425 research outputs found
Effect of glycosaminoglycans on growth factor-stimulated trophoblast invasion
Objectives: To determine the effect of glycosaminoglycans and a series of growth factors on the viability and invasion of the extravillous trophoblast cell line SGHPL4.
Methods: Cells were cultured in Hams F10 media supplemented with fetal bovine serum and L-glutamine. For viability studies cells were seeded into 96-well culture plates (104 cells/well), maintained in serum free medium for 24h and then incubated with glycosaminoglycans (heparin, heparin sulphate and hyaluronic acid; each 100ng/ml) ± growth factors (VEGF, FGFand HB-EGF). Cell viability was measured in cells using the MTS assay. Cellular invasion was assessed using the FluoroBlok invasion assay. Cells were serum-starved for 24 h, incubated with the fluorescent dye DiIC12(3) (10mg/ml) for 1 hour prior to seeding onto an artificial extracellular matrix coated 8 mm FluoroBlok porous membrane inserts (2.5 x 105 cells per insert). Growth factors ± GAGs were added to the cell suspension and the inserts were lowered into a 96-well plate containing 10% fetal calf serum.
Plates were incubated at 37°C for 24h. Invasion was determined by measurement of fluorescence of invaded cells using a fluorescent plate reader (Ex549/Em565 nm).
Results: Cell numbers were significantly increased following incubation with VEGF, FGF and HB-EGF. Cell number was also increased after incubation with each of the glycosaminoglycans tested. The largest increase was observed following incubation with heparin sulphate. Cell numbers were further increased when the GFs were incubated with HS and heparin, but not with hyaluronic acid. Invasion was increased following incubation with VEGF, HBEGF and HGF. Heparan sulphate and heparin increased invasiveness in a dose-dependent manner. In contrast, hyaluronic acid had no significant effect.
Conclusion: This study demonstrates a role for glycosaminoglycans in key features of trophoblast function
Analysis of procainamide-derivatised heparan sulphate disaccharides in biological samples using hydrophilic interaction liquid chromatography mass spectrometry
Glycosaminoglycans (GAGs) are a family of linear heteropolysaccharides made up of repeating disaccharide units that are found on the surface and extracellular matrix of animal cells. They are known to play a critical role in a wide range of cellular processes including proliferation, differentiation and invasion. To elucidate the mechanism of action of these molecules, it is essential to quantify their disaccharide composition. Analytical methods that have been reported involve either chemical or enzymatic depolymerisation of GAGs followed by separation of non-derivatised (native) or derivatised disaccharide subunits and detection by either UV/fluorescence or MS. However, the measurement of these disaccharides is challenging due to their hydrophilic and labile nature. Here we report a pre-column LC-MS method for the quantification of GAG disaccharide subunits. Heparan sulphate (HS) was extracted from cell lines using a combination of molecular weight cutoff and anion exchange spin filters and digested using a mixture of heparinases I, II and III. The resulting subunits were derivatised with procainamide, separated using hydrophilic interaction liquid chromatography and detected using electrospray ionisation operated in positive ion mode. Eight HS disaccharides were separated and detected together with an internal standard. The limit of detection was found to be in the range 0.6â4.9 ng/mL. Analysis of HS extracted from all cell lines tested in this study revealed a significant variation in their composition with the most abundant disaccharide being the non-sulphated âUAâGlcNAc. Some structural functional relationships are discussed demonstrating the viability of the pre-column method for studying GAG biolog
Heparan sulfate disaccharide measurement from biological samples using pre-column derivatization, UPLC-MS and single ion monitoring
Glycosaminoglycans are a heterogeneous family of linear polysaccharides comprised of repeating disaccharide subunits that mediate many effects at the cellular level. There is increasing evidence that the nature of these effects is determined by differences in disaccharide composition. However, the determination of GAG disaccharide composition in biological samples remains challenging and time-consuming. We have developed a method that uses derivatization and selected ion recording and RP-UPLCMS resulting in rapid separation and quantification of twelve heparin/heparin sulfate disaccharides from 5 ÎŒg GAG. Limits of detection and quantitation were 0.02â0.15 and 0.07â0.31 ÎŒg/ml respectively. We have applied this method to the novel analysis of disaccharide levels extracted from heparan sulfate and human cancer cell lines. Heparan sulfate disaccharides extracted from biological samples following actinase and heparinase incubation and derivatized using reductive amination with 2-aminoacridone. Derivatized disaccharides were analyzed used UPLC-MS with single ion monitoring. Eight HS disaccharide subunits were separated and quantified from HS and cell lines in eleven minutes per sample. In all samples the most abundant subunits present were the unsulfated ÎUA-GlcNAc, ÎUA-GlcNAc,6S and ÎUA,2S-GlcNS,6S. There was considerable variation in the proportions and concentrations of disaccharides between different cell lines. Further studies are needed to examine the significance of these differences
Can quantification of Serum Glycans predict Pre-Eclampsia?
Objectives: To determine if concentrations of placental glycans and glycan components are altered in pre-eclamspia and to determine if serum levels can predict pre-eclampsia.
Methods: Serum samples were collected from women in the third trimester of singleton pregnancy but before the onset of pre-eclampsia and also from women during unaffected pregnancies at the samegestational age. Tissues were collected from the basal plate of placentas collected at delivery following uncomplicated singleton pregnancy (term and preterm) and from pregnancies complicated by preeclampsia. Pre-eclampsia was diagnosed according to International Society for the Study of Hypertension in Pregnancy criteria. Glycan components were isolated using a combination of enzyme digestion, molecular weight filtration and ion exchange chromatography, and then derivatised prior to separation using hydrophilic interaction liquid chromatography. Components were detected using electrospray ionisation operated in positive ion mode with single ion monitoring.
Results: Specific glycan components (designated glycan 1, 2 and 3) were significantly altered in the serum from women who went on to have preeclampsia compared to those who had an unaffected pregnancy. Interestingly, levels of the same biomarkers were also elevated in nulliparous versus multiparous pregnancy. Biomarkers were also significantly altered in placental tissues from pregnancies complicated by preeclampsia
Conclusion: This study suggests that altered glycan levels may contribute to impaired placental development and that the glycome is a potential diagnostic target for pre-eclampsia, and possibly other disorders of pregnancy
When does humoral memory enhance infection?
Antibodies and humoral memory are key components of the adaptive immune
system. We consider and computationally model mechanisms by which humoral
memory present at baseline might instead increase infection load; we refer to
this effect as EI-HM (enhancement of infection by humoral memory). We first
consider antibody dependent enhancement (ADE) in which antibody enhances the
growth of the pathogen, typically a virus, and typically at intermediate
"Goldilocks" levels of antibody. Our ADE model reproduces ADE in vitro and
enhancement of infection in vivo from passive antibody transfer. But notably
the simplest implementation of our ADE model never results in EI-HM. Adding
complexity, by making the cross-reactive antibody much less neutralizing than
the de novo generated antibody or by including a sufficiently strong
non-antibody immune response, allows for ADE-mediated EI-HM. We next consider
the possibility that cross-reactive memory causes EI-HM by crowding out a
possibly superior de novo immune response. We show that, even without ADE,
EI-HM can occur when the cross-reactive response is both less potent and
"directly" (i.e. independently of infection load) suppressive with regard to
the de novo response. In this case adding a non-antibody immune response to our
computational model greatly reduces or completely eliminates EI-HM, which
suggests that "crowding out" is unlikely to cause substantial EI-HM. Hence, our
results provide examples in which simple models give qualitatively opposite
results compared to models with plausible complexity. Our results may be
helpful in interpreting and reconciling disparate experimental findings,
especially from dengue, and for vaccination
Does the neutrino magnetic moment have an impact on solar neutrino physics?
Solar neutrino observations coupled with the recent KamLAND data suggest that
spin-flavor precession scenario does not play a major role in neutrino
propagation in the solar matter. We provide approximate analytical formulas and
numerical results to estimate the contribution of the spin-flavor precession,
if any, to the electron neutrino survival probability when the magnetic moment
and magnetic field combination is small.Comment: 7 pages, 1 figure
Comparison of local frequency shifts between MDI velocity and intensity data
We analyze the frequencies of highâdegree p modes using
velocity and intensity data from MDI. The study is
carried out with the local helioseismic technique of ring
diagrams. The resultant power spectra at several different
positions on the solar disk are fitted with symmetric and
asymmetric profiles to find the centerâtoâlimb variation
(CLV). Using symmetric profiles in the anaylsis produces
significant differences in frequency between velocity and
intensity with the differences increasing from disk center
to the limb. The use of asymmetric peak profiles reduces
these differences and in the 3 mHz band the frequencies
agree reasonably well with each other
Measuring the Solar Radius from Space during the 2003 and 2006 Mercury Transits
The Michelson Doppler Imager (MDI) aboard the Solar and Heliospheric
Observatory observed the transits of Mercury on 2003 May 7 and 2006 November 8.
Contact times between Mercury and the solar limb have been used since the 17th
century to derive the Sun's size but this is the first time that high-quality
imagery from space, above the Earth's atmosphere, has been available. Unlike
other measurements this technique is largely independent of optical distortion.
The true solar radius is still a matter of debate in the literature as measured
differences of several tenths of an arcsecond (i.e., about 500 km) are
apparent. This is due mainly to systematic errors from different instruments
and observers since the claimed uncertainties for a single instrument are
typically an order of magnitude smaller. From the MDI transit data we find the
solar radius to be 960".12 +/- 0".09 (696,342 +/- 65 km). This value is
consistent between the transits and consistent between different MDI focus
settings after accounting for systematic effects.Comment: Accepted for publication in The Astrophysical Journal (2012 March 5
Our Sun. IV. The Standard Model and Helioseismology: Consequences of Uncertainties in Input Physics and in Observed Solar Parameters
Helioseismology provides a powerful tool to explore the deep interior of the
Sun: for example, the adiabatic sound speed can be inferred with an accuracy of
a few parts in 10,000. This has become a serious challenge to theoretical
models of the Sun. Therefore, we have undertaken a self-consistent, systematic
study of sources of uncertainties in the standard solar model, which must be
understood before the helioseismic observations can be used as constraints on
theory. We find that the largest uncertainty in the sound speed in the solar
interior, namely, 3 parts in 1000, arises from uncertainties in the observed
photospheric abundances of the elements; uncertainties of 1 part in 1000 arise
from (1) the 4% uncertainty in the OPAL opacities, (2) the 5% uncertainty in
the basic pp nuclear reaction rate, (3) the 15% uncertainty in the diffusion
constants for the gravitational settling of helium, and (4) the 50%
uncertainties in diffusion constants for the heavier elements. (Other
investigators have shown that similar uncertainties arise from uncertainties in
the interior equation of state and in rotation-induced turbulent mixing.) The
predicted pre-main-sequence solar lithium depletion is a factor of order 20 (an
order of magnitude larger than that predicted by earlier models that neglected
gravitational settling and used older opacities), and is uncertain by a factor
of 2. The predicted neutrino capture rate is uncertain by 30% for the Cl-37
experiment and by 3% for the Ga-71 experiments (not including uncertainties in
the capture cross sections), while the B-8 neutrino flux is uncertain by 30%.Comment: LaTeX, 38 pages (including 8 figures); ApJ, in press. Added
figures/color figurea available at
http://www.cita.utoronto.ca/~boothroy/sun4.htm
PENGARUH LOKASI DAN PROMOSI PENJUALAN TERHADAP KEPUTUSAN PEMBELIAN MORNING BREAD BANDUNG (Survei Pada Konsumen Morning Bread Bandung)
ABSTRAK
Penelitian ini bertujuan untuk mengetahui pengaruh dari lokasi dan promosi penjualan
terhadap keputusan pembelian (survei pada konsumen Morning Bread Bandung) secara
simultan maupun parsial. Metode penelitian yang digunakan adalah metode deskriptif dan
verifikatif dengan jumlah sampel sebanyak 85 responden. Pengujian instrumen penelitian
menggunakan uji validitas dan reliabilitas. Metode analisis data yang digunakan adalah
analisis regresi linier berganda, korelasi berganda dan koefisien determinasi.
Hasil penelitian menunjukkan bahwa terdapat pengaruh positif dan signifikan antara
lokasi dan promosi penjualan terhadap keputusan pembelian. Besarnya pengaruh lokasi dan
promosi penjualan terhadap keputusan pembelian secara simultan sebesar 53,6% dan sisanya
46,4% dipengaruhi variabel lain yang tidak diteliti. Secara parsial besarnya pengaruh lokasi
terhadap keputusan pembelian sebesar 17,6% dan pengaruh promosi penjualan terhadap
keputusan pembelian sebesar 36%, sehingga dapat disimpulkan bahwa promosi penjualan
memberikan pengaruh paling besar terhadap keputusan pembelian.
Kata kunci : Lokasi, Promosi Penjualan dan Keputusan Pembelia
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