571-treated
embryos and anaphase is especially prolonged (ii). Embryos treated with
10 AM MK571 eventually complete first cell division but some of them
develop abnormalities, including uneven blastomeres, as seen in the 32-
cell embryos shown (iii and iv).
A.M. Hamdoun et al. / Developmental Biology 276 (2004) 452–462
Page 10
agent that must be effluxed from the cell for anaphase to
Possible candidates for such a negative agent in sea
urchins are leukotrienes, which are derived from arach-
idonic acid oxidation by specific lipoxygenases and which
are mrp substrates (see Funk, 2001 for review). Indeed, the
active moiety of MK571 is a leukotriene analog and
MK571 can also act as a leukotriene receptor antagonist,
but the Kdfor the receptor antagonism is in the 10 nM range
(Lynch et al., 1999), whereas transport inhibition occurs in
the 10-AM range which is also where the effect on mitosis is
observed. Leukotrienes have signal transduction roles and
most work has focused on them as paracrine effectors
(Funk, 2001).
If leukotrienes are involved in regulating mitosis in the
one cell stage of the sea urchin embryo they most likely
act intracellularly, perhaps as some part of a checkpoint,
and efflux from the cell then allows progression through
the cell cycle. Silver has noted changes in leukotrienes
during the cell cycle in sand dollar eggs and posited an
effect on nuclear envelope breakdown (Silver, 2001). Perry
and Epel (1985) and Hawkins and Brash (1997) have
described lipoxygenase activities in sea urchin eggs which
similarly point to active arachidonic acid metabolism in
these cells. However, a specific 5-lipoxygenase has not
been identified.
This study points to dual roles for mrp transporters in
development. One is the well-known effect on toxicant
efflux. The other is a possible role in pumping out
endogenous compounds as part of cell regulation. Such
roles have recently been described for establishing auxin
gradients in Arabidopsis (Friml et al., 2003) and differ-
entiation in Dictyostelium (Good and Kuspa, 2000).
We thank Drs. Kathy Foltz and Forest O'Neil for their
generous assistance with screening of cDNA macroarray
libraries. Drs. Julie Alipaz and Stephen Palumbi are
thanked for providing expert guidance with sequencing
of the transporter genes and assembly of the sequences.
Ms. Nature McGinn is thanked for her assistance with
embryo imaging. Dr. Christian Leutenegger provided
technical support for q-pcr. This work was funded in part
by: UC Coastal Toxicology, Research and Teaching
Program (GC), by a NICHD Training Grant (AH) T32
HD071131, bFertilization and Early DevelopmentQ and by
the National Sea Grant Program, National Oceanic and
Atmospheric Administration, US Department of Commerce
under project number R/CZ-182 through the California Sea
Grant College (DE). The views expressed herein are those
of the authors and do not necessarily reflect the views of
NOAA or any of its subagencies. The US government is
authorized to reproduce and distribute for governmental
purposes. BML contribution number 2217.
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My Two Cents (Daily) - New Summer 2015 catalog Blu-ray slates from Scream and Kino http://t.co/qWTsXmeET5
The Wizard of Oz: An Amazing New Version of the Classic Movie
The Wizard of Oz will celebrate its 75th Anniversary in style, remastered and converted into 3D.
For a one-week engagement beginning September 20th, The Wizard of Oz will be shown with impressive visual details never before seen and in a spectacular 3D format. One of the best places to see this new vision of the film is at the TCL Chinese Theatre where it originally premiered back in 1939. [Read on here...]


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|By PR Newswire
Article Rating:
|February 25, 2013 02:00 PM EST
-- Palantir commits its innovative technology platform to help response organizations better analyze information and mobilize resources before, during and after crises
NEW YORK, Feb. 25, 2013 /PRNewswire/ -- Palantir Technologies announced a long-term Commitment to Action at the Clinton Global Initiative (CGI) Winter Meeting held last week. The CGI commitment will use cutting edge data integration and analytics to significantly improve disaster relief efforts. As part of the commitment, Palantir has combined forces with Team Rubicon and Direct Relief to help expand current efforts to better mobilize, share and allocate resources in the face of natural and other disasters.
The commitment, made through CGI, between Palantir Technologies, Team Rubicon and Direct Relief is the first of its kind - bringing together volunteers and staff with military, pharmaceutical, disaster relief and data science expertise to more efficiently meet the needs of vulnerable communities hit by calamities.
"Palantir's CGI Commitment will empower analysts and responders to leverage information so they can make better decisions regarding the critical needs in the most vulnerable areas. This will expand on the recent work with Team Rubicon in the aftermath of Sandy," said President Bill Clinton, Founder, William J. Clinton Foundation and the Clinton Global Initiative. "They did an astonishing thing for some very grateful people and I'm glad they were willing to help me here to try to do it for everybody in our country and eventually beyond our borders."
During Hurricane Sandy, Direct Relief used Palantir to integrate information from their own databases, partnering organizations, and open source datasets to conduct meteorological, social vulnerability, supply chain, and health risk analyses of areas in the path of the storm. This resulting information enabled Direct Relief to pre-position supplies and medicine before the storm hit, analyze real-time weather data during the storm, and provide emergency medical supplies where they were needed most in the wake of the storm. After the storm, volunteers and staff on the ground used Palantir Mobile to collect and send data to Direct Relief headquarters in real time. Palantir was also used to monitor infectious disease trends and route medical resources to the areas in greatest need. Team Rubicon also used the software to manage more than 10,000 volunteers and rapidly integrate photos, video, text and phone calls to address the urgent needs of victims in the Rockaways.
"The global challenge of humanitarian relief following a natural disaster is one of the most important problems of our time," said Dr. Alexander Karp, founder and CEO of Palantir Technologies. "We are delighted to join the Clinton Global Initiative, and to work with Team Rubicon and Direct Relief. Together we can make a big difference."
Palantir can be rapidly deployed to laptops and smart phones and allows dispatchers and analysts working under critical time pressure to fuse together and analyze large amounts of data from different sources. Originally developed for use by the intelligence and defense community, the platform includes robust, built-in measures to allow users to access and share information across the community while protecting privacy and civil liberties.
For Additional Information contact [email protected]
About the Clinton Global Initiative
Established in 2005 by President Bill Clinton, the Clinton Global Initiative (CGI) convenes global leaders to create and implement innovative solutions to the world's most pressing challenges. CGI Annual Meetings have brought together more than 150 heads of state, 20 Nobel Prize laureates, and hundreds of leading CEOs, heads of foundations and NGOs, major philanthropists, and members of the media. To date CGI members have made more than 2,300 commitments, which have improved the lives of over 400 million people in more than 180 countries. When fully funded and implemented, these commitments will be valued at more than $73.1 billion.
CGI also convenes CGI America, a meeting focused on collaborative solutions to economic recovery in the United States, and CGI University (CGI U), which brings together undergraduate and graduate students to address pressing challenges in their community or around the world. For more information, visit clintonglobalinitiative.org and follow us on Twitter @ClintonGlobal and Facebook at facebook.com/clintonglobalinitiative.
About Palantir
Palantir offers the world's leading data integration platform. The Palantir Platform works at any scale and empowers the entire analysis stack through its ability to access any data store in the enterprise, while enforcing robust security and civil liberties protections. Because it is a productized solution, the cost and time required for implementation is significantly less than for a custom-built solution.
Palantir is headquartered in Palo Alto, Calif., with offices in New York, Washington, D.C., and London. Additional information is available at www.palantir.com.
The world's leading Cloud event, Cloud Expo has launched Microservices Journal on the SYS-CON.com portal, featuring over 19,000 original articles, news stories, features, and blog entries. DevOps Journal is focused on this critical enterprise IT topic in the world of cloud computing. Microservices Journal offers top articles, news stories, and blog posts from the world's well-known experts and guarantees better exposure for its authors than any other publication. Follow new article posts on Twitter at @MicroservicesE
Mar. 30, 2015 12:00 PM EDT Reads: 1,483
SYS-CON Events announced today the IoT Bootcamp – Jumpstart Your IoT Strategy, being held June 9–10, 2015, in conjunction with 16th Cloud Expo and Internet of @ThingsExpo at the Javits Center in New York City. This is your chance to jumpstart your IoT strategy. Combined with real-world scenarios and use cases, the IoT Bootcamp is not just based on presentations but includes hands-on demos and walkthroughs. We will introduce you to a variety of Do-It-Yourself IoT platforms including Arduino, Raspberry Pi, BeagleBone, Spark and Intel Edison. You will also get an overview of cloud technologies s...
Mar. 30, 2015 11:00 AM EDT Reads: 2,136
SYS-CON Events announced today that SafeLogic has been named "Bag Sponsor" of SYS-CON's 16th International Cloud Expo® New York, which will take place June 9-11, 2015, at the Javits Center in New York City, NY. SafeLogic provides security products for applications in mobile and server/appliance environments. SafeLogic's flagship product CryptoComply is a FIPS 140-2 validated cryptographic engine designed to secure data on servers, workstations, appliances, mobile devices, and in the Cloud.
Mar. 30, 2015 11:00 AM EDT Reads: 1,485
Wearable technology was dominant at this year's International Consumer Electronics Show (CES) , and MWC was no exception to this trend. New versions of favorites, such as the Samsung Gear (three new products were released: the Gear 2, the Gear 2 Neo and the Gear Fit), shared the limelight with new wearables like Pebble Time Steel (the new premium version of the company's previously released smartwatch) and the LG Watch Urbane. The most dramatic difference at MWC was an emphasis on presenting wearables as fashion accessories and moving away from the original clunky technology associated with t...
Mar. 30, 2015 10:00 AM EDT Reads: 1,453
After making a doctor's appointment via your mobile device, you receive a calendar invite. The day of your appointment, you get a reminder with the doctor's location and contact information. As you enter the doctor's exam room, the medical team is equipped with the latest tablet containing your medical history – he or she makes real time updates to your medical file. At the end of your visit, you receive an electronic prescription to your preferred pharmacy and can schedule your next appointment.
Mar. 30, 2015 10:00 AM EDT Reads: 869
Containers and microservices have become topics of intense interest throughout the cloud developer and enterprise IT communities. Accordingly, attendees at the upcoming 16th Cloud Expo at the Javits Center in New York June 9-11 will find fresh new content in a new track called PaaS | Containers & Microservices Containers are not being considered for the first time by the cloud community, but a current era of re-consideration has pushed them to the top of the cloud agenda. With the launch of Docker's initial release in March of 2013, interest was revved up several notches. Then late last...
Mar. 30, 2015 09:15 AM EDT Reads: 2,245
The WebRTC Summit 2014 New York, to be held June 9-11, 2015, at the Javits Center in New York, NY, announces that its Call for Papers is open. Topics include all aspects of improving IT delivery by eliminating waste through automated business models leveraging cloud technologies. WebRTC Summit is co-located with 16th International Cloud Expo, @ThingsExpo, Big Data Expo, and DevOps Summit.
Mar. 30, 2015 09:00 AM EDT Reads: 1,635
SOA Software has changed its name to Akana. With roots in Web Services and SOA Governance, Akana has established itself as a leader in API Management and is expanding into cloud integration as an alternative to the traditional heavyweight enterprise service bus (ESB). The company recently announced that it achieved more than 90% year-over-year growth. As Akana, the company now addresses the evolution and diversification of SOA, unifying security, management, and DevOps across SOA, APIs, microservices, and more.
Mar. 30, 2015 08:30 AM EDT Reads: 2,073
GENBAND has announced that SageNet is leveraging the Nuvia platform to deliver Unified Communications as a Service (UCaaS) to its large base of retail and enterprise customers. Nuvia's cloud-based solution provides SageNet's customers with a full suite of business communications and collaboration tools. Two large national SageNet retail customers have recently signed up to deploy the Nuvia platform and the company will continue to sell the service to new and existing customers. Nuvia's capabilities include HD voice, video, multimedia messaging, mobility, conferencing, Web collaboration, deskt...
Mar. 30, 2015 01:00 AM EDT Reads: 1,489
SYS-CON Media announced today that @WebRTCSummit Blog, the largest WebRTC resource in the world, has been launched. @WebRTCSummit Blog offers top articles, news stories, and blog posts from the world's well-known experts and guarantees better exposure for its authors than any other publication. @WebRTCSummit Blog can be bookmarked ▸ Here @WebRTCSummit conference site can be bookmarked ▸ Here
Mar. 29, 2015 10:00 PM EDT Reads: 1,835
SYS-CON Events announced today that Cisco, the worldwide leader in IT that transforms how people connect, communicate and collaborate, has been named "Gold Sponsor" of SYS-CON's 16th International Cloud Expo®, which will take place on June 9-11, 2015, at the Javits Center in New York City, NY. Cisco makes amazing things happen by connecting the unconnected. Cisco has shaped the future of the Internet by becoming the worldwide leader in transforming how people connect, communicate and collaborate. Cisco and our partners are building the platform for the Internet of Everything by connecting the...
Mar. 29, 2015 07:00 PM EDT Reads: 5,259
Temasys has announced senior management additions to its team. Joining are David Holloway as Vice President of Commercial and Nadine Yap as Vice President of Product. Over the past 12 months Temasys has doubled in size as it adds new customers and expands the development of its Skylink platform. Skylink leads the charge to move WebRTC, traditionally seen as a desktop, browser based technology, to become a ubiquitous web communications technology on web and mobile, as well as Internet of Things compatible devices.
Mar. 29, 2015 06:00 PM EDT Reads: 1,871
SYS-CON Events announced today that robomq.io will exhibit at SYS-CON's @ThingsExpo, which will take place on June 9-11, 2015, at the Javits Center in New York City, NY. robomq.io is an interoperable and composable platform that connects any device to any application. It helps systems integrators and the solution providers build new and innovative products and service for industries requiring monitoring or intelligence from devices and sensors.
Mar. 29, 2015 06:00 PM EDT Reads: 1,500
Docker is an excellent platform for organizations interested in running microservices. It offers portability and consistency between development and production environments, quick provisioning times, and a simple way to isolate services. In his session at DevOps Summit at 16th Cloud Expo, Shannon Williams, co-founder of Rancher Labs, will walk through these and other benefits of using Docker to run microservices, and provide an overview of RancherOS, a minimalist distribution of Linux designed expressly to run Docker. He will also discuss Rancher, an orchestration and service discovery platf...
Mar. 29, 2015 04:15 PM EDT Reads: 2,449
SYS-CON Events announced today that Vitria Technology, Inc. will exhibit at SYS-CON's @ThingsExpo, which will take place on June 9-11, 2015, at the Javits Center in New York City, NY. Vitria will showcase the company's new IoT Analytics Platform through live demonstrations at booth #330. Vitria's IoT Analytics Platform, fully integrated and powered by an operational intelligence engine, enables customers to rapidly build and operationalize advanced analytics to deliver timely business outcomes for use cases across the industrial, enterprise, and consumer segments.
Mar. 29, 2015 03:30 PM EDT Reads: 2,197
SYS-CON Events announced today that Solgenia will exhibit at SYS-CON's 16th International Cloud Expo®, which will take place on June 9-11, 2015, at the Javits Center in New York City, NY, and the 17th International Cloud Expo®, which will take place on November 3–5, 2015, at the Santa Clara Convention Center in Santa Clara, CA. Solgenia is the global market leader in Cloud Collaboration and Cloud Infrastructure software solutions. Designed to "Bridge the Gap" between Personal and Professional Social, Mobile and Cloud user experiences, our solutions help large and medium-sized organizations dr...
Mar. 29, 2015 03:00 PM EDT Reads: 2,877
SYS-CON Events announced today that Liaison Technologies, a leading provider of data management and integration cloud services and solutions, has been named "Silver Sponsor" of SYS-CON's 16th International Cloud Expo®, which will take place on June 9-11, 2015, at the Javits Center in New York, NY. Liaison Technologies is a recognized market leader in providing cloud-enabled data integration and data management solutions to break down complex information barriers, enabling enterprises to make smarter decisions, faster.
Mar. 29, 2015 03:00 PM EDT Reads: 3,490
@ThingsExpo has been named the Top 5 Most Influential M2M Brand by Onalytica in the 'Machine to Machine: Top 100 Influencers and Brands.' Onalytica analyzed the online debate on M2M by looking at over 85,000 tweets to provide the most influential individuals and brands that drive the discussion. According to Onalytica the "analysis showed a very engaged community with a lot of interactive tweets. The M2M discussion seems to be more fragmented and driven by some of the major brands present in the M2M space. This really allows some room for influential individuals to create more high value inter...
Mar. 29, 2015 01:45 PM EDT Reads: 4,684
The list of 'new paradigm' technologies that now surrounds us appears to be at an all time high. From cloud computing and Big Data analytics to Bring Your Own Device (BYOD) and the Internet of Things (IoT), today we have to deal with what the industry likes to call 'paradigm shifts' at every level of IT. This is disruption; of course, we understand that – change is almost always disruptive.
Mar. 29, 2015 11:45 AM EDT Reads: 1,143
SYS-CON Events announced today that Akana, formerly SOA Software, has been named "Bronze Sponsor" of SYS-CON's 16th International Cloud Expo® New York, which will take place June 9-11, 2015, at the Javits Center in New York City, NY. Akana's comprehensive suite of API Management, API Security, Integrated SOA Governance, and Cloud Integration solutions helps businesses accelerate digital transformation by securely extending their reach across multiple channels – mobile, cloud and Internet of Things. Akana enables enterprises to share data as APIs, connect and integrate applications, drive part...
Mar. 28, 2015 04:15 PM EDT Reads: 1,571


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Hooves N Wheels RV Park
Weatherford, Texas
Phone: (817) 599-4686
Visit Campground Website
Sites: 70
Recent Rate: $26 5/2013
Latitude: 32.6986661999854
Longitude: -97.7707704364929
These reviews are the opinion of an RVParkReviews' member and not the views of RVParkReviews.com
Date of Stay: May, 2013 - $26.00
Park is off of the freeway. No road or /RR noise. Road and sites are dirt/gravel. Good water, power, and cell (AT&T). Staff was friendly. Not far from shopping, etc. We would camp here again. We camped at Hooves N Wheels RV Park in a Motorhome.Undo
Date of Stay: December, 2012 - $26.00
We stayed here on 2 separate occasions in December and the rate reflects a Good Sam discount. The sites were large and spacious. The owner was very friendly. This park is off the freeway enough to be considered quiet. We would stay here again when passing through the area. We camped at Hooves N Wheels RV Park in a Motorhome.Undo
65 Ratings
Weatherford, Texas
4 Ratings
Waxahachie, Texas
2 Ratings
Waxahachie, Texas
1 Ratings
Wallisville, Texas
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Flexible Rate without restrictions with free wellness path in our Spa San Lorenzo.
Gourmet Dinner in our \"Sopra le Mura\" restaurant and overnight stay.
A short break to regenerate..
2 Nights of Relax in our Palace
3 Nights /4 Days in Armony and Wellness
5 Nights of Relax in our Palace
Easter Wellness Package in Colle di Val d\'Elsa, two nights, two dinners and massage...


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Mother's Day Prayer Warriors - Ministry Videos
Embed seekfirstmedia 2023 views 2013-05-06T19:29:05 This Mother's Day we pay tribute to our mothers - the prayer warriors. The artwork was supplied by Ron DiCianni/Tapestry Productions.
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This week, VIKING Life-Saving Equipment took a step towards gaining approval for the VIKING LifeCraft, its alternative life-saving appliance (LSA) for passenger ships.
MAN Diesel & Turbo successfully demonstrated its ME-LGI concept engine in front of customers and partners at its Diesel Research Center in Copenhagen on March 17th, the company announced today.
Maersk Line, the world's largest container company, has selected Transas Marine for ECDIS implementation on more than 100 vessels, Transas announced today. The
Keppel FELS Limited (Keppel FELS), a wholly owned subsidiary of Keppel Offshore & Marine Ltd (Keppel O&M), has delivered a KFELS B Class jackup rig to Mexican national oil company,
Harkand won a multi-million contract with Maersk Oil North Sea Ltd for the provision of DSV services in the North Sea region.
The 12-month contract will be serviced by Harkand's two DSVs,
China's air force conducted its first drill above the western Pacific Ocean on Monday, the Defence Ministry said, in a move that could exacerbate tensions with


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- Kimba, Der Weisse Löwe
- Blank Manuscript Paper
- Going Over The Sea (We're going this way that way)
- Hop Little Bunnies
- Für Elise Boogie
- Stille Nacht (Jazz Version)
- Auld Lang Syne (jazzy arrangement)
- Oh Little Town Of Bethlehem (jazzy arrangement)
- Linus Jude The Dude Boogie
- Nigel Incubator Jones Boogie
- More...
- Anne Mit Den Roten Haaren
- Marco
- Mein Name Ist Drops
- Pat und Patachon
- Puschel Das Eichhorn
- Rollet, Rollt Ihr Wogen
- Schaukellied
- Schlaft Alle (Schlaflied aka Sov Alla)
- So Will Ich Immer Reisen (Sommerlied)
- Ulme - Lied
- More...
- 1000 Beautiful Things
- A Boy Was Born
- A Jazzy Beat
- A Little Prank
- A Love Story
- A Rose Is Still A Rose
- A Waltz For A Night
- Act I Opening - Part I
- Adieu A' L'automne
- Adoramus Te (arr. Tom Fettke)
- More...
- Colour Me In
- Fade Out Lines
- Falling Short
- From Eden
- I Don't Want To Change You
- It Takes A lot To Know A Man
- Stay Gold
- The Box
- The Greatest Bastard
- Trusty And True
- More...
Writer: Richard Smith, Johnny Mathis, Perry Como
The sheet music for Winter Wonderland written by Richard Smith, is available below.
In order to see the sheet music for Winter Wonderland please click on the preview button next to the score you are interested in.
Dick Smith was inspired to write this song when he saw Honesdale´s Central Park (Pennsylvania) covered in snow. It was first recorded by Richard Himber and his orchestra for RCA in 1934. The orchestra included many famous studio musicians like Artie Shaw. Singer-songwriter Johnny Mercer took the song to No.4 on the Billboard Charts; Perry Como recorded a new version in 1959 for his Christmas album. Due to its theme "Winter Wonderland" is often thought of as a Christmas song, but the Christmas season is never mentioned. As the lyrics contain the word "Sleighbells" it is not surprising then that many people associate this song with Christmas.
We have 17 arrangements of this song available for 10 instruments.
- A Kiss To Build A Dream On
- A Kiss To Build A Dream On (from Sleepless In Seattle)
- Ain't Misbehavin'
- All Of Me
- Back O' Town Blues
- Basin Street Blues
- Between The Devil And The Deep Blue Sea
- Bibbidi-Bobbidi-Boo (The Magic Song)
- Bibbidi-Bobbidi-Boo (The Magic Song) (Accordion)
- Bibbidi-Bobbidi-Boo (The Magic Song) (Piano Duet)
- Bibbidi-Bobbidi-Boo (The Magic Song) (from Cinderella)
- Blueberry Hill
- Cabaret
- Chinatown, My Chinatown
- Do You Know What It Means To Miss New Orleans
- Dream A Little Dream Of Me
- Georgia On My Mind
- Gully Low Blues
- Hear Me Talkin' To Ya
- Hello Dolly
- Honeysuckle Rose
- How Come You Do Me Like You Do
- I Ain't Gonna Give Nobody None Of This Jelly Roll
- I Can't Believe That You're In Love With Me
- I Got Rhythm
- I Still Get Jealous
- In The Shade Of The Old Apple Tree
- Is You Is Or Is You Ain't My Baby?
- It Don't Mean A Thing (If It Ain't Got That Swing)
- La Vie En Rose (Take Me To Your Heart Again)
- Mack The Knife
- Makin' Whoopee!
- Muskrat Ramble
- Nobody Knows The Trouble I've Seen
- Ol Man Mose
- On The Sunny Side Of The Street
- On Treasure Island
- Pennies From Heaven
- Save It Pretty Mama
- Shoe Shine Boy
- Someday (You'll Be Sorry)
- Song Of The Islands
- St. Louis Blues
- Stars Fell On Alabama
- Struttin' With Some Barbecue
- Swing That Music
- That's A Plenty
- The Frim Fram Sauce
- The Music Goes Round And Round
- The Whiffenpoof Song (Baa! Baa! Baa!)
- Twelfth Street Rag
- Way Down Yonder In New Orleans
- We Have All The Time In The World
- What A Wonderful World
- When I Grow Too Old To Dream
- When It's Sleepy Time Down South
- When The Saints Go Marching In
- When You're Smiling (The Whole World Smiles With You)
- Winter Wonderland
- You Are My Lucky Star
- Zip-A-Dee-Doo-Dah
- A Certain Smile
- Chances Are
- I Was Telling Her About You
- It's Not For Me To Say
- Misty
- The First Time Ever I Saw Your Face
- Winter Wonderland
- A Garden In The Rain
- All At Once You Love Her
- And I Love You So
- Catch A Falling Star
- Caterina
- Christmas Dream (from The Odessa File)
- Dance Only With Me
- Don't Let The Stars Get In Your Eyes
- Forever And Ever
- Have I Stayed Away Too Long
- Home For The Holidays
- (There's No Place Like) Home For The Holidays
- Hot Diggity (Dog Ziggity Boom)
- I Want To Give (Ahora Que Soy Libre)
- Idle Gossip
- I'm Confessin' That I Love You
- It's Beginning To Look A Lot Like Christmas
- It's Impossible (Somos Novios)
- Jesus, I Come
- Magic Moments
- Papa Loves Mambo
- Papa Loves Mambo (from Ocean's Eleven)
- Please, Mr. Sun
- Round And Round
- Santa Claus Is Comin' To Town
- The Way We Were
- There Is No Christmas Like A Home Christmas
- Till The End Of Time
- Wanted
- Winter Wonderland
- You Must Have Been A Beautiful Baby
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Fall Camp '14: Quarterbacks Preview
Mitch Leidner will lead a young group of QBs in 2014
Spring Game 2014
Football in the Spring
2014: Redshirted.
OVERVIEW: Enrolled at Minnesota in January 2014 and participated in spring practice.
HIGH SCHOOL: Attended Wyandotte High School in Kansas City, Kan. two-time all-state quarterback Kansas City-Atchison League Player of the Year 2013 Sports Highlights Unlimited Kansas City Prospect of the Year also named all-city, all-county and all-area passed for 2,527 yards and 26 touchdowns as a senior in 2013 also ran for 519 yards and nine touchdowns in 2013 rated a three-star recruit by ESPN, Rivals.com and Scout.com also a letterwinner in basketball and track.
PERSONAL: Born Dec. 16, 1995 son of Leonard McKinzy and step-mother Andrean McKinzy has five siblings.
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StartUpHire is the place to find Accounting jobs in and near Mississippi. Review the list of Accounting Mississippi career opportunities to the left. These Accounting jobs represent some of the most rewarding careers available.
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Location: Ridgeland, MS Position Summary: The revenue accountant is responsible for reviewing sales contracts, orders and invoices for compliance with software revenue recognition guidance and the company?s revenue recognition policies. The revenue accountant is also responsible1/24/2015 Ridgeland, MS Save This Job
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DESCRIPTION Be Part of the Next Generation of Flight Aurora Flight Sciences is a leader in unmanned aerial systems, with a fleet of innovative aircraft designed to empower small tactical units, and safeguard our borders. Aurora provides the capabilities and process controls of2/10/2015 Columbus, MS Save This Job


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Alexandria Education Foundation welcomes four board members
The Alexandria Public Schools Education Foundation (AEF) recently announced the election of four new members to its board of directors: Jean Bristow, Craig Olson, Bonnie Schnell and Randy Winger.
"We are excited to welcome these new members," said Greg Trumm, foundation board president. "Each one has close ties to the community and share a passion for education that will help advance the mission of the foundation."
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Dear SAIC Alumni,
Welcome to my.saic.edu, SAIC's alumni networking site. We are glad you're here and hope you will visit frequently to stay connected with the SAIC community.As one of SAIC's 15,000 alumni, you shape, enrich, and inspire the cultural landscape through your work. And this space is dedicated to celebrating the alumni community's innovation and creativity.The SAIC online alumni community offers opportunities for social and career networking. Create your own profile, upload images of your work, and search for your classmates. Join groups, register for events, and receive SAIC Alumni e-news and e-vites. Follow your fellow alumni's exhibitions, publications, performances, and achievements, and let us know about your own upcoming news, events, and announcements.We hope you will join us online, on campus, and at exhibitions and events across the world and throughout the year.If you have questions or information you would like to share with our team, please email email@example.com or call us at 312.499.4195. Best wishes,Melissa Cowley Wolf
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3/26/2015 Anida Yoeu Ali Studio Update
3/26/2015Wafaa Bilal in 56th Venice Biennale
3/23/2015Alumnae Seek to Establish Andersonville Community Art Space
3/14/2015 » 4/26/2015Rachel Adams Curating Andy Coolquitt's Work
3/28/2015Career + Co-op: Saturday Skill Share Online Presence
3/28/2015Closing Reception & Artist's Talk: Negin Sharifzadeh & Story of a Curse #1
3/28/2015 » 4/4/2015SAIC Career + Co-Op: Animation Festivals
3/28/2015Opening Reception: Shifting Impressions


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Mighty Dungeons Cheats for iPhone - iPodHome / iPhone - iPod /
This page contains Cheats for Mighty Dungeons organized by sections for iPhone - iPod. Mighty Dungeons is a Action game, made by Josh Presseisen, released on Apr 11, 2013 and published by Josh Presseisen. If you can't find a hint or secret in our list, then please check this page periodically for the latest updates.
- Developer:
Mighty Dungeons iPhone - iPod CheatsJosh Presseisen
- Publisher: Josh Presseisen
- Genre: Action
- Release Date: Apr 11, 2013
- ESRB: Not Set
- Other Platforms
Stuck on this game? Set a specific, clearly defined question and let others answer your question or view answers to previously asked questions. If you think you are an Mighty Dungeons expert then please try to help others with their questions.
Don't forget to Bookmark this page for the latest Mighty Dungeons Cheats. At this moment there are no cheats or walkthroughs available for Mighty Dungeons. If you know cheats, secrets, hints or glitches for this game then please Submit them.
Mighty Dungeons - Gameplay Video
- Apr 14, 2013, Views: 52
Mighty Dungeons - Official Launch Trailer
- Apr 24, 2013, Views: 64
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