text:
Page 1
Expression, purification and characterization of the secreted
luciferase of the copepod Metridia longa from Sf9 insect cells
Galina A. Stepanyuka,b, Hao Xua, Chia-Kuei Wua, Svetlana V. Markovab, John Leea, Eugene
S. Vysotskia,b*, and Bi-Cheng Wanga
aDepartment of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, USA
bPhotobiology Lab, Institute of Biophysics Russian Academy of Sciences, Siberian Branch, Krasnoyarsk,
Metridia luciferase is a secreted luciferase from a marine copepod and uses coelenterazine as a
substrate to produce a blue bioluminescence (λmax = 480 nm). This luciferase has been successfully
applied as a bioluminescent reporter in mammalian cells. The main advantage of secreted luciferase
as a reporter is the capability of measuring intracellular events without destroying the cells or tissues
and this property is well suited for development of high throughput screening technologies. However
because Metridia luciferase is a Cys-rich protein, E. coli expression systems produce an incorrectly
folded protein, hindering its biochemical characterization and application for development of in
vitro bioluminescent assays. Here we report the successful expression of Metridia luciferase with its
signal peptide for secretion, in insect (Sf9) cells using the baculovirus expression system.
Functionally active luciferase secreted by insect cells into the culture media has been efficiently
purified with a yield of high purity protein of 2–3 mg/L. This Metridia luciferase expressed in the
insect cell system is a monomeric protein showing 3.5-fold greater bioluminescence activity than
luciferase expressed and purified from E. coli. The near coincidence of the experimental mass of
Metridia luciferase purified from insect cells with that calculated from amino acid sequence, indicates
that luciferase does not undergo posttranslational modifications such as phosphorylation or
glycosylation and also, the cleavage site of the signal peptide for secretion is at VQA-KS, as predicted
from sequence analysis.
Although the phenomenon of bioluminescence is widespread most luminous species are marine
inhabitants. Many of these marine systems use imidazopyrazinone derivatives as the luciferin
and the one most commonly isolated is coelenterazine. Coelenterazine and its derivatives are
used by many bioluminescent proteins such as Renilla luciferase [1], the Ca2+-regulated
photoproteins [2,3], luciferase from the scyphozoan medusa Periphylla [4], squid
photoproteins [5], etc. The best-characterized examples of such proteins are Ca2+-regulated
photoproteins and Renilla luciferase, the ones responsible for bioluminescence of animals
belonging to the classes Hydrozoa and Anthozoa respectively. Crystal structures are now
available for a number of coelenterazine-dependent proteins, including several structures of
different ligand-dependent conformational states of photoproteins [6–10], Renilla luciferase
*Corresponding author: Dr. Eugene S. Vysotski, Mailing address: Photobiology lab, Institute of Biophysics, Russian Academy of
Science, Siberian Branch, Krasnoyarsk 660036, Russia, Fax: 7 (3912) 433400; Phone: 7 (3912) 494430; E-mail: <email@example.com> or
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting
proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could
affect the content, and all legal disclaimers that apply to the journal pertain.
NIH Public Access
Author Manuscript
Protein Expr Purif. Author manuscript; available in PMC 2009 October 1.
Published in final edited form as:
Protein Expr Purif. 2008 October ; 61(2): 142–148. doi:10.1016/j.pep.2008.05.013.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 2
[11], and its natural "substrate" the Ca2+-regulated coelenterazine-binding protein [12]. The
spatial structures of the photoproteins for example, have allowed rational engineering using
site-directed mutagenesis to produce novel spectral and enzymatic properties, which could find
usefulness in imaging and other applications [13–15].
Among coelenterazine-dependent bioluminescent proteins are also found several secreted
proteins. These are luciferases from the deep-sea shrimp Oplophorus gracilirostris [16], and
the marine copepods Gaussia and Metridia [17,18]. The bioluminescence of marine copepods
originates as a secretion from epidermal glands in response to various stimuli [19]. Metridia
luciferase catalyzes an oxygen oxidation of coelenterazine to produce light (~ 480 nm) and
requires no other cofactors for the bioluminescent reaction. Metridia luciferase (GenBank
accession no. AAR17541) is a small protein (~ 23 kDa) containing an N-terminal signal peptide
for secretion (17 amino acids) [18].
The secreted luciferases are very convenient bioluminescent reporters [18,20–25] because they
enable monitoring of intracellular events with high sensitivity, without destroying the cells or
tissues. Secretion therefore, is a property well suited for development of high throughput
screening technologies. Already the secreted luciferase from Gaussia shows much promise as
a bioluminescent label for in vitro hybridization assays [26]. However for in vitro assay
development it is important that high purity luciferases need to be available in sufficient
Most secreted proteins contain Cys residues and disulfide bonds [27] and Metridia luciferase
is no exception. The amino acid sequence of Metridia luciferase includes two closely similar
motifs of 31 residues containing ten conserved Cys residues, repeating as C-x3-C-x2-C-x5-
Cx11-C and C-x2-C-x3-C-x8-C-x11-C in the first and second motifs respectively (Fig. 1B)
[18]. Dithiothreitol (DTT)1 addition results in complete loss of activity indicating that active
luciferase requires disulfide bonds. The expression of functionally active Cys-rich proteins
with numerous S-S bonds in E. coli cells is non-trivial. It has long been noted that proteins
containing disulfide bonds are rare in the cytoplasm of any organism, attributable to the
reductive environment of the cytoplasm. The E. coli periplasm apparently is more suitable for
the expression of recombinant proteins containing the disulfide bonds due to the presence of
disulfide oxidoreductase and disulfide isomerase [28]. However the main disadvantage of the
E. coli periplasm as a folding compartment is that its enzyme system is not always efficient in
catalyzing disulfide bond formation in eukaryotic proteins that contain numerous disulfide
Expression in insect cells with the use of a baculovirus expression system does not have these
shortcomings and today it is a very powerful tool for expression of recombinant eukaryotic
proteins. The eukaryotic insect host cells perform most of the posttranslational modifications
of proteins occurring in mammalian cells as well as providing a secretory pathway through the
endoplasmic reticulum that allows the correct formation of disulfide bonds. Besides, this
expression system provides easy scalability and high levels of protein expression. Also it has
been shown that most of the expressed recombinant eukaryotic proteins are as functionally
active as their authentic counterparts.
In this paper we describe the expression of Metridia luciferase in insect (Sf9) cells using the
baculovirus expression system. Functionally active luciferase is secreted by the insect cells
into the culture media because the Metridia cDNA was cloned with its own signal peptide for
secretion and as a result, the luciferase can be efficiently purified as a monomer with a yield
1Abbreviations used: MLuc164, Metridia luciferase; GSSH, oxidized glutathione; GSH, reduced glutathione; DTT, dithiothreitol; a.m.u.,
atom mass unit; RLU, relative light units; pfu, plaque-forming unit; MOI, multiplicity of infection.
Stepanyuk et al. Page 2
Protein Expr Purif. Author manuscript; available in PMC 2009 October 1.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 3
of high purity protein of 2–3 mg/L. We also show that this luciferase produced and purified
from insect cells, has 3.5-fold greater bioluminescence activity than luciferase expressed and
purified from E. coli cells. Using LC–MS we demonstrate also that the experimental mass of
Metridia luciferase purified from insect cells is in close agreement with that calculated from
its amino acid sequence. This indicates that luciferase does not undergo posttranslational
modifications such as phosphorylation or glycosylation and that the cleavage site of signal
peptide for secretion is exactly at VQA-KS, as predicted from sequence analysis.
Materials and methods
PCR cloning of the Metridia luciferase cDNA for baculovirus expression system
The oligonucleotide primers were designed to introduce the C-terminal His6-tag followed by
a TEV-specific protease site into the MLuc164 coding sequence (Fig. 1A). The cloning strategy
also generated a BamHI/XhoI restriction site on the flanking ends of the gene for directional
cloning into the baculovirus donor vector. The coding sequence of the full-length MLuc164
cDNA gene containing the N-terminal signal peptide for secretion, was amplified from a cDNA
library vector TriplEx2-MLuc164 [18] using two-step PCR. The gene-specific forward S-
ML164s (5'-GCCGGGATCCATGGATATAAAGGTTGTCTTTACTCTTGTTTTCTC-3')
and reverse, A-ML164s primers (5'-
3') were used to amplify an approximately 714 bp sequence by polymerase chain reaction using
"PCR super-mix" (Invitrogen). PCR was performed for 27 cycles (denaturation: 25 sec at 95°
C; annealing: 30 sec at 56°C; extension: 45 sec at 68°C). For the second PCR, the first PCR
product was used as a template and the full-size gene was amplified under a similar condition
with forward S-ML164s primer and second reverse A2-ML164s primer (5'-
GCCGCTCGAGTCAGTGATGGTGATGGTGATGGATGCCCTG-3'). The amplified
product was purified using centrifuge spin-columns (Qiagen) and cloned into the pFastBac-1
vector (Invitrogen) after appropriate double digestion with BamHI/XhoI restriction enzymes
(Roche). The correct sequence was verified by restriction endonuclease digestion and
To generate a recombinant bacmid DNA the recombinant plasmid pFastBac-MLuc164 was
transformed into Max Efficiency DH10Bac competent cells (Invitrogen) and the gene of
interest was transposed into the bacmid through lacZ gene disruption. White clones containing
the recombinant bacmid were selected on Luria-agar plates containing 50 mg/ml kanamycin,
7 mg/ml gentamicin, 10 mg/ml tetracycline, 100 mg/ml Bluo-gal, and 40 mg/ml IPTG. After
36 h incubation at 37°C high molecular weight DNA was isolated from the overnight cultures
using Qiagen anion exchange resin. The recombinant bacmids were analyzed by performing
PCR to verify successful transposition with S-ML164s/A2-ML164s gene-specific primers.
Transfection of Sf9 cells with recombinant bacmid-MLuc164
The minipreparations of recombinant bacmid DNA were transfected into Sf9 nsect cells using
CELLFECTIN lipid reagent (Invitrogen). For each transfection 1.0 × 106 cells/ml with viability
>97% were seeded in a 35 mm cell culture treated plate and allowed to attach for at least 1 h.
The lipid reagent and bacmid DNA (1 µg) were diluted separately into 100 µl of SF-900 II
SFM media without antibiotics, and then combined to form lipid-DNA complexes. The lipid-
DNA complexes were diluted to 1 ml with SFM and laid over the washed Sf9 cells. The cells
were incubated for 5 h at 27°C, rinsed by SFM media, and incubated for another 72 h.
Recombinant baculovirus was harvested from the supernatant and amplified in 50-ml cell
Stepanyuk et al.Page 3
Protein Expr Purif. Author manuscript; available in PMC 2009 October 1.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 4
Techniques for plaque assay and virus titration were according to the Bac-to-Bac Baculovirus
expression system protocol (Invitrogen).
Expression and purification of MLuc164 from E. coli cells
Plasmid pET22b-MLuc164 containing the MLuc164 gene without signal peptide and any
purification tags, was used to transform the E. coli expression strain RIPL (DE3) pLysS
(Novagen). The signal peptide for secretion was removed from Metridia luciferase sequence
[18] for correct comparison of luciferase properties produced in different expression systems.
Eukaryotic signal peptides do not allow secretion of proteins in bacteria and therefore, if the
complete Metridia luciferase sequence was to be expressed in E. coli cells, it would contain
an additional 17 amino acid residue peptide at N-terminus. For protein production, the
transformed E. coli was cultivated with vigorous shaking at 37°C in LB medium containing
200 µg/ml ampicillin and induced with 1 mM IPTG when the culture reached an A600 nm of
0.8 – 1.0. After addition of IPTG, the cultivation was continued for 3 h. For purification of
luciferase, the cell paste was resuspended in 1 mM EDTA, 20 mM Tris-HCl pH 7.0 (1:20, w/
v) and disrupted with ultrasound (1 min × 6) at 0°C. Then the mixture was centrifuged and the
supernatant discarded. The pellet was washed sequentially with 0.9% NaCl, 1% Triton X-100,
5 mM EDTA, 20 mM Tris-HCl pH 7.0. All the washing procedures were performed with
centrifugation at 4°C (24,000 rpm). The final pellet was resuspended in 6 M urea in 20 mM
Tris-HCl pH 8.8, incubated overnight at 4°C, and then centrifuged. The 6 M urea extract was
loaded on a DEAE-Sepharose Fast Flow (Amersham Biosciences) column equilibrated with 6
M urea, 20 mM Tris-HCl pH 8.8. The proteins were eluted with a linear salt gradient (0 – 0.5
M NaCl) in the same buffer.
The MLuc164 sample in 6 M urea obtained after chromatography on DEAE Sepharose Fast
Flow, was refolded by a 20-fold dilution in 1 mM EDTA, 20 mM Tris-HCI pH 8.8 containing
5 mM GSH and 0.5 mM GSSG. The mixture was incubated overnight at 4°C with stirring and
then loaded on an anion exchange HiTrap Q column (Amersham Biosciences). The luciferase
sample was eluted by a NaCl linear gradient (0 – 0.5 M) in 1 mM EDTA, 20 mM Tris-HCl pH
8.8. The collected peak was loaded on HiLoad Superdex 75 (Amersham Biosciences) gel
filtration column in 0.2 M NaCl, 1 mM EDTA, 20 mM Tris-HCl pH 7.5 and active luciferase
monomer fractions were collected.
Expression and purification of MLuc164 from Sf9 cells
Sf9 cells cultured in suspension using HyQ SFX serum-free medium (HyClone), were infected
with plaque-pure recombinant virus at a multiplicity of infection (MOI) of 0.2 plaque-forming
units (pfu) and harvested at 72 h post-infection. Cells were pelleted at 2000 g for 10 min at 4°
C and the supernatant containing Metridia luciferase was kept at 4°C until purification.
Since the recombinant luciferase contained a His6-tag at the N-terminus, the protein was
purified using a Ni-NTA resin (5-ml HisTrap column, Amersham Biosciences). Routinely, 3-
liter batches of media containing the recombinant Metridia luciferase were concentrated by
ammonium sulfate precipitation (60%, m/v). The insoluble particles were spun down (6,000
rpm, 30 min) and dissolved in the Ni-binding buffer (0.2 mM NaCl, 10 mM imidazole, 50 mM
Na2HPO4/KH2PO4 buffer pH 7.1). The mixture was passed over a 5-ml His Trap Ni–column
(Amersham Biosciences) and eluted with an imidazole gradient (0 – 0.5 M) in 0.2 M NaCl, 50
mM Na2HPO4/KH2PO4 buffer p H 7.1. The luciferase peak was collected and dialyzed against
TEV protease cleavage buffer (1 mM EDTA, 20 mM Tris–HCl pH 8.0 containing 2 mM GSH
and 0.2 mM GSSG). The His6-tag was cleaved by incubation of the luciferase sample with
TEV protease at ratio 50:1 (w/w) overnight at room temperature; then the sample was loaded
on a Ni-NTA resin column and the flow-through collected. The collected sample was dialyzed
against 1 mM EDTA, 20 mM Tris-HCl pH 8.5 and loaded on an anion exchange HiTrap Q
Stepanyuk et al. Page 4
Protein Expr Purif. Author manuscript; available in PMC 2009 October 1.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 5
column (Amersham Biosciences). The luciferase was eluted by a NaCl linear gradient (0 – 0.5
M) in 1 mM EDTA, 20 mM Tris-HCl pH 8.5. The eluted peak was passed through a Superdex75
gel filtration column equilibrated by 0.2 M NaCl, 1 mM EDTA, 20 mM Tris-HCl pH 7.5.
Typically, at the final purification step the yield of high purity luciferase was 2 mg per liter of
insect cell culture.
Bioluminescence assay
The bioluminescence activity of luciferase was measured as the maximum intensity in relative
light units (RLU) at room temperature using a Turner luminometer (Turner, USA) on rapid
injection of 10 µl of 2 µM coelenterazine in methanol into a cuvette containing 5 µl of the
luciferase sample in 0.5 ml buffer (0.3 M NaCl, 20 mM Tris-HCl pH 7.5).
The specific activities of Metridia luciferases obtained with different expression systems were
determined as the integrated bioluminescence signal per mg of protein using a Luminoscan
Ascent plate luminometer (Thermo Electron Co, Finland) by injection of 50 µl of freshly
prepared coelenterazine (7.2 µM in 0.3 M NaCl, 20 mM Tris–HCl pH 7.5) into the wells
containing 100 µl luciferase at concentration 5 ng/ml in 0.3 M NaCl, 20 mM Tris–HCl pH 7.5.
Coelenterazine solution in buffer was prepared by dilution from stock solution of
coelenterazine in methanol with the concentration determined from the extinction coefficient
(methanol, ε434 nm = 8900 M−1 cm−1) [1]. The protein concentration was determined by the
Dc protein assay kit (Bio-Rad) with bovine serum albumin as a standard.
Spectral measurements
Bioluminescence spectra were measured with an AMINCO spectrofluorimeter (Thermo
Spectronic, USA) using a slit width of 8 nm and a scan rate of 16 nm/sec. All spectra were
corrected for spectral sensitivity of the instrument with the program supplied by AMINCO and
for intensity decay during the scan period. All measurements were carried out at room
SDS-PAGE and Western blot analysis
The media samples harvested at 72 h post-infection were mixed with SDS sample buffer and
heated to 100°C for 5 min. The samples were separated by 12–20% SDS-polyacrylamide
gradient gel electrophoresis and then transferred on PVDF membrane. The membrane was
blocked overnight at 4–8°C with 3% BSA in PBS containing Tween 20 (0.5%) and incubated
at room temperature for 30 min with mouse polyclonal IgG anti-His6 antibodies (Qiagen). The
appropriate secondary alkaline phosphatase-conjugated anti-mouse IgG antibody (Pierce) was
added and complexes were detected by enhanced color reaction.
Liquid chromatography–mass spectrometry
The precise molecular masses of the pure Metridia luciferase samples were determined using
HPLC-Atmospheric Pressure Ionization mass spectrometry (LC-MS) at the Chemical and
Biology Science Mass Spectrometry facilities of University of Georgia.
The luciferase samples were analyzed by separation on C-4 HPLC column (Applied
Biosystems HPLC system) followed by spraying of the eluted fractions into the mass
spectrometer (Perkin Elmer Sciex API I plus quadrapole mass spectrometer). The mass
spectrometer was calibrated with polypropylene diol, which produces precisely known
fragments up to 2100 a.m.u.
Stepanyuk et al.Page 5
Protein Expr Purif. Author manuscript; available in PMC 2009 October 1.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 6
Metridia luciferase expressed in E. coli cells
Previous studies have shown that on expression in E. coli, the luciferase lacking the secretion
sequence accumulates in insoluble inclusion bodies and therefore a refolding step is required
to recover the functionally active protein [18]. It was found that the best conditions for refolding
from inclusion bodies and forming disulfide bonds are those that mimic the endoplasmic
reticulum of eukaryotic cells [29–31], i.e., a mix of reduced and oxidized thiols (5 mM GSH,
0.5 mM GSSH) in 1 mM EDTA, 20 mM Tris-HCl pH 8.8. Although adding of the mix of
reduced and oxidized thiols in refolding buffer results in 25-fold increase of luciferase
bioluminescence activity, nevertheless only a minor part of the total protein attempted to be
refolded could be purified as functionally active monomeric luciferase. Besides the inability
of E. coli cells to provide properly folded protein and correct formation of disulfide bonds,
expression MLuc164 in E. coli results in nonspecific binding of metal ions by the Cys residues
of luciferase, evidenced by a surprisingly brown color of the inclusion bodies. After
solubilization of inclusion bodies in 6 M urea, purification by anion exchange chromatography
on DEAE-Sepharose Fast Flow column, and refolding, the MLuc164 retains the red-brown
color (Fig. 2A). The absorbance spectrum showed maxima at 412 nm and 320 nm characteristic
of proteins with an Fe-S cluster (Fig. 2B) [32]. The elemental chemical analysis of MLuc164
revealed the presence of iron at a ratio of 1:0.6 (protein/iron) and some zinc traces. In addition,
we found that adding Fe3+ caused an irreversible aggregation of luciferase indicated by gel
filtration where an iron-enriched luciferase fraction eluted in the void volume (Fig. 3). The
aggregated luciferase contained only 5–10% bioluminescence activity of the monomeric
fraction. Inclusion of chelation agents such as EDTA or an iron specific chelator (deferoxamine
mesylate) to the protein sample buffers and during the washing step of the inclusion bodies,
did not eliminate the contamination of luciferase by metals. Incubation of the luciferase sample
(1 mg/ml) in 6 M guanidine-HCl with slowly added HCl to a final concentration 0.5 M at 4°
C, merely resulted in partial decoloration of the sample but did not completely remove iron,
indicated by the remaining absorbance bands at 320 nm and 412 nm, though with lower
intensity than the sample before treatment.
Several different approaches have been tested to improve MLuc164 expression in E. coli cells.
We attempted to increase the Mluc164 solubility by fusing the luciferase with MBP (maltose-
binding protein) tag-protein. This increases the luciferase solubility but does not decrease its
aggregation. The use of the special E. coli strain Rosetta-gami (Novagen) with reduced
thioredoxin reductase activity designed for proper disulfide bond formation in the E. coli
cytoplasm, yielded the luciferase in inclusion bodies, which could be solubilized and refolded
but was completely inactive. The expression with secretion of Mluc164 into the E. coli
periplasm gave extremely low protein yield.
Apparently, Metridia luciferase expressed in E. coli cells must aggregate through
intermolecular disulfide bond formation. This process might be accelerated by binding the
metal ions because the aggregated luciferase fraction contains iron. It is reasonable to assume
that instead of intramolecular bridging the free Cys residues into S-S bonds, there are some
specific (or unspecific) mechanisms in E. coli cells, which promote binding the iron to Cys
residues but which apparently are not required for Mluc164 bioluminescence, causing only its
aggregation and misfolding. For instance, the first Cys-rich repeat (C-x3-C-x2-C-x8-C-x11-
C) of Metridia luciferase sequence has some characteristic features peculiar to the C-x3-C-x2-
C conserved binding motif of the iron-sulfur cluster of the radical-SAM enzyme superfamily
Stepanyuk et al. Page 6
Protein Expr Purif. Author manuscript; available in PMC 2009 October 1.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 7
Metridia luciferase expressed in insect cells
As a consequence of the problem of producing functionally active Metridia luciferase from E.
coli expression systems, we turned to the Bac-to-Bac system for expression of Metridia
luciferase along with its secretion sequence in the Spodoptera frugiperda (Sf9) cell line.
Exploiting the fact that the luciferase is a naturally secreted protein, we cloned the full-length
luciferase cDNA including the signal sequence for secretion, to the pFastBac-1 vector. The
amount of Metridia luciferase being expressed by the Sf9 cells was monitored by the
bioluminescence activity of a sample of the media, which should directly correspond to the
amounts of protein expressed. For this purpose, samples were taken at 0, 24, 48, 72 and 96 h
post-infection and the bioluminescent activity of the supernatant separated from the cell pellet
was measured. The appearance of MLuc164 activity in the culture media indicated that the
natural signal peptide successfully provides the luciferase secretion from these insect cells
themselves. The highest bioluminescence activity with the lowest cell breakdown, was found
in the supernatant of cells infected with virus at an MOI of 0.2 pfu for 72 h. These optimal
conditions were selected for large-scale luciferase expression and its following purification.
The purification procedure comprised concentration of the luciferase from the cell culture
media by ammonium sulfate precipitation, Ni-affinity chromatography, anion exchange
chromatography, and gel-filtration (Table 1). Because MLuc164 contains disulfide bonds, all
procedures were carried out under non-reducing conditions, i.e. without including DTT or β-
According to SDS-PAGE (Fig. 4A,B), the luciferase obtained is of high purity. Western-blot
analysis using anti-His antibody indicates that this is actually the required protein with
molecular mass ~ 24 kDa (Fig. 4C). Large-scale baculovirus expression of MLuc164 routinely
yielded approximately 2–3 mg luciferase of high purity per liter of culture media.
Metridia luciferase expressed in insect cells in contrast to the luciferase expressed in E. coli
cells, is a fully active monomeric protein with no evidence of aggregation as confirmed by gel
filtration (Fig. 3). The absorption spectrum of MLuc164 expressed in Sf9 cells shows only one
maximum at 280 nm without the maxima at 412 nm and 320 nm observed in the absorption
spectrum of luciferase purified from E. coli (Fig. 2). Also elemental chemical analysis revealed
no traces of iron or zinc in the MLuc164 purified from insect cells.
Molecular masses of MLuc164 purified from Sf9 and E. coli cells
The molecular mass of MLuc164 purified from Sf9 cells determined by the LC–MS method is
23,797 (± 2.4) Da, 10 a.m.u. less than calculated (Table 2, Fig. 5B). The near coincidence of
experimental mass with that calculated from amino acid sequence indicates that luciferase does
not undergo posttranslational modifications such as phosphorylation or glycosylation. The
discrepancy of 10 a.m.u. can be accounted for by the formation of several S-S bonds (4 or 5)
accompanied by removal of the corresponding hydrogens. Moreover, the coincidence of
molecular masses unambiguously shows that the cleavage site of signal peptide for secretion
is at VQA-KS, i.e. between Ala17 and Lys18, as predicted from the amino acid sequence
analysis of Metridia luciferase [18].
In contrast, the molecular mass of MLuc164 obtained from E. coli cells is higher by 120 a.m.u.
than the calculated mass (Table 2, Fig. 5A). Since luciferase purified from E. coli is
contaminated by iron (as well as some zinc traces revealed in the protein sample by elemental
chemical analysis), it would be reasonable to assume that higher molecular mass of this
luciferase sample is a result of the presence of this metal ion.
Stepanyuk et al.Page 7
Protein Expr Purif. Author manuscript; available in PMC 2009 October 1.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 8
Bioluminescence properties of MLuc164 purified from Sf9 and E. coli cells
Metridia luciferase expressed in Sf9 cells reveals 3.5 times higher specific bioluminescence
activity compared to monomeric MLuc164 expressed and purified from E. coli cells (Fig. 6).
The bioluminescence of both luciferases is also activated more than 100-fold in the presence
of NaCl but with a slightly shifted optimum for NaCl concentration for luciferase obtained
from insect cells (0.5 M) than for MLuc164 purified from E. coli (0.3 M) (Fig. 7). Besides,
MLuc164 expressed in insect cells displays higher tolerance towards ionic strength (1 – 2 M
of NaCl) than does luciferase from E. coli, which might indicate a more rigid and stable protein
structure. It is likely that the effect of NaCl on bioluminescence activity of MLuc164 results
from chloride ion interaction as has been reported for Gaussia luciferase [34], not unexpected
from the high identity of amino acid sequence of the two proteins [18]. Activation of
Metridia luciferase purified both from insect and E. coli cells by bivalent cations such as
Mg2+ or Ca2+ is not so evident. At the same time, both Metridia luciferases reveal a similar
pH optimum for bioluminescence, around 7.3–7.4 (Fig. 7) and bioluminescence emission
maxima (Fig. 8) that are very close to those of natural Metridia luciferase [18]. It should be
noted that while the pH optima for bioluminescence of Metridia luciferases correspond to those
of Gaussia luciferase [33], the light emission maxima (483 and 486 nm for luciferases purified
from E. coli and insect cells respectively) are red-shifted from the maximum of Gaussia
luciferase (473 nm) [34]. This spectral shift could be a result of differences in the amino acid
residue composition of the active sites of Metridia and Gaussia luciferases, as also found for
the two Ca2+-regulated photoproteins, aequorin and obelin, where one residue difference in
the coelenterazine-binding cavity appears to result in a more than 10 nm difference in their
bioluminescence maxima [14].
The work described here shows that secreted Metridia luciferase expressed in a baculovirus
insect cell system is functionally active and has a higher specific bioluminescent activity as
compared with monomeric MLuc164 expressed in E. coli cells. In addition, we found that
Metridia luciferase from insect cells is a stable monomeric protein with molecular mass closely
corresponding to that calculated from sequence, thereby indicating the lack of any post-
translation modifications. In addition, the Metridia luciferase purified from insect cells has no
tendency for aggregation even at high protein concentration and shows no contamination by
metal ions, in contrast to luciferase expressed in E. coli. Also we show that the baculovirus
expression system is convenient for both production and purification of the large amounts of
Metridia luciferase necessary for crystallization and spatial structure determination,
biochemical characterization, or development of in vitro analytical assays.
This work was supported by the National Institutes of Health (Grant 1P50 GM62407), University of Georgia Research
Foundation and Georgia Research Alliance, the Russian Foundation for Basic Research and Taiwan National Science
Council (Grant 06-04-89502), and the program for "Molecular and Cellular Biology" of Russian Academy of Sciences.
1. Hori K, Charbonneau H, Hart RC, Cormier MJ. Structure of native Renilla reniformis luciferin. Proc.
Natl. Acad. Sci. USA 1977;74:4285–4287. [PubMed: 16592444]
2. Vysotski ES, Lee J. Ca2+-regulated photoproteins: Structural insight into the bioluminescence
mechanism. Acc. Chem. Res 2004;37:405–415. [PubMed: 15196050]
3. Shimomura O. The discovery of aequorin and green fluorescent protein. J. Microsc 2005;217:1–15.
[PubMed: 15655058]
4. Shimomura O, Flood PR. Luciferase of the scyphozoan medusa Periphylla periphylla. Biol. Bull
Stepanyuk et al.Page 8
Protein Expr Purif. Author manuscript; available in PMC 2009 October 1.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 9
5. Isobe M, Kuse M, Yasuda Y, Takahashi H. Synthesis of 13C-dehydrocoelenterazine and model studies
on Symplectoteuthis squid bioluminescence. Bioorg. Med. Chem. Lett 1998;8:2919–2924. [PubMed:
6. Head JF, Inouye S, Teranishi K, Shimomura O. The crystal structure of the photoprotein aequorin at
2.3 Å resolution. Nature 2000;405:372–376. [PubMed: 10830969]
7. Liu ZJ, Vysotski ES, Chen CJ, Rose J, Lee J, Wang BC. Structure of the Ca2+-regulated photoprotein
obelin at 1.7 Å resolution determined directly from its sulfur substructure. Protein Sci 2000;9:2085–
2093. [PubMed: 11152120]
8. Deng L, Markova SV, Vysotski ES, Liu ZJ, Lee J, Rose J, Wang BC. Crystal structure of a Ca2+-
discharged photoprotein: implications for mechanisms of the calcium trigger and bioluminescence. J.
Biol. Chem 2004;279:33647–33652. [PubMed: 15155735]
9. Deng L, Vysotski ES, Markova SV, Liu ZJ, Lee J, Rose J, Wang BC. All three Ca2+-binding loops of
photoproteins bind calcium ions: the crystal structures of calcium-loaded apo-aequorin and apo-obelin.
Protein Sci 2005;14:663–675. [PubMed: 15689515]
10. Liu ZJ, Stepanyuk GA, Vysotski ES, Lee J, Markova SV, Malikova NP, Wang BC. Crystal structure
of obelin after Ca2+-triggered bioluminescence suggests neutral coelenteramide as the primary
excited state. Proc. Natl. Acad. Sci. USA 2006;103:2570–2575. [PubMed: 16467137]
11. Loening AM, Fenn TD, Gambhir SS. Crystal structures of the luciferase and green fluorescent protein
from Renilla reniformis. J. Mol. Biol 2007;374:1017–1028. [PubMed: 17980388]
12. Stepanyuk GA, Liu ZJ, Lee J, Markova SV, Frank LA, Vysotski ES, Wang BC. Crystal structure of
coelenterazine-binding protein from Renilla muelleri at 1.72 Å: Why it is not a calcium-regulated
photoprotein. Photochem. Photobiol. Sci 2008;7:442–447. [PubMed: 18385886]
13. Malikova NP, Stepanyuk GA, Frank LA, Markova SV, Vysotski ES, Lee J. Spectral tuning of obelin
bioluminescence by mutations of Trp92. FEBS Lett 2003;544:184–188. [PubMed: 14596937]
14. Stepanyuk GA, Golz S, Markova SV, Frank LA, Lee J, Vysotski ES. Interchange of aequorin and
obelin bioluminescence color is determined by substitution of one active site residue of each
photoprotein. FEBS Lett 2005;579:1008–1014. [PubMed: 15710383]
15. Loening AM, Wu AM, Gambhir SS. Red-shifted Renilla reniformis luciferase variants for imaging
in living subjects. Nat. Methods 2007;4:641–643. [PubMed: 17618292]
16. Inouye S, Watanabe K, Nakamura H, Shimomura O. Secretional luciferase of the luminous shrimp
Oplophorus gracilirostris: cDNA cloning of a novel imidazopyrazinone luciferase. FEBS Lett
2000;481:19–25. [PubMed: 10984608]
17. Bryan BJ, Sent-Gyorgyi CS. Luciferases, fluorescent proteins, nucleic acids encoding the luciferases
and fluorescent proteins and the use thereof in diagnostics, high throughput screening and novelty
items. International Pub. 1999No. WO 99/49019
18. Markova SV, Golz S, Frank LA, Kalthof B, Vysotski ES. Cloning and expression of cDNA for a
luciferase from the marine copepod Metridia longa. J. Biol. Chem 2004;279:3212–3217. [PubMed:
19. Evstigneev, PV.; Bitukov, EP. Bioluminescence of Marine Copepods. Greze, BN., editor. Kiev,
Ukraine: Naukova Dumka; 1990.
20. Thompson EM, Nagata S, Tsuji FI. Vargula hilgendorfii luciferase: a secreted reporter enzyme for
monitoring gene expression in mammalian cells. Gene 1990;96:257–262. [PubMed: 2269435]
21. Inouye S, Ohmiya Y, Toya Y, Tsuji FI. Imaging of luciferase secretion from transformed Chinese
hamster ovary cells. Proc. Natl. Acad. Sci. USA 1992;89:9584–9587. [PubMed: 1409669]
22. Tanahashi A, Ohmiya Y, Honma Y, Katsuno S, Ohta Y, Nakamura H, Honma K. Continuous
measurement of targeted promoter activity by a secreted bioluminescence reporter Vargula
hilgendorfii luciferase. Anal. Biochem 2001;289:260–266. [PubMed: 11161320]
23. Tannous BA, Kim DE, Fernandez JL, Weissleder R, Breakefield XO. Codonoptimized Gaussia
luciferase cDNA for mammalian gene expression in culture and in vivo. Mol. Ther 2005;11:435–
443. [PubMed: 15727940]
24. Suzuki T, Usuda S, Ichinose H, Inouye S. Real-time bioluminescence imaging of a protein secretory
pathway in living mammalian cells using Gaussia luciferase. FEBS Lett 2007;581:4551–4556.
[PubMed: 17825828]
Stepanyuk et al. Page 9
Protein Expr Purif. Author manuscript; available in PMC 2009 October 1.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 10
25. Wurdinger T, Badr C, Pike L, de Kleine R, Weissleder R, Breakefield XO, Tannous BA. A secreted
luciferase for ex vivo monitoring of in vivo processes. Nat. Methods 2008;5:171–173. [PubMed:
26. Verhaegen M, Christopoulos T. Recombinant Gaussia luciferase. Overexpression, purification, and
analytical application of a bioluminescent reporter for DNA hybridization. Anal. Chem
2002;74:4378–4385. [PubMed: 12236345]
27. Fahey RC, Hunt JS, Windham CC. On the cysteine and cystine content of proteins. Differences
between intracellular and extracellular proteins. Mol. Evol 1977;10:155–163.
28. Kadokura H, Katzen F, Beckwith J. Protein disulfide bond formation in prokaryotes. Annu. Rev.
Biochem 2003;72:111–135. [PubMed: 12524212]
29. Hwang C, Sinskey AJ, Lodish HF. Oxidized redox state of glutathione in the endoplasmic reticulum.
Science 1992;257:1496–1502. [PubMed: 1523409]
30. Wetlaufer DB, Branca PA, Chen G-X. The oxidative folding of proteins by disulfide plus thiol does
not correlate with redox potential. Protein Eng 1987;2:141–146. [PubMed: 3507696]
31. Rudolph R, Lilie H. In vitro folding of inclusion body proteins. FASEB J 1996;10:49–56. [PubMed:
32. Cosper MM, Cosper NJ, Hong W, Shokes JE, Broderick WE, Broderick JB, Johnson MK, Scott RA.
Structural studies of the interaction of S-adenosylmethionine with the [4Fe-4S] clusters in biotin
synthase and pyruvate formate-lyase activating enzyme. Protein Sci 2003;12:1573–1577. [PubMed:
33. Sofia HJ, Chen G, Hetzler BG, Reyes-Spindola JF, Miller NE. Radical SAM, a novel protein
superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms:
Functional characterization using new analysis and information visualization methods. Nucleic Acids
Res 2001;29:1097–1106. [PubMed: 11222759]
34. Inouye S, Sahara Y. Identification of two catalytic domains in a luciferase secreted by the copepod
Gaussia princes. Biochem. Biophys. Res. Commun 2008;365:96–101. [PubMed: 17981153]
Stepanyuk et al.Page 10
Protein Expr Purif. Author manuscript; available in PMC 2009 October 1.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 11
Figure 1.
(A) Schematic representation of the Metridia luciferase gene (MLuc164) and the construct
used for its expression. (B) Alignment of two conservative repeats R1 and R2 in Metridia
luciferase [18]. The shaded boxes indicate the conserved Cys residues. The residues in gray
show the identical amino acids.
Stepanyuk et al.Page 11
Protein Expr Purif. Author manuscript; available in PMC 2009 October 1.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 12
Figure 2.
(A) MLuc164 purified from E. coli cells at protein concentration 42 mg/ml. (B) Normalized
absorption spectra of MLuc164 purified from insect Sf9 (dashed line) and E. coli (solid line)
cells respectively. The peak at 412 nm and shoulder at 320 nm are characteristic for proteins
with an iron-sulfur cluster [32].
Stepanyuk et al.Page 12
Protein Expr Purif. Author manuscript; available in PMC 2009 October 1.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 13
Figure 3.
The elution profile of Metridia luciferases purified from E. coli (black line) and insect Sf9 (gray
line) cells on a HiLoad Superdex 75 column. The area highlighted in the dark gray color shows
the collected fractions containing the active monomers of MLuc164. V0, void volume of the
Stepanyuk et al.Page 13
Protein Expr Purif. Author manuscript; available in PMC 2009 October 1.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 14
Figure 4.
SDS-PAGE and Western-blot analysis of Metridia luciferase expressed in and purified from
Sf9 cells. (A) MLuc164 after purification on the Ni-HisTrap column (line 2); (B) MLuc164
after gel filtration on HiLoad Superdex 75 column (line 2); (C) Western-blot analysis of
MLuc164 (sample of 72 h post-transfected culture media) (line 2); negative control (line 3).
Molecular mass markers (Kaleidoscope prestained standards, Bio-Rad) are shown always in
line 1.
Stepanyuk et al.Page 14
Protein Expr Purif. Author manuscript; available in PMC 2009 October 1.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 15
Figure 5.
LC–MS spectra of pure Metridia luciferase samples purified from E. coli (A) and Sf9 insect
(B) cells. Each peak is labeled with its mass in Daltons.
Stepanyuk et al.Page 15
Protein Expr Purif. Author manuscript; available in PMC 2009 October 1.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript


text:
Trailer Report: New 'I, Frankenstein,' 'Hobbit' Previews Notch Top Views
The first trailer for I, Frankenstein, the horror reboot written and directed by 30 Days of Night scribe Stuart Beattie debuted as the most viewed YouTube trailer of the week, according to Internet monitoring service Zefr.
The preview for the Aaron Eckhart-Yvonne Strahovski feature notched 2.2 million views in its opening week.
Also making a strong debut was the new preview for The Hobbit: The Desolation of Smaug. Trailer No. 2 for part two of Peter Jackson's trilogy based on the beloved J.R.R. Tolkien novel garnered 1.7 million views for the week.
The movie's first trailer was No. 4 in the rankings. Its 1.2 million views for the week gave it a lifetime total of 30.04 million.
Sandwiched between the two Hobbit trailers was the first trailer for 300: Rise of an Empire, with 1.5 million views (30.3 million lifetime).
Need for Speed, which debuted at No. 1 last week, fell to fifth with 1.03 million YouTube views (8.3 million lifetime).
The bottom five were all returning trailers: Curse of Chucky, The Hunger Games (trailer No. 2), Frozen and Jackass Presents: Bad Grandpa, which recorded between 846,000 and 642,000 views each.
Rounding out the top 10 was the first trailer for Hunger Games: Catching Fire, which bounced up from 44th to 10th. The preview for the Thanksgiving sequel has recorded 50.6 million lifetime views.
Never miss a story. Stay up-to-date with THR's Daily Headlines.
Must Reads
Kids' Choice Awards 2015: Red-Carpet Photos
Kids' Choice Awards 2015: Best and Worst Moments (Photos)
'Game of Thrones' Star Emilia Clarke on Playing Sarah Connor, Turning Down 'Fifty Shades' and Moving on From Nudity
L.A.'s Soho House Turns 5: Membership Rejections, Success Secrets Revealed in Oral History
Gavin Polone on TV's Dirty Secret: Your Agent Gets Money for Nothing (Guest Column)
Reality Power: The Declining Clout of the Broadcast Unscripted Chief
Why More 'Star Wars' Actors Haven't Become Stars
From Christian Bale to Benedict Cumberbatch: Why British Actors Nab So Many American Roles
The Uncensored, Epic, Never-Told Story Behind 'Mad Men'

subset: commoncrawl


text:
We at the Good Hotshot enjoy helping our friends in the small business sector out which is why we've been really excited to tell you about 3 SEO Quotes.com.
Before we get into the nuts & bolts on why you need these guys, first lets have a heart to heart about the SEO (search engine optimization) industry as a whole.
We've got a lot of friends who are small business owners and literally all of them have had horrendous experiences when hiring SEO providers. I'm not quite sure if it's something in the water, but SEO people are typically very slick sales folks and they're even better at getting small business owners to fork over a lot of money without really showing anything for it besides "just wait a bit, it takes time for your rankings to start coming up…"
Back to 3 SEO Quotes. This company was started by industry leading SEO experts who have owned their own highly successful SEO firms in past. The one thing they have in common is they all want to help the little guy out and offer a way to 'hold SEOs accountable,' for what they do.
3 SEO Quotes does just what their name says, and so much more. When a small business contacts them, they immediately contact the business to discuss their situation. Once they figure out what they need to, they then shop the deal to some pre-screened providers they've known, and worked with for awhile to give them a chance to compete for the small business owners business. Once 3 SEO Quotes get their quotes back, they discuss them with the small business in great depth and decide who would be a good fit to discuss next steps.
If ,and when the small business owner selects an SEO provider 3 SEO Quotes acts as their personal advocate and consultant through the entire campaign.
So far, 3 SEO Quotes has a Denver SEO office located near 11th and Colorado. To learn more, check them out at 3seoquotes.com.


text:
Hartwell Residential Home
117 Jumble Lane, Ecclesfield, Sheffield S35 9XJ
Hartwell Residential Home has thirty-one rooms available for residential care, in a relaxing environment highly-suited to the practice of efficient care.
A day care and respite care service is also available to the local community, meaning carers are able to contribute to the lives of many families with their in-depth expertise.
Residents enjoy a versatile accommodation, with an activities programme and regular outings to place of interest, together with in-house entertainment and a nutritious food menu.
Group: brighterkind
Person in charge: Maria Whittaker (Homes Manager)
Local Authority / Social Services: Sheffield City Council (click for contact details)
Type of Service: Care Home only (Residential Care) – Privately Owned , 34 residents
Registered Care Categories*: Old Age
Single Rooms: 32
Shared Rooms: 1
Rooms with ensuite WC: 23
Facilities & Services: Day Care • Respite Care • Convalescent Care • Own Furniture if required • Smoking not permitted • Close to Local shops • Minibus or other transport • Lift • Wheelchair access • Gardens for residents • Phone Point in own room/Mobile • Television point in own room
Latest CQC* Report on Hartwell Residential Home: click here
*Care Quality Commission (CQC) is responsible for the registration and inspection of social care services in England.
Bed Vacancies
Current Bed Vacancies Posted by Hartwell Residential Home
Please Contact Us for Latest Vacancies
Current Recommendations
There is currently no 'Recommendation Score' for Hartwell Residential Home.
There are currently no recommendations for Hartwell Residential Home
Care Home Idol 2014
There are currently no Care Home Idol entrants who have performed at Hartwell Residential Home
Home •
Care Home Search •
Care Homes by Name •
Care Home Providers •
Care Home Associations •
Care Home Fees Advice
Products & Services for Care Homes • Care Data • Care News & Events • Care Home Jobs
Care Training Courses • iPhone & Android Apps
About carehome.co.uk • Our Services / Advertise • Terms & Conditions, Privacy Policy & Cookies • Follow us on Twitter • Follow us on Facebook
© 2015 carehome.co.uk


text:
Recent articles
Prosthetic technology
7-year-old Lakewood girl gets a new 'robohand' through 3D printing technology
LOS ALAMITOS >> Faith Lennox's parents said the 7-year-old was so excited to try on her new arm that she barely slept during the night.A birth defect led to Faith's left arm being amputated just below the elbow when she was 9 months old. Faith, who lives in Lakewood with her family, used an electronic hand and prosthetic arm when she was 3, but grew out of it in two years. Her parents didn't replace it because their insurance company was hassling them about...
L.A., San Bernardino metro areas rank in top 30 for people who identify as LGBT, study says
LONG BEACH >> People living in the greater Los Angeles metropolitan area who are comfortable identifying as LGBT are the eighth largest such group in the nation, according to a first-of-its-kind study from Gallup and the
Recreation and leisure
Pickleball: What is it, and why it has started in Lakewood?
LAKEWOOD >> It's called the sweet sport with the sour name.Pickleball — one of the fasting growing sports in the country — is a combination of tennis, badminton and ping pong. The game uses rules similar to tennis and is played with a paddle larger than the one used in ping pong and a plastic, perforated ball resembling a Wiffle ball.The game is low-impact and popular with active seniors as well as adults and...
Long Beach Center to offer pop-up store for masculine women, transgender men
LONG BEACH >> Mary Going wanted to wear a suit when she married her girlfriend, but said the shopping experience was humiliating."Nothing fit. It was too big. No matter how much money I spent, it wouldn't work," said the Oakland resident, who went to several fancy department stores and designer boutiques in San Francisco preceding her 2008 nuptials to Martha Rynberg, 39. "Many of the sales people told me to shop in the little boys department or rolled...
Rivers and streams
Los Angeles River study examining levels of contaminated fish in Long Beach
LONG BEACH >> Activists and conservation biologists, who have been fishing a portion of the Los Angeles River to determine its ecological health, are scheduled to return to the area in March for the final portion of a four-part study.The study is examining the river's 1-mile stretch between Willow Street and Pacific Coast Highway, an estuary filled with marine life. While other studies have looked at the levels of...
Armed forces
How you can help build a Long Beach-centric exhibit on the Pearl Harbor attack
long beach >> The 75th anniversary of the Japanese attack on Pearl Harbor and its immediate and dramatic impact on the city will be the subject of an extensive, upcoming exhibit at the Historical Society of Long Beach.On Sunday, the historical society will collect materials for possible use in "Long Beach Remembers Pearl Harbor: A...
Art photography
Photo exhibit by Lisa Hartouni at Hot Java explores immigration detention centers, social justice
long beach >> Lisa Hartouni's photograph of the Mardi Gras Motel in Inglewood shows that this locale might have been a festive place to stay, but in 1987, the facade betrayed its true purpose.Women and children rally outside the hotel, protesting the Immigration and Naturalization Services' practice of detaining minors -- some of them U.S. citizens -- at the private facility in an attempt to lure their parents, some of...
Latin Grammy Award-winning singer Laura Pausini to play L.A.'s Greek Theatre
Italian pop singer Laura Pausini, who's sold more than 70 million albums worldwide, said her awards are kept in a special place — her childhood home in Solarolo, Italy."My dad (Fabrizio Pausini) is big fan of Elvis Presley. So the house where I grew up is called Pausiniland," she said with a laugh. "My dad keeps all my awards, dresses I've worn in concerts. That's where it's all on display."Four special fans from...
Queen Mary's 80th anniversary celebration includes new plans for museum, science center
LONG BEACH >> The city's most famous queen — which celebrated its 80th birthday along with thousands of visitors on Friday — has ambitious plans to partner with Long Beach and Los Angeles unified school districts to convert part of the ship into a 65,000-square-foot museum and science and learning center, officials said.The Exhibit Hall would be turned into a 45,000-square-foot museum, while the adjacent R Deck would become 20,000 square feet of classroom...
Documentary 'Letter to Anita' looks at effects of Florida's anti-gay campaign
long beach >> Ronni Sanlo is a survivor from Anita Bryant's war against gay and lesbians.Sanlo's story is told in the documentary "Letter to Anita" that will be screened Saturday as part of the 2014 Long Beach QFilms Festival. The long-running LGBT film festival will take place Friday to Sunday at the Art Theatre of Long Beach, and the neighboring Long Beach LGBTQ Center.In 1977, Bryant — the...


text:
|Project Name
|Price (USD)
|Project Name
|Price (USD)
|Taxas holdem source code
|We are looking for a source code package relating to a card game app such as Texas holdem. We will be...preferred UI to apply onto your app before we pay for a complete app package. Please, feel free...free to contact us if you have your own app and/or are interested in selling one to us. Thank you.
|Mobile Phone, iPhone, Android, HTML5
|Mar 30, 2015
|Mar 30, 2015Ends - 6d 18h
|develop simply app android with source code
|I need an android application with source code that it checks some changes on some data from a database...database mysql. This app is setting up via user authentications that check some data on my sql table. I need...NOT BID because i have some days to delivery the app fully functional . We require flexibility and reliability
|PHP, Mobile Phone, Android, MySQL
|Mar 31, 2015
|Taxas holdem source code -- 2
|We are looking for a source code package relating to a card game app such as Texas holdem. We will be...preferred UI to apply onto your app before we pay for a complete app package. Please, feel free...free to contact us if you have your own app and/or are interested in selling one to us. Thank you.
|Mobile Phone, iPhone, Android, HTML5
|Mar 31, 2015
|source code needed for iOS app.
|...to buy an iOS source code The app link is the following. https://itunes.apple.com/us/app/flinch/id862550306...2550306?mt=8. Instant pay for the exact app. Thanks.
|Mobile Phone, iPhone
|Mar 28, 2015
|Mar 28, 2015Ends - 4d 2h
|Android app
|i need an app that i can install in my childs phone and i can track viber messages from that phone. ...send or recive from viber. I dont need any special app coz it will be used just for my childs (personal...if u like u can send me just apk file and not source code.
|Mobile Phone, Android
|Mar 31, 2015
|viber source code with manual for modifing.
|We need to source code of VIBER for iPhone & Android phones. Only experienced freelancers/teams
|PHP, Mobile Phone, iPhone, Android, Amazon Web Services
|Mar 28, 2015
|Mar 28, 2015Ends - 4d 7h
|APP Modification on existing APP-source Code, Anpassen einer SmartPhone APP, Quellcode wird zur Verfügung gestellt
|...end: We want you to offer us an APP-modification. The app-source-code is for you availiable in our GIT...HTML-5, to enshure an operatability of the app on android, IOS and windows divices. You have to integrate...integrate and document all modifications in the source-code of the app and create additionaly a config-file to
|Mar 27, 2015
|Mar 27, 2015Ends - 3d 7h
|deploying android security app
|I have source code for an android app which runs when we open the android device . It uses voice recognition...registered email address . I need help in deploying the app into nexus 9 tablet .Necessary changes much be made
|Mobile Phone, Android, MySQL
|Mar 26, 2015
|Mar 26, 2015Ends - 2d 20h
|Android App with a frame to a website
|I'm looking for some source code to do this, don't need the APK fully done. It's really basic but I...I got no time. Just an App for Android that does the following: I have a responsive website, and...includes that website as frame. So you open the "App" and it loads the website in a frame. It's a VirtueMart
|Mobile Phone, Android
|Mar 26, 2015
|Mar 26, 2015Ends - 2d 3h
|Android app development
|Hello, I need a Android launcher app with google single sign in with a home screen resembling...resembling a home theatre with some app intents placed permanently. Need to embed server apps to access videos...PDFs that points to a directory(using FTP). Source code required to modify the folder that is pointed
|Java, Mobile Phone, Android
|Mar 25, 2015
|Mar 25, 2015Ends - 1h 44m
|redesign and repogrammering a android app
|...instead of Datex-1. Then you will redesign the Android app we have to fit the new system and make some changes...skills in programming. I will provide the old source code. Questions? Please ask.
|PHP, Java, Mobile Phone, Android, MySQL
|Mar 29, 2015
|Mar 29, 2015Ends - 5d 16h
|POS Android with source
|...business from anywhere with WiFi & 3G Mobile App Customers can discover, order, and pay ahead
|Mobile Phone, iPhone, Android, iPad, Anything Goes
|Mar 28, 2015
|Mar 28, 2015Ends - 4d 3h
|Buy Android/IPhone Mobile Game or App
|...like to buy fully functional android/iphone game or app with the source code and copyright. If any one willing...willing to sell the game or app please contact me, Thanks, Azghar
|iPhone, Android
|Mar 27, 2015
|Mar 27, 2015Ends - 3d 8h
|Need Social Media Andorid/IPHone App Source Code
|...social media APp in Andrid/IPhone ready with them then i would like to purchase the source code for same
|Mobile Phone, iPhone, Android, PhoneGap
|Mar 30, 2015
|Mar 30, 2015Ends - 6d
|need a chat app something like line are viber source code
|im looking for on unity chat app source code something like viber are line but i would definetly prefer...you only if you know you have a good chat app source code nothing like whatsapp.
|Mobile Phone, iPhone, Android, Objective C, HTML5
|Mar 31, 2015
|modify Android app and install on server and create apk file for test
|hello, i need great android programmer who can edit android app for me , change logo, install in...default language in app create apk for test. i will send you link for app code source
|Mobile Phone, Android
|Mar 26, 2015
|Mar 26, 2015Ends - 2d 9h
|TinderEstate (iPhone or Android App)
|Develop Mobile App (either for iPHone OR Android) Code ownership must be transferred and be free to
|Android, iPhone
|Mar 25, 2015
|Mar 25, 2015Ends - 9h 14m
|Readymade POS Web preferably wordpress + Android with source
|Cloud-based point of sale system Set up the sleek, Android tablet and other hardware Process Transactions...business from anywhere with WiFi & 3G Mobile App Customers can discover, order, and pay ahead
|PHP, Mobile Phone, iPhone, Android, Wordpress
|Mar 28, 2015
|Mar 28, 2015Ends - 4d 2h
|Update Java Source Code
|Hi, I am having the source code of a java project and it's a little outdated. I am also having the compiled...(Same source) 2 months later. I am searching for someone that is able to update my source code from
|Java, XML
|Mar 25, 2015
|Mar 25, 2015Ends - 7h 36m
|Android application for Chat Rooms
|Hi, I need a simple app which can host chat rooms (see attached image) For the exact design and concept...please download and use "Keyja" app from the playstore. I need a smiliar app to that with slight modifications...modifications. You can just copy and edit its source code. Thanks!
|Mobile Phone, Android
|Mar 27, 2015
|Mar 27, 2015Ends - 3d 12h
|Best Seller Android App Makeover with revenue share
|...#1 best selling app for birders called iBird Pro. It's in need of a makeover for Android 5 (Lollipop) using...not look like the current app. Which means it will be easier to build the app since the current UI matches...have us check it off, then write code. We use bitbucket for source control and stress weekly updates
|Android, iPhone, Java, Mobile Phone, SQLite
|Mar 20, 2015
|Mar 20, 2015Ends - 4d 10h
|instagram Clone works with shared hosting
|...An instagram Clone App android version only and send me the source code. Android studio or eclipse.
|Mobile Phone, Android
|Mar 27, 2015
|Mar 27, 2015Ends - 3d 9h
|Ecommerce Iphone and Android App
|...need a mobile app with 1 touch QR scan ordering. So basically the ability to download the app and make a...is set up, a member can scan a QR code on a member card, that code will take them to product page, and... So step by step for user: Download app. from app store Set up account and instant payment options
|Mobile Phone, Android
|Mar 26, 2015
|Mar 26, 2015Ends - 2d 1h
|Telegram server source code
|...Telegram server source code 2.Need a set of complete and Telegram communications source code 3.think
|C Programming, Java, Python
|Mar 31, 2015
|Xamarin iPhone and Android App
|Very simple app for iPhone and Android to display a text box to prompt for a URL a button to load...URL's web page a web viewer Then send me the source to run on Xamarin Studio 5.8.1 Starter (Free) edition
|iPhone, Android, C# Programming
|Mar 27, 2015
|Mar 27, 2015Ends - 3d 5h
|Sudoku Android App
|Hi everyone! I need a developer for an android Sudoku app. 1. Puzzles will be supplied by me. 2...User can play as a guest or login/register. 3. App communicates with server via API's. So you need to...selected bidders will receive full documentation. 5. App is multilingual. Text should be kept on external
|Mobile Phone, Android
|Mar 31, 2015
|Convert android app in Ios application
|...already have an app code for android and i want someone to integrate my app from android to Ios ...
|Mobile Phone, iPhone
|Mar 27, 2015
|Mar 27, 2015Ends - 3d 15h
|Android Launcher Apps
|The following with source code required : This app will be used for 1080p android device. The layout...Google login single sign in link to the launcher app has to be created. 2. Calendar intent has to be...soon as the device is started.) 3. Newspaper App - Point to the directory of the FTP Server to access
|PHP, Java, Mobile Phone, Android
|Mar 30, 2015
|Mar 30, 2015Ends - 6d 6h
|Edit a source code of a website
|...programmer and coder to make some editing on a source code of a script for a social site .. the site is
|PHP, Javascript, Website Design, Boonex Dolphin, HTML5
|Mar 29, 2015
|Mar 29, 2015Ends - 5d 13h
|Write an Android and iPhone app
|Need an app created that will automatically answer cell phone calls. Details when we talk. This must...be an ORIGINAL app with ORIGINAL SCRIPT and THEMES. NO re-skins or templates or code from previous projects
|Mobile Phone, iPhone, Android
|Mar 11, 2015
|Mar 11, 2015Ends - 17h 11m
|Write an Android application
|I need someone who have the game source code for spin the bottle game
|Mobile Phone, Android, Game Development
|Mar 26, 2015
|Mar 26, 2015Ends - 2d 1h
|Create an Android Maps KML Parser
|Hello. I am creating an app that uses AirBNB's AirMapView: https://github.com/airbnb/AirMapView ...layer. Write me a library that I can add to Android Studio that would let me feed it a KML, and it...com/matiash/Simple-KML/ All I need is the source code for the library that I can add to my project
|Java, XML, Mobile Phone, Android, Google Earth
|Mar 31, 2015
|Whmcs client area APP for Android & ios
|...with xCode and Android with eclipse. here is some features we need to have in this app: See overview...invoices and pay the invoices Adding new order in app payment gateway ( Paypal,Credit Card ) Push notifications(on...administrator. We need to be able to customize this app anytime we need and change and update the screens
|iPhone, Android, Software Architecture, iPad
|Mar 25, 2015
|Mar 25, 2015Ends - 15h 25m
|Android Developers for a Job portal app
|We are looking for a team to develop an android application for a unique job portal and it's corresponding...along with his skill sets and a link to user written code etc Select the no of months to hire and submit
|PHP, Android, Cloud Computing, Amazon Web Services, RESTful
|Mar 30, 2015
|Mar 30, 2015Ends - 6d 11h
|RE of windows app
|...have a windows freeware and looking for its source code. Only serious bidders please. You can find the
|C Programming, .NET, Windows Desktop, Software Architecture
|Mar 29, 2015
|Mar 29, 2015Ends - 5d 9h
|Build a Website for downloadin video and audio files from sites like youtube
|...have extra work that all to be converted to Android,iPhone app and browser extension.
|PHP, Website Design, MySQL, jQuery / Prototype, Bootstrap
|Mar 25, 2015
|Mar 25, 2015Ends - 1h 18m
|I need the software in c# only with Source Code
|I need the software in c# only with Source Code i need a software which will install automatically
|PHP, Software Architecture
|Mar 31, 2015
|programming Plugins and apps
|...make application with source code ENIGMA2 Plugins XBMC Plugins Android APP IOS APP The principle of
|PHP, Linux, Mobile Phone, Android, Software Architecture
|Mar 28, 2015
|Mar 28, 2015Ends - 4d 15h
|Implement Echoprint Open Source Server - Phase 1: Web Server
|...an open source music fingerprint and resolving framework powered by the The Echo Nest. The code generator...microphone or file into Echoprint codes) is open source (MIT licensed) and free for any use. The server...component that stores and resolves queries is open source (Apache 2 licensed) and free for any use. The data
|Java, Python
|Mar 30, 2015
|Mar 30, 2015Ends - 6d 15h
|Have you built a browser on the Chromium Source Code?
|...Chromium Source Code? I am looking for a developer who has built a browser on the Chromium Source Code / Web
|C Programming, Google Chrome
|Mar 31, 2015
|Social Media Application in XAMARIN tool
|...someone has done Android social Media application then please bid here. i need source code of the application...application. note first i will check your app on my device ..
|.NET, Mobile Phone, iPhone, Android, C# Programming
|Mar 30, 2015
|Mar 30, 2015Ends - 5d 23h
|Android App for Bluetooth ESC/POS Receipt Printers
|I am looking for someone to build me an Android application that will automatically and immediately print...differentiate between general emails, a 16 digit code is included within the subject line (as a filter)...correct 16 digit code need to be printed. This application will be installed on Android phones and use
|Mobile Phone, Android, Google App Engine
|Mar 29, 2015
|Mar 29, 2015Ends - 5d 20h
|Android app
|Android app developing
|Mar 27, 2015
|Mar 27, 2015Ends - 3d 11h
|Complete nearly finished IOS Notification app
|I have a small IOS app that is mostly coded already and needs completing. The app is simply installed...notifications. I already have a working Android version. You can view the Android version here: https://play.google...smartmon.teamnotifications. You can login to the app using user:appdev2@mailinator, pass:a The documentation
|Mobile Phone, iPhone, English (US)
|Mar 26, 2015
|Mar 26, 2015Ends - 2d 2h
|Create Obj-C library of project, based on C# source
|Hello, We have C# library of our In-App-Support Service, source code located here: https://github.com/tagirovgeorge/Dome
|.NET, Mobile Phone, iPhone, C# Programming, Objective C
|Mar 31, 2015
|Build a Website Need web based accounting software with source code
|...ready made asp.net accounting software with source code immediately. Thanks Ramesh D
|ASP.NET, C# Programming, Microsoft SQL Server
|Mar 31, 2015
|Mar 31, 2015Ends - 6d 22h
|Android app
|Social Media App. Project will be described module by module.
|Mobile Phone, Android
|Mar 25, 2015
|Mar 25, 2015Ends - 2h 18m
|Create ios and android Tinder App copy win twist
|Hi there, I'm looking for somebody to code a tinder style app with a difference. If you can please give...give me a quote for a copy to build a tinder style app then we can discuss details. Thank you.
|Mobile Phone, iPhone
|Mar 28, 2015
|Mar 28, 2015Ends - 3d 23h
|Need design in software in.net I will give source code
|Hi I need design in my software I want to do it with design UI I have life time work if I will be happy I will make happy by giving life time work Thanks Ds
|ASP, C Programming, C# Programming, HTML, ASP.NET
|Mar 25, 2015
|Mar 25, 2015Ends - 7h 56m
|Development of cross-platform loyalty app
|...The requirements are below. APP DESCRIPTION: This is a loyalty App that will be used by staff in a... they will use a phone or tablet to scan the QR code. They can then assign points to the card ID. This...REQUIREMENTS: - The app only has to work on Android 4.x and above at this time - The app will need to be
|Android, Mobile Phone
|Mar 29, 2015
|Mar 29, 2015Ends - 5d 10h


text:
Featured in
Plain Background Group
That sounds like a really good idea. It looks too good not to.
lol, thanks
– Grinch/R. Pross
Oh STOP!
– Grinch/R. Pross
well,,,,,,,,,, ok,……..
– Grinch/R. Pross
Delicious capture!
nom, nom, nom… just what i could do with now! X
Me too!! Makes me wish I had it here at work!!! lol
– Grinch/R. Pross
Huge congratulations! this image is so superb it has been featured in the Plain Backgrounds group! (24th Aug 2012) Forum topic here
Sally X
Thank you so much for the honor of this feature Sally!!
– Grinch/R. Pross
Looks delicious!
LOL, Thanks, it was!!!!
– Grinch/R. Pross
Thank you for adding your work to The Best of RedBubble!
Simply grand! Cute title! MMMMMM!


text:
I have fw 20.0.B.0.85 for D2114 PT-BR. if you want...i'm uploading rith now in google drive
Thanks, I'll try. Do you know any way to root this phone? Thanks.
XDA Developers was founded by developers, for developers. It is now a valuable resource for people who want to make the most of their mobile devices, from customizing the look and feel to adding new functionality.
Are you a developer?
Copyright © xda-developers. Hosted by Leaseweb


text:
Nicotine can harm developing brains, experts say
Having a baby is a life-changing event, so it's completely normal to feel a bit overwhelmed. However, if your spirits are down despite your new bundle of joy, it may be a sign of postpartum depression. Take our online assessment to help determine your risk.
Disclaimer |
Privacy Notice |
Copyright 2013 - Hardin Memorial Hospital, All rights Reserved.


text:
Is it bad to not wake up properly until midday with a 3 wo?(32 Posts)
apachepony Fri 25-Jan-13 13:55:41
Brices Fri 25-Jan-13 22:03:48
BreastmilkDoesAFabLatte Fri 25-Jan-13 22:03:56
WinkyWinkola Fri 25-Jan-13 22:06:04
BluelightsAndSirens Fri 25-Jan-13 22:06:23
SamSmalaidh Fri 25-Jan-13 22:15:04
NotSoNervous Sat 26-Jan-13 09:03:37
MannUp Sat 26-Jan-13 09:56:29
Join the discussion
Registering is free, easy, and means you can join in the discussion, get discounts, win prizes and lots more.Register now
Already registered with Mumsnet? Log in to leave your comment or alternatively, sign in with Facebook or Google.
Talk: Customise | Unanswered messages | Getting started | Acronyms | FAQs
Threads: Active | I'm on | I'm watching | I started | Last 15 minutes | Last hour | Last Day
Please login first.


text:
Within City
Within 1 mile
Within 2 miles
Within 5 miles
Within 10 miles
Within 20 miles
Sort by
closest first
best match
most reviewed first
highest rated first
alphabetical order
"As an engineer and sports car enthusiast, I consider myself a discriminating consumer of tires and wheels. I value quality products, fair pricing and doing business locally. For ..."
"Extremely honest and reliable. Excellent customer service. I highly recommend GG's for your auto repairs."
"This establishment is a rip off from word go. They quoted me twice for different work that I had done elsewhere for more then half off. When I ..."
"Golden Nozzle Car Wash is a very convenient place for me. It is close to home and it does a good job at cleaning my car. I also ..."
"The service here is great! They are fast and efficient. I always get my oil changed here. You can get a coupon which will save you money, plus ..."
"I would (and have) highly recommend 3j's to anyone for tires, repairs or maintenance. Honest and accommodating group."
"This company sells big truck tires. They specialize in the Goodyear brand, and they also sell recaps. My husband bought 4 recaps for the rear and two new ..."
"My parents have been buying cars from Ken's for the past 15 years at least. They have never had any problems. They have always help my parents when ..."
"Go see Frank for exhaust and muffler work. In an area of corporate domination (midas, meineke) Frank will give a better deal just to get you in the ..."
"Whatever small repair your vehicle may need, trust the folks at Midas. They have given me good deals on tires and the offer a very reasonable price for ..."


text:
Your pot at the end of the rainbow could be vulnerable to identity thieves and hackers if you have outdated contact information. Keep your information up to date by logging in and clicking Info Center, then Personal Information. Learn more about keeping your money secure.
Secure Online Banking LoginComing Soon!


text:
- List all football matches scheduled in ASTRO Sport channels
- Set a reminder of the matches to your Google calendar
- Enables you to set your favourite teams and all the matches will be synchronize based on your favourite teams.
- Share, Vote, Like and Comment features for each matches
This application has been tested works on HTC Sensation, Samsung Galaxy S2 and Samsung Galaxy Ace. It should also works on other android devices as well.
Note :
- Please use "Enable Time Preference" wisely, otherwise you will not see any schedule in the listing.
If your match schedule is empty every time you do synchronization, kindly check if you have set "Time Preference"
because when "Time Preference" is set, "Bola Reminder" will only check match schedule based on your preferred time set in the setting (Default : Start Time = 8am, End Time = 11:59pm)
- For better schedule matching, if you want to set any team in "My Favourite", kindly DO NOT use short form such as "man city" or "man utd". Please use proper/full team name such as "Manchester City" or "Manchester United"
** Match schedule listing is subject to change.
Keyword : Bolasepak, football, soccer, malaysia, astro, reminder, schedule, jadual, sepakbola, sport channel
What's New
Jadual Liga Super 2015
# Felda United
# Johor DT
# Kelantan
# Lions XII
# Pahang
# Perak
# Sarawak
# Selangor
# Sime Darby
# Terengganu
Jadual Bola
1. Keputusan Perlawanan.
2. Jadual Perlawanan.
3. Kedudukan Liga.
Keputusan perlawanan dikemaskini secara LIVE!
(Keputusan dikemaskini setiap 3 minit)
Stadium Astro
- Video match highlights, pre and post-game interviews from the Barclays Premier League
- TV schedule and channel listings in Malaysian time
- Videos, news, statistics and standings for the top European football leagues tournaments and more
- Local sports content (news, video and features)
It is advisable to use Android version 4.0 and above to use the application.
Goal Live Scores
• Real Time scores, faster than anyone else.
• Live MatchCast Animation
• Super-fast push notifications
• Live Events
• Action from over 750 Leagues and Competitions
• Form and Head to Head for every match.
• In play Match Stats thousands of games.
• Predict the result
• League tables and form
• Fixtures and results
• Favorite teams & Leagues
• Track individual games with a single tap in MyScores
• Link directly to the match page from your notifications.
Fit Malaysia
- Organise sports games among your friends easily
- Find more players from the surrounding communities
- Search for any sports games going on around you
- Try out new sports which you have always wanted to play
- Make new friends and enjoy the joy of sports together!
Lead a healthier lifestyle through sports. Never will you have to cancel a game again due to the lack of players! Join us today! Please visit www.fit.my for more information.
Directed by Kementerian Belia dan Sukan (KBS), FitMalaysia aims to facilitate the adoption of healthy lifestyles amongst Malaysians. It is also part of the ministry's aspiration to make Malaysia a true sporting nation - a springboard to cultivate sporting success on a global playfield.
Football Live Scores
more than 1000 leagues, 10.000 teams and 100.000 players from countries all over the world.
Super fast push notifications to never miss a goal!
Interested in more background? Tons of statistics, videos and images from your favorite players/teams
All functionality is completely free.
- Schedules/fixtures overviews per country/league
- Live tables during soccer matches
- Topscores
- Live football statistics (ball possession, shots on goal etc)
- Player information + video's
- Team information + video's
- Substitutions
- Match voting
- Match videos
- Commenting and chats
- ....and many more
You are missing something? Tell us and we will build it for you within a week!
Live scores directly from the source, the 1st live score service on the Internet, powered by LiveScore.com since 1998.
This application comes directly from LiveScore Ltd., the leaders and inventors of real-time delivery of live sport data and owners of the no.1 ranked football / soccer web site globally.
- Live football / soccer scores from leagues, cup competitions and championships worldwide
- More than 1000 live football / soccer games followed live every week during the season
- Detailed match information
- Live tennis, basketball, ice hockey & cricket scores
- Live league tables for football, hockey and basketball
- Select your own refresh period
- User friendly
- Time & Date localized
- Very fast and responsive even on slow connection
- Minimal data transfers
- Bandwidth friendly with our data compression
- Lightweight
- Free
Android OS 2.0.1 (or higher) required.
Jadwal Bola
Dilengkapi dengan Berita Sepak Bola Terkini
3D Johor Darul Takzim LWP
To change the Live Wallpaper image you should do double click on the image. Also you can do it from the options menu too, where you can also configure the speed, transparency...
To INSTALL: You should open the application and press the menu button and select the option to "Set Live Wallpaper". You can also do this from the android menu, looking for the option of Live Wallpapers and then selecting it.
Tags: Liga Super Malaysia, Liga Perdana Malaysia, Kelab Bola Sepak Darul Takzim, Stadium J-Corp,
Pasir Gudang, Harimau Duitan, Harimau Selatan, Southern Tigers, The Bees, Bumblebee, Johor DT, JDT
ScoreCenter LIVE
ScoreCenter LIVE lets you stay up-to-date with sports matches anytime, anywhere. From the most popular sports such as the soccer, baseball and basketball, to football, ice hockey, and even formula 1. Don't miss out on ScoreCenter LIVE, the "all-in-one sports app" that supports over nine sports and more than 100 tournaments and leagues.
1. Everything you want to know about a match!
ScoreCenter will let you in on the ins and outs of sports matches.
Detailed sports information provided by ScoreCenter includes basic information such as scoring players, ball occupancy rate, shots, to more advanced information like player selection/replacement and formations. A "box score" function lets you check team and player records in real-time in sports matches. For the fans that couldn't make it to the stadium, ScoreCenter is the next best thing, and for those at the stadium, it will make the experience all the more exciting.
2. It's all about team spirit!
Games are always so much more fun with some company! You'll never watch a game alone when you're connected with ScoreCenter. Use the "Support Your Team" function to view match predictions and share information. Find other fans and support your team together! Try the "Match Prediction" function.
3. Get notifications on game news!
No need to check again and again for that final score. ScoreCenter's notification function creates a summary of important game schedules, news, and outcome, and alerts you as soon as new information becomes available.
4. Get social media feeds!
Now you don't have to log into Twitter or Facebook to see sports new feeds. ScoreCenter's social media channel lets you view Twitter, Facebook, and YouTube news feeds on your favorite teams all in one place.
5. Over 100 sports and leagues
The major sports that "ScoreCenter LIVE" support are as shown below.
: Soccer, Baseball, Basketball, Volleyball, Football, Ice Hockey, Tennis, Golf, Formula-1
New additions will be made in the future. If there is a sport or league you'd like to see added to ScoreCenter, send an e-mail to our customer service center.
Football Live Scores
Added football full league and top scorers for the Premier, Liga, Serie A, Ligue 1, Bundesliga, Eredivisie, Portuguese SuperLiga and Irish Premier L.
Added push notifications! Do you wish to receive push notifications for a match? You will receive a notification when either team score during the match!
Added live football/soccer scores for the USA Major League Soccer, Japan League 1 and League 2, Korean K-League, China Super League, Russia Premier Liga, South Africa Premier Soccer League, Singapore S-League, Poland Ekstraklasa, Slovenia 1. Liga.
The app shows the scorers of each match, yellow cards, red cards, penalties and so forth.
The app automatically saves the countries you are more interested in and orders them following your choices.
The live football/soccer scores are updated live 24/7
Keywords: football scores, soccer, live scores, free, champions league, soccer scores, liga, serie a, football results, football scores app
Champions League
Live score
Pre-match odds for the whole tour
Easy navigation and informative result's tables.
Opportunity of choosing favorite teams for tracking results of their games.
Opportunity of push notifications before the match begins and after past matches with results.
Time of match beginning sets according to your timezone.
Football Schedule - Live Score
Soccer Live
We include this kind of competitions:
- UEFA Champions League
- Euro League
- Premier League
- La Liga
- Bundesliga
- Serie A
- French League
and a lot more competitions around the world.
Enjoy it ;)
The Soccer Livescore App
Detailed feature list:
- Simple soccer / football live scores
- Game details, lineups, statistics, last results of teams, Head 2 Head, form and momentum chart!
- Team / Squad profile, team / squad statistics
- Player profile, picture, player statistics
- Expert Tips
- User prediction
- See other user profiles including Badges, their points, prediction accuracy, Favourite Teams
- Add games to watchlist and get push notifications (GCM) on games start, half time, full time, red cards, goals and line-ups when available
- Match Fixtures and historical results
- League standings
- Top scorers
- Favourite Teams, if you have selected any, a service checks every day at your chosen time if one of your teams is playing
- Filter out unwanted leagues, custom ordering
- Show game tables from yesterday, today, tomorrow
- In-game chat & facebook/Google+ connect (both optional)
- Attend to our Bet-Challenges and win goodies (when & how see in the News & Update section in the app)
- Widget with scores and red cards(Android 4.0 and above)
- Lockscreen Widget (Android 4.2 and above)
- Tablet UI
- Share game details with your friends including scores, key events, stats and lineups
FourFourTwo Malaysia/Singapore
– Completely re-designed with interactive features
– Video interviews with players
– Amazing high resolution pictures
More from developer
MY-Traffic is a community traffic monitoring for Malaysian. It has features :
- Graphical way to view road incidents data extracted from PLUS and LLMTraffic
- LIVE traffic cam from PLUS and LLM (Lembaga Lebuhraya Malaysia)
- Statistics Reports (Accident, Roadblock & Speed Traps statistics)
- User can contribute their traffic incidents and this incident will automatically alerted other traffic users.
- Each incidents contributed will be rated "Helpful" or "Not Helpful" by other users to make sure it's a reliable
- User with most rated reliable incident will be appeared in "Top Contributor" hall of fame.
Keyword : trafik, traffic jam, jalan, malaysia, google map, traffic map, traffic congestion,kuala lumpur,traffic cam, live camera, kl traffic cam,plus traffic, llmtrafik
Promo background image is used with owner permission http://sergey1984.deviantart.com/
Movie Reminder - Malaysia
For example, if you are big fan of Tom Hanks and you are not sure what or when movies related to Tom Hanks will be aired on ASTRO or local cinema, then Movie Reminder is the perfect application for you.
Today tab : Shows all today's movie schedule
Favourite tab : Shows only matched movie schedule based on "My Favourite" list
Cinema tab : Shows only matched cinema schedule based on "My Favourite" list
"Like" and "Comment" features (Touch on movie poster to show these features)
Note : You will not see any movie schedule listing until you have set your favourite actor or movie title.
Bola Reminder - Indonesia
- List all football matches scheduled in SCTV, RCTI, TVOne, ANTV, GlobalTV, MNCTV channels
- Set a reminder of the matches to your Google calendar
- Enables you to set your favourite teams and all the matches will be synchronize based on your favourite teams.
- Share, Vote, Like and Comment features for each matches
This application has been tested works on HTC Sensation, Samsung Galaxy S2 and Samsung Galaxy Ace. It should also works on other android devices as well.
Note :
- Please use "Enable Time Preference" wisely, otherwise you will not see any schedule in the listing.
If your match schedule is empty every time you do synchronization, kindly check if you have set "Time Preference"
because when "Time Preference" is set, "Bola Reminder" will only check match schedule based on your preferred time set in the setting (Default : Start Time = 8am, End Time = 11:59pm)
- For better schedule matching, if you want to set any team in "My Favourite", kindly DO NOT use short form such as "man city" or "man utd". Please use proper/full team name such as "Manchester City" or "Manchester United"
** Match schedule listing is subject to change.
Keyword : jadwal bola, sepakbola,SCTV, RCTI, TVOne, ANTV, GlobalTV, MNCTV


text:
Classic boat show celebrates 25 years
The 2012 Chain of Lakes Classic Boat Show, sponsored by the Minnesota Lakes Maritime Museum, is celebrating its 25th year on Saturday, July 14.
The event is held at Arrowwood Resort and Conference Center near Alexandria, and will feature a variety of watercraft on land and in the water from manufacturers such as Chris Craft, Gar Wood, Larson, Alexandria Boat Works and others.
Are you a newspaper subscriber but you don't have a Digital Access account yet? https://secure.forumcomm.com/?publisher_ID=40&event=subscriber/lookup.
You will need your subscription account number and phone number. Not sure if you have an account? Email us at firstname.lastname@example.com and we can help you.


text:
Category:Ejected English ministers of 1662
This category is for Church of England ministers who were ejected or otherwise resigned their livings or charges as a direct consequence of the Act of Uniformity 1662. It includes ministers who held livings at the time, but were not episcopally ordained, under the Commonwealth.
The exclusion resulting put an end to "Puritanism" considered as a reform movement in the Church of England, marking a watershed after which it is more accurate to talk of Nonconformism. The total number of these excluded clergy was estimated at 2,450, by Edmund Calamy, of whom 1300 became nonconforming ministers.
See also Category:Ejected Welsh ministers of 1662
Pages in category "Ejected English ministers of 1662"
The following 130 pages are in this category, out of 130 total. This list may not reflect recent changes (learn more).
- William Bagshaw
- Nathanael Ball
- Francis Bampfield
- Matthew Barker
- John Bartlet (divine)
- William Bartlet
- William Bates (minister)
- Richard Baxter
- Thomas Baylie
- William Benn (divine)
- William Blackmore (minister)
- James Bradshaw (1613–1685)
- James Bradshaw (minister)
- Haslefoot Bridges
- John Brinsley the Younger
- Thomas Brooks (Puritan)
- John Bryan (minister)
- Anthony Burges
- Cornelius Burges
- Ralph Button
- Richard Byfield


text:
The Top 10 In Action Sports
01Welcome to Qasr Al-Farid: The Rock With A Door That Will Rock Your World
02Already Got MTB Withdrawal Symptoms? Here Are The Crazy Downhill and Enduro Highlights From Crankworx New Zealand
0318 Things That Will Give Anyone Who Did Duke Of Edinburgh A Serious Flashback
0416 Things That Will Give 90s Skater Kids A Serious Flashback
05This 10 Year Old Kid Just Nailed A Dub Cork 1080... On His Second Attempt
06The 10 Most Spectacular Road Trips on Earth
07Water Slides, Plane Wings and Danny MacAskill: This is Road Cycling Like Never Before...
0824 Reasons Why Snowboarding Was Better In The Early Noughties
09Could Scientists Be About to Build a Terminator? They're Closer Than You Think…
10Win a Holiday for 2 in Chamonix with Three #holidayspam
Flying Finns: Episode 4 - California Dreamin'
A Snowboarding video by keltron - 16,839 views
Sleeping in the kitchen and getting changed in the car park is nothing for these guys as they head to Mammoth for some Californian shred.
It also means they can take a trip to San Diego for some much needed skate and surf time!
What Do You Think?
Get the best in action sports straight to your inbox


text:
Phone 020 7021 0888 : International +44 20 7021 0888 (Lines open 24/7)
Our company regularly use the services of London Translations and from the courtesy and professionalism exhibited by your customer service agents, to the exceptionally quick turnaround time and accuracy of the translations, we have yet to find fault in your service.
Turnaround and services were great. Everything was prompt and the projects went smoothly. We were pleased with the results; the work was completed in a very timely manner, the cost was reasonable and the translation accurate
The translations are excellent, and were delivered with quite amazing speed, meaning our multi-lingual website was operational ahead of schedule. Not only that, but your level of customer service is excellent - most attentive and professional.
All in all a very professional approach with a personal touch. Your team were flexible and always kept to deadlines (often at short notice). Impressively accurate translations to match our specialist brief.
I would like to strongly recommend the friendly, efficient and speedy service of London Translations Ltd. I went to them with a rush job and a plea for a one-day turn around and they did it for me straight away, which was perfect.
We were very impressed with the delivery, the flexibility and the approach. Everything was done according to brief with a polite, yet business-like manner.
I would personally like to thank you for the technical translation work you undertook to be completed in a relatively short time.
From the initial phone call we made to you a couple of days before New Year you immediately grasped what we needed.
We have complex, multi-language requirements in our research projects and the translations received are done very quickly and are of high quality.
Alex Cazacu who looked after the Draper work at London Translations was very efficient in ensuring requirements were fulfilled on time and to budget.
We have always found London Translations to be fast responding, accurate offering superb standard of service at a fair price.