Cell adhesion molecules (CAMs) are expressed ubiquitously. Each of the four families of CAMs is comprised of glycosylated, membrane-bound proteins that participate in multiple cellular processes including cell-cell communication, cell motility, inside-out and outside-in signaling, tumorigenesis, angiogenesis and metastasis. Intercellular adhesion molecule-2 (ICAM-2), a member of the immunoglobulin superfamily of CAMs, has six N-linked glycosylation sites at amino acids (asparagines) 47, 82, 105, 153, 178 and 187. Recently, we demonstrated a previously unknown function for ICAM-2 in tumor cells. We showed that ICAM-2 suppressed neuroblastoma cell motility and growth in soft agar, and induced a juxtamembrane distribution of F-actin in vitro. We also showed that ICAM-2 completely suppressed development of disseminated tumors in vivo in a murine model of metastatic NB. These effects of ICAM-2 on NB cell phenotype in vitro and in vivo depended on the interaction of ICAM-2 with the cytoskeletal linker protein α-actinin. Interestingly, ICAM-2 did not suppress subcutaneous growth of tumors in mice, suggesting that ICAM-2 affects the metastatic but not the tumorigenic potential of NB cells. The goal of the study presented here was to determine if the glycosylation status of ICAM-2 influenced its function in neuroblastoma cells.
Because it is well documented that glycosylation facilitates essential steps in tumor progression and metastasis, we investigated whether the glycosylation status of ICAM-2 affected the phenotype of NB cells. We used site-directed mutagenesis to express hypo- or non-glycosylated variants of ICAM-2, by substituting alanine for asparagine at glycosylation sites, and compared the impact of each variant on NB cell motility, anchorage-independent growth, interaction with intracellular proteins, effect on F-actin distribution and metastatic potential in vivo.
The in vitro and in vivo phenotypes of cells expressing glycosylation site variants differed from cells expressing fully-glycosylated ICAM-2 or no ICAM-2. Most striking was the finding that mice injected intravenously with NB cells expressing glycosylation site variants survived longer (P ≤ 0.002) than mice receiving SK-N-AS cells with undetectable ICAM-2. However, unlike fully-glycosylated ICAM-2, glycosylation site variants did not completely suppress disseminated tumor development.
Reduced glycosylation of ICAM-2 significantly attenuated, but did not abolish, its ability to suppress metastatic properties of NB cells.
Cell adhesion molecules (CAMs) participate in diverse functions in normal cells and tumor cells. These functions include cell-cell and cell-matrix adhesion, migration, oncogenesis, metastasis, tumor suppression or progression, and signal transduction [1–5]. Based on known functions, four major families of CAMs have been identified: integrins, cadherins, selectins, and immunoglobulin (Ig) superfamily. CAMs of the Ig superfamily contain one or more domains similar to the C2 type domains characteristic of a subset of Ig molecules (immunoglobulin-like C2 domain [IgLD]) .
Intercellular adhesion molecule-2 (ICAM-2), a 55-60 kDa transmembrane glycoprotein, belongs to the Ig superfamily of CAMs [7, 8]. In normal tissue, neovascular endothelial cells express the highest level of ICAM-2. Established vasculature and some leukocytes express a relatively low level of ICAM-2. Functionally, the extracellular domain (ED) of ICAM-2 mediates binding of ICAM-2 on vascular endothelial cells to β2-integrins on the surface of leukocytes, an initial step in immune responses [9, 10]. ICAM-2 also facilitates neutrophil binding to and migration through vascular endothelium as a component of immune reactions [11, 12]. In tumor cells, we recently identified a unique role for ICAM-2 when we demonstrated that ICAM-2 suppressed the metastatic phenotype neuroblastoma (NB) cells .
Structurally, ICAM-2 wild type (WT) protein in normal cells  and NB cells is identical (Figure 1). ICAM-2 WT is expressed as a 275-amino acid protein containing two Ig-like domains (IgLD-1 and IgLD-2) in the extracellular domain (ED) at the N-terminus of the protein, a 26-amino acid transmembrane domain (TD), and a 26-amino acid cytoplasmic domain (CD). The first 21 amino acids comprise a signal peptide (sp) that routes the nascent protein through the endoplasmic reticulum, for post-translational processing. Processing includes deletion of the sp, to yield a mature protein of 254 amino acids. The mature WT protein consists of a 202 amino acid ED, and the TD and CD noted above. The CD binds to actin cytoskeletal linker ERM proteins (ezrin, radixin, moesin) and to α-actinin [15, 16]. The functional significance of the interactions of ICAM-2 with actin linker proteins in tumor cells is largely unknown, with the exception of our recent observation that the interaction of ICAM-2 with α-actinin is essential to ICAM-2-mediated suppression of the metastatic potential of NB cells .
Schematic structure of ICAM-2 and ICAM-2 variants. Schematic representation of the structures of ICAM-2 and hypo-glycosylated ICAM-2 glycosylation site variants (gsv). ICAM-2 wild type (WT) contains six glycosylated amino acids in its extracellular domain (ED), at residues 47, 82, 105, 153, 176 and 187. The ED also contains two immunoglobulin-like domains (designated IgLD-1 and -2). Short transmembrane (TD) and cytoplasmic (CD) domains are also present. The N-terminus of the nascent protein contains a 21-amino acid signal peptide (sp) that is not present in the 254 amino acid mature protein. Variants were constructed by substituting alanines (A) for asparagines (N) at the indicated amino acid residues. For example, gsv1,4,5 contains alanines at mutation sites 1, 4, and 5 (amino acids 47, 153, and 176); therefore, gsv1,4,5 is not able to be glycosylated at these three sites. Variant gsv1-6 has alanines at all six glycosylation sites, and is non-glycosylated when expressed in SK-N-AS cell transfectants.
The ED of ICAM-2 contains six N-glycosylation sites (asparagines [Asn] residues; amino acids # 47, 82, 105, 153, 178 and 187) , a frequent post translational modification of CAM proteins and a modification known, in general, to influence CAM structure and function and to impact the phenotype of tumor cells [18–20]. For example, altered N-glycosylation of β1-integrin decreases cell migration , and aberrant N-glycosylation of E-cadherin increases discohesion of oral cancer cells . The effect of glycosylation of ICAM-2 on tumor cell function is unknown.
Our previous studies, however, document that fully glycosylated ICAM-2 suppresses the development of disseminated tumors in a murine model of metastatic NB. Importantly, and consistent with preclinical data, we also showed that primary human NB cells that express endogenous ICAM-2 have morphologic and histologic properties of NB cells recognized clinically to have limited metastatic potential . These observations suggest a functional role for ICAM-2 in primary tumor cells. Therefore, in order to identify domains of ICAM-2 that contribute significantly to its function in tumor cells, we generated four ICAM-2 constructs, encoding glycosylation site variants (gsv) of this protein. We expressed each construct in SK-N-AS NB cells, which have no detectable endogenous ICAM-2 and which display a metastatic phenotype in in vitro assays and in vivo models. We altered the glycosylation status of ICAM-2 by substitution of alanines for asparagines, to prevent glycosylation at specific residues that comprise N-linked glycosylation sites in situ; and then determined whether substitutions modulated the ability of ICAM-2 to suppress metastatic properties of NB cells. The data show that hypo-glycosylated variants of ICAM-2 have a significant impact on NB cell phenotype, but to a lesser extent than that seen with ICAM-2 WT.
The human neuroblastoma (NB) cell line SK-N-AS (American Type Culture Collection, Manassas, VA) was maintained in Dulbecco's Modified Eagle Medium (DMEM; Hyclone, Fisher Scientific, Savannah, GA) with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) and 2 mM L-glutamine (Hyclone, Fisher Scientific, Savannah, GA) at 37°C and 10% CO2. Parent SK-N-AS cells and stable transfectants were cultured under the same conditions. Human dermal microvascular endothelial cells (HDMVEC) were obtained from Lonza, Inc. (Allendale, NJ), and the human bone marrow endothelial cell line, HBMEC-28, was provided by Dr. E. van der Schoot (Sanquin Blood Supply Foundation, The Netherlands) . HDMVEC cells were grown in gelatin-coated culture flasks in Endothelial Cell Grown Medium (EGM) supplemented with 10% heat-inactivated FBS. HBMEC-28 cells were propagated in medium prepared using the EGM™ BulletKit™ (Lonza) according to the directions of the manufacturer.
The plasmid encoding human ICAM-2 was generated as published . Briefly, cDNA encoding ICAM-2 was generated from RNA isolated from human umbilical vein endothelial cells (Clonetics, San Diego, CA). Primers for full length ICAM-2 were: forward (5'GCTTCCGCTGCCTGGATTCT3') and reverse (5'AAGTCCAGGTGTTGTATTC3'). Amplification was performed at 95°C for 1 min; then 30 cycles of 94°C for 30 sec, 55°C for 30 sec, 72°C for 1 min, followed by 72°C for 7 min. The resulting cDNA was isolated after electrophoresis in agarose gels, sequenced in its entirety by automated sequencing methods (St. Jude Hartwell Center for Bioinformatics, Memphis, TN), and subcloned into the BamH1 restriction site of the expression plasmid pIRESneo2 (Clontech, San Jose, CA) to generate pIRES.ICAM-2. Plasmid transfections were carried out using FuGene6 (Roche Diagnostics, Indianapolis, IN). Forty-eight hours after transfection, 750 μg/ml geneticin (G418; Roche Diagnostics Corporation, Indianapolis, IN) was added to select transfected cell populations. The use of a vector containing an internal ribosomal entry site (IRES) between the ICAM-2 cDNA and the gene encoding the G418 resistance protein eliminated the need to isolate and characterize multiple individual clonal cell lines. The ICAM-2 cDNA sequence was verified as error-free in the resulting cell line (SK-N-ASpIRES.ICAM-2) by the St. Jude Hartwell Center.
The objective of this study was to evaluate the impact of the glycosylation status of ICAM-2 on the function in NB tumor cells. Therefore, mutagenesis by overlapping PCR, using Pfu polymerase (QuickChange mutagenesis kit; Stratagene-Agilent Tech, Wilmington, DE) and pIRES.ICAM-2 as a template, was performed to replace some or all of the six asparagine glycosylation sites with alanine residues at positions 47, 82, 105, 153, 176, and 187, each of which is glycosylated in the endogenous wild-type protein. Alanine was chosen for substitution at all six or of a subset of glycosylation sites to prevent glycosylation [24–26]. Plasmids encoding mutated variants of ICAM-2 cDNA were identified by restriction enzyme analysis and sequences confirmed by direct DNA sequencing. Transfection of plasmids encoding ICAM-2 variants into SK-N-AS cells, selection of transfected cells, and culture conditions were the same as for SK-N-ASpIRES.ICAM-2 transfectants. Plasmids and transfected cell lines are designated as glycosylation site variants (gsv) followed by the positions of the amino acids (asparagines) that were replaced with alanine. For example, gsv1,4,5 is non-glycosylated at amino acids 47, 153 and 176 (Figure 1). In Figures 2, 3, 4, 5, 6, 7 and 8, transfected SK-N-AS cell lines expressing variant forms of ICAM-2 are referred to as the specific variant expressed: gsv1,4,5; gsv1,2,5; gsv4,5; and gsv1-6. SK-N-AS cells expressing ICAM-2 WT are labeled in the Figures as "WT". SK-N-ASpIRESneo2 control transfectants are labeled "neo".
To determine if the growth rates of all SK-N-AS transfectants expressing ICAM-2 variants were comparable, 1×106 cells were plated in 75 cm2 flasks, and cell numbers determined daily thereafter, using a hemocytometer. Doubling times were calculated using http://www.doubling-time.com/compute.php.
Each well of a 6-well plate was plated with 1mL 0.8% Sea-plaque agarose in DMEM with 10% FBS, and cooled to 4°C overnight. NB cells were resuspended in soft agar (0.4%) in DMEM with 10% FBS at a density of 30,000 cells in 10 mL, and 1 mL of cell suspension was overlaid in the wells containing 0.8% agar. Plates were then incubated at 37°C for 14-21 days. Colonies of >20 cells were identified microscopically and the number of colonies/well recorded. Six replicate wells of each transfectant were included in at least three independent experiments, as published previously [13, 28].
To assess the motility of transfectants expressing ICAM-2 variants, we performed scratch or wound healing assays . Cells were plated at an initial density of 1×106 cells per well in 6-well plates and allowed to grow to confluency. A centerline was marked on the underside of each well along its horizontal axis, to designate loci at which images would be acquired at each time point. Vertical linear defects (scratches) were introduced into cell monolayers using a 200 μL pipet tip. Each well received 3 scratches. Images were acquired at 0, 24, 48, and 72 hours post scratch, at 100× magnification at each intersection of the scratch wound (vertical defect) and the marked horizontal centerline using a Dr-5 digital camera (Southern Microscope) mounted t22232o a Nikon Eclipse TS100 inverted microscope. For each time point, 5 measurements were taken per well in each of 3 wells and the average the horizontal width of the linear defect in pixels calculated using Adobe Photoshop CS4 (version 11.0.2).
Cells (SK-N-AS cells transfected to express ICAM-2 WT or variants gsv1,4,5; gsv1,2,5; gsv4,5; or gsv1-6) were plated in 175 cm2 tissue culture flasks (Corning) with standard growth medium (high glucose DMEM + 10%FBS + 2 mM L-glutamine) and grown to near-confluence. Cells were washed 3 times with ice-cold PBS (pH 8.0) and surface proteins biotinylated for 30 minutes at room temperature using 0.5 mg/ml EZ-Link® Sulfo-NHS-LC-Biotin (21335; Thermo Scientific/Pierce) reconstituted in PBS (pH 8.0). The biotinlyation reaction was quenched by washing the cells 3 times with 100 mM glycine in PBS (pH 7.2). Cells were then lysed with 0.5% NP-40 + complete Mini protease inhibitor (Roche) and homogenized. Biotinylated protein preparations were then incubated with 100 μl Pierce streptavidin® resin (6% crosslinked beaded agarose 50% aqueous slurry) at room temperature for 20 minutes with gentle rocking, and then centrifuged to separate membrane-associated, biotin-avidin precipitated proteins from unlabeled proteins. After unbound proteins (supernatant) were removed, beads were washed 3 times with cold lysis buffer, boiled in sample buffer, and subjected to SDS-PAGE. Following electrophoresis, standard immunoblots were performed.
Cleavage of N-linked glycans was carried out using Endo H (#P0702L; New England BioLabs, Ipswich, MA) and PNGase F (#P0704S; New England BioLabs, Ipswich, MA) according to the manufacturer's instructions. In brief, 20 μg of protein from control cells (HBMEC-28) or NB cells expressing ICAM-2 WT or a variant were denatured at 100°C for 10 minutes with glycoprotein denaturing buffer (5% SDS, 0.4M DTT in PBS). After cooling, protein solutions were centrifuged and incubated for an additional hour at 37°C with 2 μl of either Endo H enzyme (plus G5 Reaction buffer, pH 5.5; New England Biolabs) or PNGase F enzyme (plus G7 reaction buffer, pH 7.5 plus 10% NP-40 ; New England Biolabs). Control samples were subjected to the same denaturing and digesting conditions, but substituting equivalent volumes of water for enzyme. Samples were then subjected to SDS-PAGE and immunoblot analyses.
Co-immunoprecipitations (IP) were done as described previously . The antibody used for IP of α-actinin was MAB1682 (Millipore, Fisher Scientific, Savannah, GA). Antibodies used for immunoblots (IB) for ICAM-2, α-actinin, and actin were AF244 (R&D Systems, Minneapolis, MN) and sc-1512 (Santa Cruz Biotech, Santa Cruz, CA), 4968 (Cell Signaling Technology, Danvers, MA), and A5316 (Sigma-Aldrich, St. Louis, MO), respectively. AF244 is a goat polyclonal IgG that recognizes the extracellular domain of ICAM-2. C-20 is a goat polyclonal IgG that recognizes the intracellular C-terminus of the ICAM-2 protein. Secondary HRP-conjugated anti-goat (AF244, sc-1512), anti-rabbit (4968) and anti-mouse (A5316) antibodies were purchased from Santa Cruz Biotech. The ECL system (Pierce ECL Chemiluminescent Substrate; Thermo Scientific) was used to detect presence of horseradish peroxidase (HRP) enzyme. Immunoblot result in Figure 2D was quantitated using Adobe Photoshop CS4 (San Jose, CA) by multiplying area by integrated intensity of the band of interest and normalizing that result to that of a control protein (actin) band.
Actin fibers were visualized by fluorescence microscopy using standard methods . Briefly, cells were plated in chamber well slides (2 × 105 cells per chamber) and allowed to adhere. Approximately 18 hours after plating, cells were washed with PBS and fixed with 3.7% (w/v) paraformaldehyde in PBS for 10 minutes at room temperature. Remaining attached cells were washed with PBS, and permeabilized with 0.2% (v/v) Triton X-100 for 1 minute. Actin fibers were stained with 10 μg fluorescein isothiocyanate (FITC)-conjugated phalloidin (P5282; Sigma-Aldrich, St. Louis, MO) in 1 mL phosphate buffered saline (PBS) for 30 minutes. Phalloidin-stained fibers were visualized by confocal fluorescence microscope equipped with narrow band filters, by standard methods (Figure 5A) or by the Zeiss Axio Observer Z.1 microscope platform in conjunction with Zen 2011 Blue imaging software (Carl Zeiss)(insets in Figure 5A). DAPI (4',6-diamidino-2-phenylindole; appears as blue fluorescence) was used to detect DNA.
Flow cytometric analyses were used to verify the cell membrane localization of ICAM-2 and ICAM-2 variants. Cells (1 × 106) were trypsinized, washed twice with PBS, and incubated for 30 min with goat serum (G9023, Sigma-Aldrich) diluted 1:10 in PBS. Cells were then incubated for 60 minutes on ice with mouse monoclonal anti-human ICAM-2 antibody (CBR-IC2/2, sc-23935 PE; Santa Cruz Biotech). A separate aliquot of cells was incubated with mouse isotope-matched IgG (sc-2025, Santa Cruz Biotech, Santa Cruz, CA) as a negative control. All samples were then washed twice with FACS buffer (1% BSA in PBS) and incubated for 60 minutes on ice with phycoerythrin (PE, sc-3798, Santa Cruz Biotech, Santa Cruz, CA)-conjugated goat anti-mouse IgG. Data were acquired on an LSR-II cytometer (BD Biosciences, San Jose, CA) and analyzed using FlowJo v 7.6.1 (TreeStar) software. Only intact cells, as identified by forward and side light scatter, were analyzed. Propidium iodide (5 μg/mL PBS; stored at 4°C in the dark) staining was also used as a parameter to exclude non-viable cells.
Transfected cells were seeded at a density of 15,000 cells/cm2 and maintained under standard culture conditions for 24-48 hours. Photomicrographs were acquired at 20× magnification using a Dr-5 digital camera (Southern Microscope) mounted to a Nikon Eclipse TS100 inverted microscope and archived with TSView software (version 7.1.04, Tucsen Imaging Technology Co./Southern Microscope, Inc., Haw River, NC).
Cells were washed three times with PBS at 4°C, and resuspended in Cell Lysis Buffer (10 mM HEPES, 10 mM NaCl, 1 mM KH2PO4, 5 mM NaHCO3, 1 mM CaCl2, and 0.5 mM MgCl2 solution ) + 1 mM EDTA + protease inhibitor (complete mini protease inhibitor tablets; Roche Applied Science, Indianapolis, IN) solution. Cells were allowed to swell for 5 minutes on ice and then homogenized using a Fisher Scientific Misonix XL2000 Ultrasonic Homogenizer (Fisher Scientific), and centrifuged at 7,500 rpm for 5 minutes. The resulting supernatant suspension contained both plasma membrane and cytosolic proteins. This suspension was then re-centrifuged at 25,000 rpm for 30 minutes to separate the plasma membrane (pellet) from the soluble protein fraction. The crude plasma membrane pellet was then resuspended in 40 μl PBS and standard assays done to determine the protein concentration of these suspensions. Samples were stored at -80°C until ready for use. Suspensions were verified as membrane-enriched by immunoblot analysis for the plasma membrane marker (ATP1A1; Novus, Novus Biologicals, Littleton, CO) .
Female Es1 e /SCID mice (6-8 weeks old) were obtained from St. Jude Children's Research Hospital Animal Resources Center (ARC) (Memphis, TN), with the approval of Dr. Phil Potter . The in vivo animal studies were carried out in accordance with a protocol approved by the St. Jude Children's Research Hospital Animal Care and Use Committee (Approval Number: 374). All mice were maintained in the Animal Resources Center at St. Jude and monitored daily for tumor growth. Mice were euthanized as soon as any animal that appeared to be in distress or discomfort. Animals were housed in an AAALAC accredited vivarium at St. Jude Children's Research Hospital Animal Resources Center under the direction of Dr. Richard Rahija. Bedding and chow were autoclaved and cages changed twice weekly.
When injected intravenously into the tail veins of Es1 e /SCID mice, SK-N-AS NB cells (5 × 105 cells) produce disseminated tumors in 100% of mice. The number of cells administered was not toxic to Es1 e /SCID mice and no discomfort or death was seen at the time of injection. In this model, there is no "primary tumor" but the pattern of tumor development recapitulates the clinical dissemination of high-risk NB; tumors develop in multiple anatomic locations including lung, liver and bone marrow [33, 34]. Groups of 10 (neo and WT transfectants) or 5 mice (gsv1,4,5; gsv1,2,5; gsv4,5; gsv1-6 transfectants) were followed for 300 days after intravenous injection of tumor cells. Any animal that appeared to be in distress was euthanized and the day of euthanasia was recorded as the day of death.
Statistical analyses were performed using GraphPad Prism software, version 5.02 (GraphPad, San Diego, CA); graphs were generated using the same software. Two-tailed t tests were used to analyze results of soft agar assays and two-way ANOVA followed by Bonferroni post tests was used to analyze scratch assays. Soft agar assays and scratch (wound healing) assays were repeated a minimum of three times, with each experiment including six replicates of each cell line. Kaplan Meier survival data were used to calculate the 95% confidence interval for time-dependent fractional survival, using a log-rank (Mantel-Haenszel) test to compare survival of mice receiving vector control cells (neo), ICAM-2 WT, or gsv transfected cells.
The schematic structure of ICAM-2 (Figure 1) reflects the complete 275 amino acid glycoprotein. The indicated domains of this extensively glycosylated protein were discussed in the Background section. The heavy glycosylation status of the ICAM-2 WT protein dictates its migration in SDS-PAGE gels as a diffuse band rather than as a discrete band of specific molecular mass. In this study, as the degree of glycosylation decreases when glycosylation sites are mutated, variants are predicted to migrate at different rates even though the number of amino acids remains unchanged (bottom panel of Figure 1). The non-glycosylated variant gsv1-6 has a predicted molecular mass of 30 kDa.
SK-N-AS cells express no detectable endogenous ICAM-2 protein . We transfected these cells to stably express ICAM-2 WT or hypo-glycosylated variants of ICAM-2. RT-PCR analysis of positive control human dermal vascular endothelial cells (HDMVEC) and transfectants expressing ICAM-2 WT or ICAM-2 glycosylation variants (gsv1,4,5, gsv1,2,5, gsv4,5 gsv1-6) demonstrated similar levels of ICAM-2 transcript (Figure 2A, lanes 1,3-7). As expected, neo transfectants (SK-N-AS cells transfected with pIRESneo2 control plasmid) (lane 2) expressed no detectable ICAM-2 RNA. Immunoblot analyses (Figure 2C) done with antibody AF244 (R&D Systems) confirmed protein expression in all cell lines except SK-N-ASpIRESneo. Fully glycosylated endogenous ICAM-2 expressed by HBMEC-28 cells (lane 1) and by transfected ICAM-2 WT cells (lane 2) migrated in SDS-PAGE gels with the expected molecular mass of 55-60 kDa . Immunoblots also demonstrated the expected decrease in apparent molecular mass of hypo-glycosylated ICAM-2 variants compared to full length ICAM-2. As the extent of glycosylation decreased, immunoreactive bands appeared less diffuse and of lower mass. The non-glycosylated variant expressed by gsv1-6 cells migrated at ~30 kDa, as expected for a protein of 254 amino acids (275 amino acid nascent protein – the 21 amino acid signal peptide). Notably, differences in level of glycosylation preclude direct comparison of levels of protein expression among the transfectants. Furthermore, the unexpected area/intensity of the immunoreactive band in the lane containing lysate from gsv4,5 cells suggest the possibility of N-glycan heterogeneity among the differentially glycosylated ICAM-2 variants. Immunoblots for actin and α-actinin served as loading controls.
Transfected SK-N-AS cells express ICAM-2 transcripts and proteins. A) RNA from control human dermal microvascular endothelial cells (HDMVEC) generated RT-PCR products of the predicted 631 base pairs. All ICAM-2 transfectants contained readily detectable ICAM-2 RNA. B) Bar graph depicting quantitation of the RT-PCR products shown in "A". RNA expression levels were within 20% among the transfectants. This level of variability was not statistically significant. C) Immunoblots of whole cell lysates (40 μg protein/lane) demonstrated that control HBMEC-28 cells and WT transfectants expressed immunoreactive protein having an apparent molecular mass of 55-60 kDa. ICAM-2 variants expressed proteins of appropriately lesser apparent masses. Transfectants expressed equivalent levels of actin and α-actinin. D) Deglycosylation of proteins in whole cell lysates and subsequent immunoblots for ICAM-2 confirmed that all variants displayed the expected apparent molecular mass of ~30kDa. The "nonspecific" band that appears at ~36kDa in all lanes marked + PNGase F is the PNGase F protein itself. E) Quantitation of ICAM-2 variants (lanes indicated as + PNGase F) was done to compare relative amounts of ICAM-2 protein expressed by transfectants. Results were normalized to the level of actin expression for each cell line. F) ICAM-2 WT and variants localized to cell membranes. Experimental details are included in Methods. G) Membrane localization of ICAM-2 WT and gsv4,5 was confirmed by fluorescence activated cell sorting (FACS) of intact cells, incubated with an antibody recognizing the extracellular domain (ED) of ICAM-2 (CBR-IC2/2) and a PE-conjugated secondary antibody. Non-intact cells were gated out using light scatter parameters and propidium iodide uptake. FACS profiles shown are for PE-positive cells generated using negative control IgG (blue line) or anti-ICAM-2 (red line). H) Biotinylation experiments also demonstrated that ICAM-2 WT and variants localized to cell membranes. Experimental details are included in Methods.
Next, we examined ICAM-2 N-glycan sensitivity to endoglycosidase H (Endo H) and N-glycosidase F (PNGase F) since modification of glycosylation status can be detected using these enzymes. Endo H hydrolyzes high mannose and hybrid N-glycans synthesized in the endoplasmic reticulum (ER), while PNGase F hydrolyzes all N-linked glycans including complex glycans synthesized in the Golgi. ICAM-2 expressed by all transfectants and HBMEC-28 cells were sensitive to PNGase F and displayed increased electrophoretic mobility following a 1 hour incubation with PNGase F (Figure 2D), with non-glycosylated forms of ICAM-2 migrating at ~30 kDa. (The "nonspecific" band that appears at ~36kDa in all lanes marked + PNGase F is the PNGase F protein itself.) In contrast to results with PNGase F, results following incubation of cell lysates with Endo H differed for forms of ICAM-2 that were glycosylated at position 47 compared with those that were not glycosylated at this position. ICAM-2 WT and gsv4,5 and ICAM-2 from control HBMEC-28 cells were Endo H resistant, while gsv1,4,5 and gsv1,2,5 were Endo H sensitive. Potentially, N-glycans of ICAM-2 modify protein conformation thereby impacting protein stability, in addition to their documented roles in ligand recognition. Comparison of results from gsv4,5 with gsv1,2,5 and gsv1,4,5 suggests that N-glycans at amino acid 47 may be the most critical with respect to protein conformation and stability. Interestingly, quantitation of this experiment (Figure 2E) by densitometric scanning demonstrated that the level of gsv1,2,5 expressed was ~60% that of WT protein and the level of gsv1,4,5 was ~83% that of WT protein. Possibly this observation reflects a decreased protein stability of these variants, each of which contained an amino acid substitution at position 47 that prevented glycosylation at this site. Further, the robust level of expression of variant gsv4,5, the only variant that did not contain an amino acid substitution at position 47, is consistent with the hypothesis that N-glycosylation at this position may be critical to ICAM-2 protein structure and stability. Our data comparing Endo H and PNGase F sensitivities of ICAM-2 WT and variants suggest that N-glycosylation site at amino acid 47 may be the most critical N-glycan structure with respect to protein stability.
Next, we showed that similar to the WT protein, hypo-glycosylated ICAM-2 variants were associated with the plasma membrane (Figure 2F, 2G, 2H). We first isolated membrane-enriched fractions of each transfectant and performed immunoblots for ICAM-2 and also for the plasma membrane marker ATP1A1 (Na+/K+ transporting ATPase subunit alpha 1; Novus Biologicals, Littleton, CO). Membrane-enriched fractions contained readily detectable levels of ATP1A1 and also ICAM-2 proteins of the expected molecular mass using an antibody that recognizes the CD domain of ICAM-2 (sc-1512; Santa Cruz Biotech) (Figure 2F). To corroborate these data, we performed immunofluorescence staining using intact transfected cell lines and an antibody that recognized the ED of ICAM-2 (CBR-IC2/2-PE). Flow cytometric analysis of WT, neo and each of the four gsv transfectants demonstrated measurable levels of antibody reactivity (cell surface expression in intact cells) for ICAM-2 protein only for cells expressing WT and gsv4,5 (Figure 2G), even though gsv1,4,5, gsv1,2,5, and gsv1-6 were clearly seen by immunoblot. The CBR-IC2/2 antibody was generated using full length ICAM-2 (Santa Cruz Biotech). This antibody recognizes the LFA-1 binding domain of ICAM-2 which includes amino acid residues #42-63 within extracellular IgLD-1 domain [17, 35–38], and inhibits binding of LFA-1 to ICAM-2. Our FACS results show that the CBR-IC2/2 did not react with any of the three variants containing an amino acid substitution at position 47 (gsv1,4,5, gsv1,2,5 and gsv1-6), suggesting that substitution at this position destroyed antibody recognition. However, since gsv4,5 contained the endogneous Asn at position 47, we used CBR-IC2/2 to confirm membrane localization of the expressed variant. The data are consistent with the previous report [17, 35–38] that antibody CBR-IC2/2 recognizes amino acid residues within IgLD-1 and that an Asn residue at position 47 is essential for that recognition.
Next, cell surface protein biotinylation was performed to confirm cell membrane surface localization of all ICAM-2 variants. As shown in Figure 2H, biotinylation of proteins of intact cells showed readily detectable ICAM-2 proteins with the expected electrophoretic mobility for all cell lines. Data in Figures 2F, 2G, and 2H indicate that ICAM WT and variants localize to cell membranes.
Taken together, the data indicate that variable glycosylation does not affect the subcellular localization of ICAM-2 and that variants localize to the plasma membrane similar to the endogenous protein. As expected, control-transfected SK-N-AS cells showed no detectable ICAM-2 expression.
Having confirmed expression of transfected cDNAs and membrane localization of encoded proteins, we further characterized transfectants with respect to cell morphology and cell doubling time. Photomicrographs (Figure 3) demonstrate that all six transfected SK-N-AS cell lines and the untransfected SK-N-AS parent cell line grow as attached monolayers. In general, all seven also showed similar nascent neuronal morphology, were similar in size when detached, and had doubling times of 35.0 ± 4.4 hours. The single exception to this was the gsv1,2,5 cell line, which had a doubling time of 59 hours. Interestingly, in spite of being similar in size when detached, the images in Figure 3 suggest that gsv1,4,5 and gsv1,2,5 transfectants were more adhesive than the ICAM-2 variant transfectants, and appeared smaller in size when attached and grew as aggregates more similar to WT transfectants rather than the more diffuse monolayers of untransfected and control neo transfectants. In contrast, gsv1-6 transfectants appeared morphologically more similar to neo controls than to WT transfectants.
Morphology of ICAM-2 variants derived from SK-N-AS neuroblastoma cells. Cells plated at low density (~20% confluence) in CoStar tissue culture flasks were cultured at 37°C at 5% CO2 in humidified chambers, and allowed to proliferate to ~70% confluence. SK-N-AS cells transfected to express WT or gsv ICAM-2 have nascent neuronal morphologies. In comparison to SK-N-ASpIRES.ICAM-2 transfectants (WT), gsv transfectants were morphologically more pleomorphic and discohesive. All transfectants grew as attached monolayers. When detached and sized using a Coulter Counter, no differences in cell size were observed.
Of particular interest, SK-N-ASpIRESneo cells and gsv transfectants appeared more discohesive and pleomorphic than cells expressing ICAM-2 WT. Also, SK-N-ASpIRESneo cells and gsv transfectants had relatively non-smooth borders compared to cells expressing ICAM-2 WT. Cells expressing gsv4,5 demonstrated a "drunken honey comb" appearance, a description frequently appropriate to malignant cells with decreased cohesiveness and non-smooth borders. Cells expressing gsv1-6, grew in smaller clusters than other transfectants. The most consistent findings were the relatively discohesive and pleomorphic characteristics of SK-N-AS cells expressing gsv transfectants, compared to ICAM-2 WT, possibly indicative of a more motile phenotype than that of cells expressing the WT protein.
To address this possibility and to more completely characterize the functional phenotype of the gsv transfectants, we characterized each transfectant using assays that reflect specific aspects or cellular functions involved in metastasis. These assays included cell motility (wound healing or scratch assays), anchorage-independent growth in soft agar, subcellular localization of actin fibers (F-actin), and development of disseminated tumors in an in vivo model of metastatic NB.
Cell motility is a primary determinant of metastatic potential [39–41]. The actin cytoskeleton, in turn, is a primary determinant of cell motility. Therefore, we compared the relative motility of the parental cell line and all six transfectants in scratch (wound healing) assays (Figures 4A, 4B). We also assessed the subcellular distribution of F-actin by staining actin fibers with FITC-conjugated phalloidin (Figure 5A) and the actin fiber distribution of cells at the leading edge of migration in wound healing assays (Figure 5B) to evaluate potential differences between neo and WT transfectants.
ICAM-2 WT and glycosylation variants inhibit NB cell migration in scratch assays. Expression of WT and gsv ICAM-2 inhibited SK-N-AS cell motility in vitro. A) Scratch assays to evaluate cell motility were performed using standard methods, and images acquired at 24 hour intervals. At each time point, the distance remaining across the scratch was calculated in pixels. Only wells in which initial scratches were of similar width were included in analyses. B) Analysis by two-way ANOVA followed by Bonferroni post test showed that at 72 hours post scratch, all variants except gsv4,5 migrated more slowly than transfectants that expressed no detectable ICAM-2 (neo) (P < 0.05* [gsv1-6] and P < 0.001*** [gsv1,4,5 and gsv1,2,5]). At 72 hours post scratch, all gsv transfectants migrated more rapidly than transfectants expressing ICAM-2 WT (P<0.01 – P<0.001, P values not indicated on graph).
The cellular distribution of F-actin in control transfectants (neo) differs from that of transfectants expressing ICAM-2 WT. A) Control neo transfectants harbored transverse actin fibers. ICAM-2 WT induced juxtamembrane actin fiber distribution. ICAM-2 gsv expression did not induce a juxtamembrane localization of actin fibers. Each transfectant was plated at ~60% confluence and maintained under standard conditions at 37oC to allow the cells to adhere to chamber slides. Approximately 18 hours after plating, cells were fixed with 3.7% paraformaldehyde and incubated with FITC-conjugated phalloidin. Actin fibers were then visualized using confocal fluorescence microscopy by standard methods. Inset photomicrographs were acquired at 20x magnification and using a Zeiss Axio Observer Z.1 microscope platform in conjunction with Zen 2011 Blue imaging software (Carl Zeiss) (Photomicrographs of control cell lines (neo and WT) were published previously ). B) The distribution of actin fibers in cells at the leading edge of migration in scratch (wound healing) assays was similar to actin fiber distribution in stationary sub-confluent cultures of each cell line. Scratch assays were performed as described for experiments depicted in Figure 4. At 72 hours "post scratch", cells were fixed and actin fibers visualized using FITC-phalloidin staining as described above, and fluorescence photomicrographs acquired using Nikon TE2000-U microscope with X-cite 120 mercury vapor short arc in conjunction with Q-capture ProV 5.1.1.14 software (Nikon Instruments, Inc.). Care was taken to acquire images of the control neo transfectants in areas where residual gaps from the original scratch were still visible, to confirm acquisition of the leading edge of migration.
First, in standard scratch assays the width of the scratched area that remained free of cells at 24 hour intervals after introducing the scratch was quantitated in pixels. Results were expressed as the degree to which the scratch had closed, at 24 hour intervals post scratch (% scratch closure). SK-N-ASpIRESneo cells which express no detectable ICAM-2 completely closed the scratch within 72 hours (Figure 4A). In contrast, SK-N-ASpIRES.ICAM-2 cells migrated more slowly (P < 0.001, compared to SK-N-ASpIRESneo cells), with only 56% of the initial scratch filled at 72 hours (Figure 4B). In general, cells expressing the gsv ICAM-2 constructs migrated more slowly than control SK-N-ASpIRESneo cells and more rapidly than SK-N-ASpIRES.ICAM-2 (WT) cells. P values ranged from < 0.05-0.001 when cell lines expressing gsv constructs were compared with either control cell line. The single exception was the lack of a significant difference in the motility of cells expressing gsv4,5 and cells expressing no detectable ICAM-2 (neo, in Figure 4) at any time point. Simple differences in cell doubling times do not account for the apparent lack of function (failure to inhibit migration) of gsv4,5, as the doubling time for these cells did not differ from other transfectants.
gsv4,5 was the only construct evaluated that did not contain an Asn → Ala substitution at amino acid 47, suggesting that glycosylation at this site minimizes or circumvents the ability of ICAM-2 to inhibit NB cell motility. Possibly, glycosylation at position 47 inhibits adhesion, but this effect may be modulated by glycosylation at other sites in the WT protein. However, since ICAM-2 WT is glycosylated at this site and the WT protein significantly inhibits NB cell motility, the data are not consistent with a straightforward correlation between glycosylation of this specific residue and cell motility, as reflected by this assay. The data demonstrate that NB cell motility was most affected by fully glycosylated ICAM-2, but that three of the hypo-glycosylated ICAM-2 constructs retained an attenuated function compared to the endogenous fully-glycosylated protein.
Second, we performed phalloidin staining assays to determine the subcellular distribution of actin fibers. The ICAM-2-mediated decrease in NB cell motility suggested a possible involvement of the actin cytoskeletal network, a primary regulator of cell motility; and our previous work demonstrated a correlation between transverse distribution of actin fibers and a relatively motile metastatic phenotype in a panel of NB cell transfectants. As a preliminary assessment of actin cytoskeletal function, we examined the subcellular localization of actin fibers in cells expressing ICAM-2 variants. Fluorescence photomicrographs of F-actin in control cell lines (neo and WT cells in Figure 5)  stained with FITC-conjugated phalloidin demonstrated the expected contrast in distribution of actin fibers; relatively motile SK-N-ASpIRESneo cells harbored transverse actin fibers, while relatively non-motile SK-N-ASpIRES.ICAM-2 cells had juxtamembrane actin fibers. If the scratch assay and F-actin distribution accurately reflect cell motility, then scratch assay results would predict that cells expressing gsv4,5 would have transverse actin fibers and the other three constructs would have juxtamembrane fibers. FITC-conjugated phalloidin staining clearly demonstrated that this prediction did not hold true, and that cells expressing all four ICAM-2 variants had transverse actin fiber distribution (Figure 5 and data not shown). This assay does not provide a simple preliminary screen to assess the impact of ICAM-2 WT or gsv on NB cell motility in vitro.
We then also addressed whether the observed differences in actin fiber distribution could be attributed to differences in cell density. We plated neo transfectants at twice the original density and ICAM-2 cells at half the original density shown in the insets of the two upper panels in Figure 5A. Altering cell density did not affect actin fiber distribution. Actin fiber distribution appeared to be unrelated to cell density. As an alternative approach to examine the effect of cell density on F-actin distribution in the neo and ICAM-2 WT transfectancts we again performed a scratch assay as shown in Figure 4, but just prior to complete closure of the scratch by neo transfectants (~72 hours post scratch), we fixed both neo and ICAM-2 WT transfectants and incubated the cells with FITC-conjugated phalloidin. The question to be addressed was whether the F-actin distribution of cells at the leading edge of migration would be similar to its distribution in non-migrating cells. Fluorescence photomicrographs in Figure 5B show that both non-migrating and migrating SK-N-ASpIRESneo cells display transverse actin fibers and both non-migrating and migrating SK-N-ASpIRES.ICAM-2 cells display juxtamembrane actin fibers.
We next assessed anchorage-independent colony growth in vitro, an assay that often reflects tumorigenic potential in vivo in preclinical models. Of note, while somewhat controversial, multiple published studies have also demonstrated correlations between growth in soft agar and metastatic, rather than tumorigenic potential [42–44]. Our previously published data demonstrate that ICAM-2 transfectants with decreased metastatic potential in vivo also produce fewer colonies in soft agar in vitro. A plausible explanation consistent with observed correlations between anchorage-independent growth in vitro and metastatic potential in vivo is that relatively motile cells show decreased sensitivity to loss of anchorage dependency and therefore a greater capacity to initiate colony growth. The aggregation of sparsely plated cells precedes colony growth, and cells that more readily aggregate (relatively motile cells) produce more viable colonies in soft agar. Therefore, it was of interest to determine whether hypo-glycosylated ICAM-2 variants suppressed growth in soft agar to the same extent as ICAM-2 WT. As expected, cells expressing fully-glycosylated ICAM-2 WT formed fewer colonies in soft agar (9.6 ± 1.4) than control transfectants (227 ± 10.1 colonies) (P< 0.0001***, Figure 6). The number of colonies produced by all variants was intermediate to the number of colonies produced by cells expressing the WT and cells expressing no detectable ICAM-2. Hypo- and non-glycosylated variants produced significantly fewer colonies than control transfectants (P < 0.0001), but significantly more colonies than SK-N-ASpIRES.ICAM-2 cells (P < 0.05 - < 0.0001). The single exception to this observation was that cells expressing gsv4,5 produced the similar number of colonies (16.7 ± 2.6) as SK-N-ASpIRES.ICAM-2 cells. Hypo-glycosylated ICAM-2 suppressed anchorage-independent growth, but the degree of suppression was less than that induced by ICAM-2 WT. We concluded that all forms of ICAM-2 were functional in this assay, and that the cells expressing each of the four variants needed to be characterized using an in vivo model of metastatic NB.
WT and gsv ICAM-2 suppressed anchorage-independent growth in vitro . All four glycosylation site ICAM-2 variants suppressed anchorage-independent growth. Soft agar assays were performed using standard methods. Number of colonies of >20 cells were visualized 14-21 days after plating and results analyzed using a 2-tailed t test and GraphPad Prism software. P = 0.0124*. P < 0.0001***. Not shown in this Figure are control experiments demonstrating that the growth in soft agar of parental SK-N-AS cells was equivalent to control SK-N-ASpIRESneo transfectants.
Prior to undertaking these in vivo experiments, we addressed a more mechanistic aspect of ICAM-2 function in NB cells. Our previous work demonstrated that the α-actinin binding site in the cytoplasmic domain (CD) of ICAM-2 is essential to ICAM-2-mediated suppression of disseminated tumor development in an in vivo model of metastatic NB . When the α-actinin binding site of ICAM-2 is intact, ICAM-2 associates with α-actinin and actin simultaneously and suppresses the metastatic properties of NB cells. Having observed that the four gsv transfectants retained the function of the WT protein in either scratch or anchorage-independent growth assays or both, we postulated that the glycosylation status of ICAM-2 would not affect its association with α-actinin and actin in NB cells. To address this hypothesis, we performed immunoprecipitations (IP) with an antibody to α-actinin, and then immunoblots (IB) to determine whether gsv ICAM-2 variants associated with α-actinin and actin. IP/IB results for positive (WT) and negative (neo) control cell lines (Figure 7A) demonstrate the anticipated interaction of ICAM-2, α-actinin and actin compared to minimal interaction of α-actinin and actin when ICAM-2 was not expressed. IP/IB results (Figure 7B) showed that all ICAM-2 variants associated with α-actinin and actin.
ICAM-2 WT and variants co-precipitated with α-actinin. A) IP/IB experiments with control cell lines demonstrate the expected association of ICAM-2, α-actinin and actin in lysates from SK-N-ASpIRES.ICAM-2 cells (labeled as WT), but not SK-N-ASpIRESneo cells (neo). Results for these control cell lines were published previously . B) ICAM-2 glycosylation site variants associated simultaneously with α-actinin and actin. Immunoprecipitations (IP) were performed using whole cell lysates and a mouse monoclonal antibody to α-actinin (MAB1682, Millipore). Following protein separation by electrophoresis, immunoblots (IB) were performed with antibodies to α-actinin (sc-7454, Santa Cruz Biotech), ICAM-2 (AF244, R&D Systems), or actin (4968, Cell Signaling). The presence of ICAM-2 WT and variants in each preparation was confirmed by immunoblot analysis of input preparations (a representative blot is shown in Figure 2C) and also by immunoblot analysis of the proteins remaining in the supernatant following precipitation (not shown), to confirm the presence of sufficient/excess ICAM-2 protein or variant in each preparation used for immunoprecipitation experiments.
In interpreting the IP/IB data, we note that the data are qualitative and do not necessarily reflect the ratio in which these proteins associate for several reasons. Firstly, immunoblots for glycosylated ICAM-2 give an area x density signal approximately 300% - 400% greater than that for the same amount of de-glycosylated ICAM-2 (Figure 2C). Secondly, α-actinin induces actin self-association , resulting in variable numbers of actin molecules associated with a single α-actinin molecule. Thirdly, the necessary use of mono- and polyclonal antibodies likely allows binding of secondary antibodies to a variable number of recognition sites on primary antibodies. However, the data do clearly indicate that all four variants associate with α-actinin, an interaction essential to ICAM-2-mediated suppression of metastatic potential. Based on this mechanistic consideration and on cell function assays (Figures 4 and 6), we would predict that all hypo-glycosylated ICAM-2 variants retain at least partial function, in suppressing metastatic properties of NB cells. Next, we tested this hypothesis in a murine model of metastatic NB. Of note, based on co-IP data in Figure 7, ICAM-2 WT and gsv4,5 appear to interact more readily with α-actinin than do the other three hypo-glycosylated ICAM-2 variants. We next evaluated the function of ICAM-2 WT and variants in an in vivo model of metastatic neuroblastoma.
To determine the extent to which N-linked glycosylation impacts the metastatic phenotype of ICAM-2 expressing NB cells in vivo, we injected each of the SK-N-AS NB cell transfectants intravenously into 6-8 week old Es1 e /SCID mice, and observed the mice for tumor development. As expected, Kaplan-Meier survival plots demonstrated that mice receiving control cells (N = 10) that express no detectable ICAM-2 (Figure 8, neo [solid red line]) developed tumors at multiple anatomic sites and required euthanasia within 2-3 months. Also as expected, mice receiving cells that express ICAM-2 WT (N = 10) developed no tumors and survived significantly longer (300 days, P < 0.001 compared to controls). Interestingly, expression of hypo-glycosylated ICAM-2 delayed, but did not prevent, development of disseminated tumors in this in vivo model. More specifically, when mice were injected with control neo transfectants, tumors became evident by non-invasive imaging within 30-60 days (data not shown). Euthanasia of mice in this group was required as early as day 38. In contrast, mice injected with gsv4,5 transfectants did not develop detectable tumors or require euthanasia until day ~200. At day 300, the termination date of the experiment, no tumor was evident in a subset of this group of mice. The similarity in function of WT and gsv4,5 in anchorage-independent growth assays (Figure 6), interaction with α-actinin (Figure 7), and the in vivo survival assay (Figure 8) suggest the hypothesis that glycosylation of ICAM-2 at amino acid 47 contributes significantly to the function of ICAM-2 in NB cells. However, overall, mice receiving cells expressing each of the glycosylation site variants survived longer than control cells (P ≤ 0.0002), but not as long as mice receiving cells expressing ICAM-2 WT (P ≤ 0.05). Hypo-glycosylation attenuated but did not abolish the ability of ICAM-2 to suppress metastatic properties of NB cells.
ICAM-2 variants significantly delayed but did not completely inhibit development of disseminated tumors in an in vivo model of metastatic neuroblastoma. Mice received intravenous injections of SK-N-AS cells expressing no detectable ICAM-2 (neo, N = 10), ICAM-2 WT (N = 10), or one of four hypo-glycosylated forms of ICAM-2 (N = 5/group). Kaplan-Meier survival plots were analyzed by log-rank (Mantel-Cox) test using GraphPad Prism 5 software (Version 5.02). Mice receiving cells expressing hypo-glycosylated ICAM-2 survived longer than mice receiving cells expressing no detectable ICAM-2, but not as long as mice receiving cells expressing ICAM-2 WT.
While in vitro cell motility (scratch assay) most accurately predicted cell phenotype in vivo, no single in vitro assay consistently predicted results in vivo for all ICAM-2 variants. In general, the morphology, anchorage-independent growth, and motility of cells expressing hypo- or non-glycosylated variants of ICAM-2 were intermediate to characteristics of negative and positive control transfectants expressing no detectable ICAM-2 or the WT protein. In vivo results for all transfectants were also intermediate to these control cell lines, in that expression of gsv transfectants significantly delayed but did not prevent the occurrence of disseminated disease in a model of metastatic NB. In addition, our results from Endo H sensitivity studies suggest that glycosylation at position 47 may be critical to protein folding or conformation, and that glycosylation at this position likely affects protein stability. Potentially, a decrease in ICAM-2 protein stability leads to lower levels of membrane-associated ICAM-2, which, in turn, impacts the novel role of ICAM-2 in suppressing metastatic properties. We conclude that the glycosylation status of ICAM-2 significantly affects the function of this protein in SK-N-AS NB cells.
We are indebted to Dr. Mary K. Danks for her support and discussions. We thank Dr. Phil M. Potter for his support, helpful discussions, and for allowing us to use the mouse strain developed in his laboratory. We are also grateful to Joanna S. Remack, Doris A. Phelps, and Rebecca A. Bush for their outstanding technical assistance. We thank Dr. E. van der Schoot (CLB, Sanquin Blood Supply Foundation, The Netherlands) for her gracious gift of the HBMEC-28 bone marrow endothelial cells. This work was supported by the NIH Cancer Center Core Grant (CA21765); and the University of Alabama at Birmingham-Comprehensive Cancer Center- Young Supporter's Board-New Faculty Development Award (YSB-NFDA).
JMF, PLG, SBB, SB and KJY conducted the study. JMF, LNC and KJY analyzed the data. KJY designed the study and wrote the manuscript. All authors read and approved the final manuscript.


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Clarence Road in Norwich NR1 consists mainly of flats and terraced houses. Properties on Clarence Road typically have values around £150,000 - £200,000, with larger terraced houses and townhouses valued at around £200,000.
Map showing Clarence Road in Norwich.


The minimum amount Kumbuka Children's Centre requires you to raise, in order to pay for your skydive using a portion of your sponsorship is £300.
A minimum of £50 goes to Kumbuka Children's Centre.
If you are raising all sponsorship online, you are required to bring on the day a copy of your online sponsorship page showing a minimum of £250 and we will invoice Kumbuka Children's Centre for the jump.
If you are raising all sponsorship offline, you are required to bring on the day a personal cheque made payable to Kumbuka Children's Centre for a minimum of £50; £200 jump balance (by cash or card taken from sponsor money) and the sponsorship forms.
If you are raising sponsorship using a combination of online and offline we suggest you raise a minimum of £50 online and the outstanding jump balance of £200 offline (on the day you need to bring in the £200 jump balance by cash or card).
All sponsorship money you raise will go to the charity (there is no minimum required with this scheme). You send this directly to Kumbuka Children's Centre.
A minimum of £121 goes to Kumbuka Children's Centre.
If you are raising all sponsorship offline, you are required to bring on the day a personal cheque made payable to Kumbuka Children's Centre for a minimum of £121; £129 jump balance (by cash or card taken from sponsor money) and the sponsorship forms.
If you are raising sponsorship using a combination of online and offline we suggest you raise a minimum of £121 online and the outstanding jump balance of £129 offline (on the day you need to bring in the £129 jump balance by cash or card).


text:
Trump's Twitter Ban Was Unfair, but Not for the Reason You Think
Photograph: PM Images/Getty Images
If Twitter had implemented its rules uniformly, other world leaders would have been banned too.
In January 2021, after former US president Donald Trump tweeted in support of an insurrection on the Capitol, his account was frozen and he was locked out. But across the world, leaders have tweeted in support of genocide and threatened violence, yet none of them have been banned from the platform. Less than six months later, in June 2021, Nigerian president Muhammadu Buhari posted a tweet threatening violence against Biafran separatist groups in the country's southwest. Buhari's tweet was removed, but his account remained live.
Almost two years after Donald Trump was banned from Twitter, Elon Musk has released a series of documents—dubbed the Twitter Files—arguing that the site got it wrong. The leaked documents show the way the platform made decisions before Musk took over, focused on the former president and other controversial moderation decisions.
In the most recent file dump published through Bari Weiss, the founder and editor of media organization The Free Press, Musk released several documents that revealed how Twitter's policy and trust and safety teams came to the decision to ban Trump in the wake of the insurrection on January 6, 2021.
In an argument outlined over Twitter, Weiss alleges that the decision to ban Trump was unprecedented, deviating from the site's reactions to other heads of state who also incited or supported violence with their tweets. Weiss cited examples from leaders in Ethiopia, India, Nigeria, and Iran that, she asserted, showed restraint on Twitter's part when deciding whether or not to keep prominent political figures on the platform, even after violations. Twitter has not released the documents detailing the decision to keep other public figures on the site.
While Weiss interpreted the reluctance to use such measures against other world leaders as evidence that Trump was treated particularly unfairly, the documents may also reveal the opposite: that the company consistently underestimated the danger its platform posed in contexts outside the US, and only acted forcefully against threats to American democracy. If Twitter had implemented its rules uniformly across the world, Trump's ban would have extended to other leaders, too.
"Vulnerable communities in far away countries are less important than the relationships with leaders like [India's Narendra] Modi or others," says an employee at an organization that was a part of Twitter's trust and safety council, which was disbanded earlier this month. The employee asked for anonymity because they are concerned their organization may be targeted by harassment and threats like those faced by former Twitter staffers.
Some of this discrepancy may come down to how different governments react to moderation by social platforms. After Twitter removed Buhari's threatening tweet against Biafran separatists, the company was slapped with a ban. But instead of banning Buhari in turn, the company later negotiated with the government to be reinstated by agreeing, among other things, to open a local office, pay local taxes, and register as a broadcaster. Nigeria is now considering legislation to regulate platforms.
"I think there are a lot of calculations that go into the trade-off about whether to take enforcement actions, and of course access to markets is one of them," says Kian Vesteinsson, senior research analyst for tech and democracy at Freedom House, a nonprofit research and advocacy group focused on democracy and political freedoms.
This question of market access has likely also come into play in India, the company's third largest market. Despite filing a lawsuit to protect tweets and accounts that the right-wing Bharatiya Janata Party (BJP) has ordered removed, Twitter has, as Weiss noted, refused to ban Modi and other BJP members from the platform. The BJP government has already begun to increase pressure on social media companies, requiring them to have in-country representatives that can be held legally accountable if companies do not comply with government takedown requests.
When making the decision to ban Trump, Twitter staff likely didn't have to consider whether the company would face a situation where it would be shut out of its home market in the US.
But this is not always the case. For instance, Twitter has technically been banned in Iran since 2009, and yet the company let a tweet by Ayatollah Ali Khamenei calling Israel a "tumor" that needs to be "eradicated" remain on the platform. Khamenei's account was temporarily suspended in 2019, and on the same day Trump was banned, tweets from Khamenei's accounts were removed—but his account remained active. At the time, Twitter did not respond to questions about how it had applied its policies to reach this decision.
"At a very high level, tech companies have not been clear or consistent about how they treat speech by politicians and other influential political figures," says Vesteinsson. "But it's been clear for years that technology companies need to step it up when it comes to protecting people in the global south and in markets that might not be deemed as valuable to these companies."
But even in instances where market access may not be a driving force, such as Ethiopia, where the company boasts only 34,000 users, a tweet from Ethiopian Prime Minister Abiy Ahmed supporting the genocide against ethnic Tigrayans was left up, accompanied by an annotation that it had violated the company's policies, but that it had been preserved as a public record of a world leader. Ahmed's Twitter account remains active with more than a million followers.
The employee involved in Twitter's trust and safety council tells WIRED that they believe that instances like Prime Minister Ahmed's in Ethiopia are given "less regard, overall, by these Western platforms because they are not considered as important." And for platforms, maintaining good relationships with foreign governments may take precedence over applying their own rules.
The employee also says that the choice to leave up accounts in the global south that violate the company's policies may also come down to a lack of investment. "During sensitive political events, they don't put as many resources into it, they don't prioritize them," the employee says. "And even when things are happening, when we call their attention to these issues, it's dismissed."
But Vesteinsson notes that, while platforms have historically been negligent of non-Western countries, the Files themselves are inherently selective, which can be misleading. "What I see here is internal teams carefully deliberating over content policy and enforcement," he says. "We don't have that same lens into how Twitter considered actioning posts from world leaders like Ahmed or Modi. We don't have that transparency in these recent disclosures. What we really need here is for platforms like Twitter to invest in greater transparency over their operations and decisionmaking."
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Vittoria Elliott is a reporter for WIRED, covering platforms and power. She was previously a reporter at Rest of World, where she covered disinformation and labor in markets outside the US and Western Europe. She has worked with The New Humanitarian, Al Jazeera, and ProPublica. She is a graduate of... Read more
Platforms and power reporter
Topicstwittercontent moderationcensorshipDonald TrumpSocial Mediadisinformation
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Twitter Promised Them Severance. They Got Nothing
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redpajama_set_name: RedPajamaCommonCrawl


Last weekend the kids and I went to a holiday cookie decorating party at our good friends' house. I dislike showing up to a party (or any gathering) empty handed so here was my chance to try something out I had been wanting to make for a few weeks now.
I got this idea from Starbucks Peppermint Brownie Cake Pops. The kids and I loved them, but at $1.50 a pop, I knew I could make something like this for far less at home. Probably not exactly like them, but close enough.
Press out the air so the bag doesn't pop or explode when you start beating the bag.
Grab the rolling pin (or whatever heavy item you so choose) and smash those candy canes.
The candy canes don't have to be perfectly crushed. I liked having some really small, fine pieces and then some larger pieces.
You may have another method you use for crushing candy canes, but I liked this way.
Follow directions on box of brownies - mix in canola oil (or whatever oil you choose), egg, water.
Pour into greased pan & bake for 26 minutes at 350 degrees.
*This part really depends on which brownie mix you choose or if you have your own brownie recipe.
Put a handful of crushed candy cane in the mix and stir.
*I did bake them 2 minutes less than the box called (24 minutes rather than 26 minutes) - not that big of a deal and I wanted them to be soft and not cross that line to brownie "rock"
Take the brownies out of the over and let them cool, approximately 15 minutes.
Honesty - this is the hardest step ever! I don't have patience for waiting. I want to sample the brownies but they are so darn hot, ouch!
Grab small handfuls of brownie and roll into balls. You decide the size.
I wanted something a little smaller than donut holes, so that's what I went with.
Place on baking pan while you go.
Pop into freezer for 1 to 2 hours.
Melt almond bark either either in a double boiler or in microwave. You choose. I suggest following the directions on the bag.
I haven't had good luck with melting things in the microwave. Timing is not my strength. Maybe you're better at it!
I chose double boiler method, but we don't have a double boiler - but I usually get out two smaller pans and put some water in the bottom one.
Stir often and stay close by. That's my only hint here - I've learned that from past experience. Too many times I've ruined the melted chocolate process by trying to multi-task.
Get the balls out of freezer and get toothpicks ready.
Start dipping in the almond bark.
You can dip the entire ball in the almond bark for 100% coverage, but on this day, I chose to go with the 75% (ish) coverage.
Immediately after dipping the ball into the almond bark, roll it in the crushed candy cane to cover (as much as you can) the white part.
Set on wax paper to dry.
Repeat steps 8 & 9 for each ball until you have a pile like this.
The verdict? I liked them. They were not exactly Starbucks, but good enough for something I made up. Actually, Jill, Christine and Sarah told me they liked them too. Maybe they were just being nice. I guess you'll have to try these out on your own to see what you think! Next time, I may experiment with adding in some frosting into the cooked brownie before rolling into the balls. We'll see!
And I do have a winner to announce!
Congratulations Elle!!! If you haven't read her blog yet, you should head on over here to do it now!
Please email me at runningwiththegirls at gmail dot com with your email address. Email me within 72 hours to claim your prize or I'll have to pick another winner.
This looks amazing! 2 of my students are obsessed with candy canes -- I've printed this out and think we'll all have fun making this as a before-school break treat!
This looks absolutely amazing... Mmmm..
And I am thrilled to be your winner too. Will email you right away! Thanks so much.
Wow! Those look incredible. Maybe I'll make those for my new years eve party!
I'm definitely adding these to pinterest! They look AMAZING!
Looks yummy, might try them this week with my girls.
I'm happy you liked them! Perhaps they will make another reappearance shortly.


Once you have your crystal ball, bring it home, and use a soft cloth to clean it thoroughly. After this is done, you will need to charge your crystal ball � these generally draw their energy from moonshine, so place it out at night in a pool of moonlight, and allow it to charge for several nights.
To amplify your crystal ball gazing, you can use a gemstone sphere as a compliment to the crystal ball. Simply having a gemstone sphere resting next the crystal ball can augment your diving two fold.


Ilayathalapathy Vijay's latest film Thalaivaa is gearing for its audio launch on June 22nd which falls on Birthday of the star actor. GV Prakash scored tunes for Thalaivaa and the album has 6 tracks.
Amala Paul is the lead actress in the film.
AL Vijay is shaping up the film as mass entertainer. Chandraparakash Jain is producing the film under Sri Mishri Productions Banner.


2. Template Changes (http://www.phplinkdirectory.com/forum/showthread.php?t=20790) - if you are a template maker or have a template that needs upgrading, here is a guide to the changes needed in the smarty code for your template files.
3. How to Upgrade (http://www.phplinkdirectory.com/forum/showthread.php?t=20756) - if you are running an earlier version and wish to upgrade, we have provided some detailed instructions for how to do it, though we also recommend getting professional help when possible. If you don't have access to the supporters forum, these instructions are also in a readme file called upgrade.txt in the release files.
4. Download it! (http://www.phplinkdirectory.com/forum/) - You just need to login to the phpLD user area and download the files to get started. Login on the left side of the homepage of this site, and then click on My Downloads.


Many Gun Owners emailed you with a serious concern. You responded in a flippant and asinine manner that shows clear disdain for your main customer, the American Gun Owner.
Third, The manufacturer is not the one you need to convince.
Thanks for posting this email. I just received a concerned telephone call about it, an email from the staffer who stimulated the mail.
I think a bit of clarification is in order.
The root of the matter is a HUGE series of inquiries by both email and telephone asking ArmaLite to cut off sales to New York and other states which deny its citizens the right to own our rifles, as several other firms have done. Some of the contacts have been duplicates of others and some were so redundant that they appeared to be spam. Many of them have been rude and I'm afraid one of my staffers thought that he was responding to a spammer and was more terse than he should have been. Once he realized what happened he came to me and apologized.
My purpose here is to pass on his apology from the corporate level and to answer the initial question: What is ArmaLite going to do?
1. We will not sell to those states which deny it's honorable citizens the right to own ArmaLite's.
2. We do not halt sales to individual officers even in problematic states. I am a former Police Officer myself, and the staffer who stimulated the recent anger is a currently serving one. We are well familiar with the fact that most rifles serving Police Officers are purchased by the officers themselves, and that they shouldn't be punished for the actions of their political elite.
We consider sales to those sate subdivisions which are not engaged or potentially engaged with disarming its citizens. DNR and Forestry Departments, for instance, sometimes serve in remote areas that conceal drug farms and their officers deserve good hardware.
3. We will not sell to those lower political subdivisions that deny their honorable citizens the right to own ArmaLite's. Chicago, for instance, prohibits its citizens from owning ArmaLite's within the city limits so we make no effort to sell into that city. We have many friends on the Chicago Police Department and have continued to sell to them individually.
Our observation is that most County Sheriffs disagree with banning sales of our rifles and many publicly refuse to enforce such laws. We sell to those departments and to their Deputies, but will not sell to those County departments headed by Sheriffs who would deny their citizens the same rights.
In short, Americans need not worry that ArmaLite is selling to those who betray them.
As you can see by reading posts on the topic, some readers have been harsh with their criticism of ArmaLite. It was in response to this atmosphere that my staffer reacted harshly. He's come to me and apologized and I personally am passing my own apology along with his.
But don't be mistaken, ArmaLite is strongly involved with both personal, corporate, and political efforts at the State, National, and International level to protect our civil rights. And we'll continue to support your shooting needs as the situation moves forward.
Okay, after reading Mark's response a couple times, I'm glad that they didn't "Recoil" the situation. I'll take his explanation at face value.
I have a little video up on YouTube that always gets the most enlightened of comments.
Sorry, I didn't edit the comments… Just Cut and Paste so you can see what's going on without having to actually go dip into those dank waters.
There are so many misconceptions about "The Fifty" that it's almost as if it's some mythological beast… One day, it's going to be like talking about Dragons or something.
The .50 doesn't put out head ripping shockwaves. It's only 2700 to 2800 FPS in most loadings. Which puts it at the same speed as say a .308 or a good old .30-06. In fact, the cartridge is really nothing more than a big .30-06. What gives the .50 the range and power, is that the projective is just so big and heavy. Bullets weight in from 650 to over 750 grains… which is impressive. The .50 BMG's slug alone is larger than some rifle cartridges. Firing a .50 BMG rifle is a lot of fun. It's impressive. The blast is huge. The power that it puts out is staggering. But the bullet zipping past something in a near miss isn't going to do anything to it other than making it soil its self.


You have always known that vinegar tasted great on your French fries and made your glassware sparkle, but I bet you didn't know the intense healing powers of this magic liquid.
Vinegar has been a trusted home remedy that your mother, grandmother, and their grandmothers have known. It literally can be used from head to toe. Scalp problems such as dandruff, athlete's foot, yeast infections, even headaches are no match for this remedy. It can also be used as a cosmetic to help protect and beautify your skin!
In The Healing Powers of Vinegar, author Cal Orey reveals the secret healing powers of red wine vinegar, apple cider vinegar, and other natural vinegars.


Wonderful Open Concept two story home with over 2600 square feet of living space! Rare 4 bedroom, 2.5 bath home. You will love to host family holidays in the huge great room and living room open to a spacious kitchen w/tons of cabinets & stainless steel appliances! Spacious master retreat with ceiling fan, en suite with garden tub and separate shower and his/her closets. All bedrooms have walk-in closets! You will love the storage space in this house! Imagine having your morning coffee on a beautiful lakeview lot overlooking the playground and pool. A+ Brownsburg Schools! Don't miss out on this true gem!
Directions:136th (Crawfordsville Rd) to Raceway, North on Raceway 1/4 mile to subdivision on Left. Go to Riverwood, look for sign.


Only 4 copies of the Berlinetta were produced. In 1953 Pininfarina was about to sign an exclusive contract with Ferrari. To avoid offending Enzo Ferrari, a clever scheme was found to prevent Pininfarina from having official contacts with Maserati. Under this stratgem, the Maserati A6 GCS/53 chassis were sold to Guglielmo 'Mimmo' Dei, who then commisioned Pininfarina to create their coachwork. The weight of the Berlinetta was only 740 kg and had a top speed of 235 km/h. The estimated value of the car is between € 800.000 and € 1.1000.000.


Maybe it's a holiday for you and maybe you have to go to work today. At any rate, peace, God bless, and Happy Monday!
We're gonna leave you this week with a video. I've often said that God has a sense of humor. I hope that's true, otherwise… I'm in big trouble.
Smile 😀 And have a Happy Monday!
We should have acknowledged Martin Luther King Day in some way, beyond mentioning that some people may have the day off. Here is today's Google Doodle. Click the image to learn more about the history and celebration of this day.
wowww; I did enjoy the video.
Smile and have a Happy Monday; 🙂 Have a happy week ahead!!!
Somehow I didn't get email notifications or anything about these comments. Thanks everybody for the feedback.
Paul: One of the things I like to do when I was teaching American History was play the entire "I Have a Dream" speech on MLK Day. It's only about 15 minutes or so but the only thing most people know is a 15 second sound bite.


text:
Ramboldia laeta är en lavart som först beskrevs av Stirt., och fick sitt nu gällande namn av Kalb, Lumbsch & Elix. Ramboldia laeta ingår i släktet Ramboldia och familjen Lecanoraceae.   Inga underarter finns listade i Catalogue of Life.

Källor

Sporsäcksvampar
laeta

redpajama_set_name: RedPajamaWikipedia


Several years ago, a highly regarded state university hit a trifecta of administrative disasters. The chair of the board stepped down in a conflict-of-interest scandal. The president resigned unexpectedly, in the wake of a snub by the board. And just as the search for a new president was entering its final stage, the board dismissed the entire search committee and scuttled the top candidates.
Everyone in the state could read about these upheavals regularly in newspapers and blogs. But the university's alumni magazine barely acknowledged their existence. After the search committee was disbanded—when irate faculty and maverick trustees were talking to newspaper columnists and writing op-eds, and people who cared about the institution must have been worried about its future—the alumni magazine gave the subject half a column toward the end of its campus news section.
The short shrift wasn't the fault of the editor or staff, who were excellent. The problem was that they worked in a university department whose director reported to the acting president, who in turn reported to the board. The editors weren't allowed to write more than half a column on the controversies surrounding the board: they were writing about the boss.
And that, in a nutshell, is why the words "editorially independent," which appear on this page beneath our logo, matter so much. An editorially independent magazine can provide a full explanation, with all the nuances and possibly unflattering details, about even the most troubling subject. A magazine that is not editorially independent can't. Sane people don't publish in-depth analyses of their supervisors' difficult moments.
The Yale Alumni Magazine is a rarity among alumni magazines, reader-driven because we are reader-supported. We're published not by Yale, but by a separately incorporated, alumni-based nonproft. More than half of our budget comes from reader subscriptions and donations, and most of the rest from advertising. Our statement of purpose charges us to deliver Yale to your mailbox "in all its complexity." Yale hasn't seen a three-part scandal like the state university's, but it has its share of troubling subjects; we analyze them for you with all those complex and even unflattering details. When Yale announced its plans for a liberal arts college in Singapore, we interviewed alumni and faculty who object to partnering with a nation whose citizens lack free speech (November/December 2010). On our cover this issue is another fraught topic, the current Title IX complaint against the university.
Delivering this information to you matters—because higher education matters; because Yale is one of the most influential institutions of higher education today; and because Yale's alumni have a permanent stake in and voice at Yale. Administrators come and go, and even tenured faculty may be wooed away to other jobs. But the alumni have a lifetime appointment.
And our independence also matters because critical thinking—informed intellectual exploration—is Yale's core value. To the university's lasting credit, it accepts the presence of an editorially independent alumni magazine in its midst. Yale is confident enough, and has enough respect for the intelligence of its alumni, to welcome lux et veritas when they are applied to its own operations.
As for the staff of the Yale Alumni Magazine: we could not be more honored than to have lux et veritas as our mission, and to have the Yale alumni as our readers. Thank you.


text:
Search Results for Tag: health
Saving Germany's midwives
Going into labor can be an anxious time, especially for first time mothers. In Germany, it's often the midwife who provides support and helps young women get through the ordeal.
Bianca Kasting, 32, knows this well enough. Last year, when she gave birth to her first child, a midwife was there to hold her hand.
In Germany there are around 21,000 midwives. And although the work they do is vital, they are often underpaid, and have to pay high insurance premiums. The situation has become so bad, that many midwives have joined protests around the country, or left the profession entirely.
Bianca wanted to do something to help, so she launched an online petition, and even cycled around Germany – her baby in tow – to raise awareness about the plight of midwives.
Listen to the report from Michael Hartlep, Germany:
http://blogs.dw.com/generationchange/files/Saving-Germanys-midwives.mp3
Bianca Kasting has launched an online campaign to rescue midwives. (Photo: Jennifer Fraczek)
Tuesday 20.05.2014 | 13:49
activism, Germany, health, midwives, women
Comments deactivated
Fighting cancer with dreadlocks
Kelly Dortmands has spent a decade growing her meter-long dreadlocks, but now she's planning to shave them all off. She's not doing it to change her look or to make a fashion statement. The 32-year-old is shedding her 53 dreads for a good cause – to raise money for cancer.
Cancer is currently the number one cause of death in the Netherlands. Kelly has seen it affect the people close to her, including her mother and her friend.
Kelly wanted to do something to help. She's hoping to raise 5300 euros – 100 euros per dreadlock – for the Dutch Cancer Fund.
Listen to the radio report by Laura Postma in Den Bosch, Netherlands:
http://blogs.dw.com/generationchange/files/Fighting-cancer-with-dreadlocks.mp3
Kelly has been growing her dreadlocks for ten years (Copyright: L.Postma)
Kelly decided to raise money for cancer research after her mom, pictured left, was diagnosed with the disease (Copyright: L. Postma)
Kelly is hoping to raise 5300 euros for cancer research(Copyright: L. Postma)
cancer, disease, dreadlocks, health, research
Opening hearts for epileptics in Ireland
In Dublin, Rachel Ashe was shocked when she was diagnosed with epilepsy. First by the fact that she would have to take medication for the rest of her life, but also by all the prejudice and ignorance about the disease that she encountered. So, she decided to set up her own support network – Epilepsy Ireland – and now she spends much of her time helping others with epilepsy and raising awareness about the illness that affects some 30 to 40 thousand people in Ireland. One of them is reporter Eoin Kernen, who brings us this week's Generation Change report from Dublin.
Listen to Eoin Kernen's report from Dublin:
Rachel Ashe has exceded her own expectations (Copyright: R. Ashe)
More about Epilepsy Ireland on their website and their Facebook page.
epilepsy, health, Ireland, prejudice
UK mentor brightens dying child's world
What's it like to know you're going to die soon? If you're 10 years old and terminally ill, what you probably need the most is a good friend. Shlomie, a youth orthodox Jew from northern England, is like a big brother to a terminally ill boy. Sharing the gift of quality time, says Shlomie, is a small sacrifice that makes him feel on top of the world.
Listen to the report by Andrew Edwards in Manchester:
Shlomie Abenson wouldn't miss his volunteer work for the world
children, England, health, illness, Manchester, terminally ill
Dignity in disease
Playing the piano, going for walks and doing household chores are what make Daniel in New York an everyday hero for Alzheimer's patient Byron O'Connell. Now he's making a film to encourage those dealing with the disease.
Listen to the report from Laura Postma in New York:
Pictures: J.P. Dobrin
The two are the subject of a documentary film
Byron has helped Dan see people differently
More on Daniel Davy's blog.
elderly, health, illness, New York, US
An ear for adolescent girls in Bangalore
Sinu, 30, from Bangalore, gave up a promising engineering career to give back to her community. She is now on a mission to keep adolescent girls in school by promoting awareness about the taboo topic of menstruation.
Listen to the report by Pia Chandavarkar in Bangalore:
Sinu Mundkur conducts sessions for adolescent schoolgirls in and around Bangalore, promoting awareness of menstruation.
Since menstruation is a topic that is rarely discussed in public, Sinu and her team have devised the program in such a way that the girls can relate to it and feel comfortable enough to open up about it
education, girls, health, India, school
Nigerian gives blood to save lives
How many times have you donated blood? A young man in Nigeria is so concerned about saving lives that he co-founded Club 25, enouraging others to donate blood 25 times before their 25th birthday. Women in childbirth are in particular need of fresh blood transfusions.
Listen to the report by Nonye Aghaji in Abuja, Nigeria:
Abdullahi Idris gives blood as often as he can
Abdullahi encouraged his friend, Hussein, to donate as well
Africa, blood, health, Nigeria
Lebanese student encourages others to donate blood to save lives
If you live in a country with no centralized blood bank, where do you go if you need a transfusion? For Lebanese pharmacy student, Yorgui Keyrouz, the answer was his mobile phone. Lebanon's health sector was in tatters following the country's 15 year civil war, so Yorgui started an initiative to fill the gaping hole and he's saved 15,000 lives already.
Yorgui Keyrouz started the blood bank on his mobile phone
Samar Khoury is ready to give
Listen to the report by Dalila Mahdawi:
Blood Bank in Lebanon
Wednesday 16.05.2012 | 14:15
health, Lebanon, Middle East
Young refugee from Burma organizes medical aid for others
Halfway between Mae Sot and the Friendship Bridge linking Thailand and Burma stands the house of the Back Pack Health Worker Team, an association that, since 1998, has been giving health assistance to refugees and migrant workers in Thai territory and internal displaced people inside Burma.
A refugee and young mother herself, Snow is the deputy director of the Back Pack organization.
Listen to the report by Roberto Tofani (presented by André Leslie):
Young Burmese refugee organizes medical aid for others
Snow prefered not to be photographed. Here are some impressions from the region. Photos by Robert Tofani.
The Moei River forms a natural border between Thailand and Burma
These Burmese children are pictured on the border with Thailand, on the Thai side
These kids attend a school for refugees, which is run by local NGOs
Asia, Burma, health, refugees, Thailand
Asia China health India Africa human rights environment women South Africa gender Latin America art teacher school recycling Berlin activism Canada poverty youth education culture children Portugal France politics Germany Kenya Argentina democracy orphans dance sports Afghanistan music refugees employment agriculture violence Middle East
Inventor's deposit ring puts change in a bottle
Selfie addict helps others cope with tech overdose
Schooling meets soccer in Mumbai's slums
52 Projects A Canadian proves sustainable living is possible by making one new thing a week for a year.
Africare Africare's programs focus on four principal interrelated areas: Food Security, Water, Health, and Emergency Response.
Amnesty International's Youth and Student program AI is a global movement committed to defending those who are denied justice or freedom.
Be the Cause Be the Cause is a Network of individuals who not only wish to make a difference in the world, but also wish to change their own lives in the process.
Drugs for Neglected Diseases Initiative DNDi is a collaborative, patients' needs-driven, non-profit drug research and development organization that is developing new treatments for neglected diseases.
envision Young people making a difference.
European Youth Forum For the rights of youth.
Futuro Si Initiative for children in Latin America.
Global Fund for Women Promoting women's economic security, health, education and leadership.
Global Youth Connect Empowering youth to advance human rights and create a more just world.
Hedwig and Robert Samuel Foundation The Hedwig and Robert Samuel Foundation is a non-profit organization which supports socially deprived children and youths in the areas of education and vocational training in Central America and Asia.
Internation Citizen Service (Facebook page) ICS, launched by the UK government, is a global volunteering experience which supports young people from all backgrounds to make a real difference to some of the world's poorest people.
International Peace Institute Promoting the prevention and settlement of conflict.
John Dau Foundation The John Dau Foundation is fulfilling the dream of Lost Boy and genocide survivor John Dau to provide healthcare in the war-torn region of South Sudan by building and sustaining medical clinics and training community health workers.
Nano Control Nano-Control has got involved with healthy indoor air as well as research and elimination of the risks and helps people harmed.
Open Doors An organization helping orphaned children in Romania
PCI – Positive Community Impact PCI is a nonprofit health and humanitarian aid organization dedicated to preventing disease, improving community health, and promoting sustainable development worldwide.
Singing for Change Fabian and Viv want to raise money for charities all over the world by taking singing challenges from donors.
Taking IT Global The largest online community of youth interested in global issues and creating positive change.
The Art of Non-Conformity Unconventional strategies for life, work, and travel.
The Free Child Project The mission of The Freechild Project is to advocate, inform, and celebrate social change led by and with young people around the world, especially those who have been historically denied the right to participate.
Turtle Foundation blog Volunteers on Cape Verde protect endangered giant sea turtles.
What Kids Can Do Voices and work from the next generation.
YMAD Youth Making a Difference – Worldwide
YouthNet Celebrating 16 years of supporting young people online.
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El Nuevo Partido Colombiano (NPC) era un partido político de Colombia, creado a partir de la reforma constitucional del acto legislativo de 2003 en el cual se dictaban otras medidas con respecto a los derechos políticos de los ciudadanos y la garantía a todos los colombianos el derecho a fundar, organizar y desarrollar partidos y movimientos, y la libertad de afiliarse a ellos o de retirarse. Fue fundado el 9 de julio de 2003 por Armando Benedetti y Gina Parody y otro grupo de senadores y representantes a la cámara. Representó el primer intento de alcanzar la unidad en entre las repartidas fuerzas del congreso de corte uribista, se crea con el fin de de fortalecer el gobierno nacional de Álvaro Uribe Vélez, unir sectores culturales de Colombia para expandir la base de la seguridad democrática en las elecciones regionales de 2003.

Historia

A partir del éxito de la política del expresidente Álvaro Uribe, un grupo de parlamentarios conformado por los representantes Gina Parody, Luis Fernando Velasco, Armando Benedetti y Nancy Patricia Gutiérrez, decide conformar un Partido por fuera del liberalismo, que respaldaría el Uribismo bajo una sola bandera: el Nuevo Partido.

La alta favorabilidad y proyecto del expresidente Álvaro Uribe, logran atraer a los senadores Mauricio Pimiento, Oscar Iván Zuluaga, Andrés González Díaz y Rafael Pardo y a los representantes Manuel Enríquez Rosero, Sandra Ceballos, Carlos Ignacio Cuervo, Carlos Enrique Soto, Adriana Gutiérrez y Ricardo Arias Mora. A pesar de esta unión, la iniciativa no logra adherir en su totalidad a cuatro Senadores uribistas, el primero de ellos Germán Vargas Lleras que gracias al poco entendimiento con uno de los fundadores, Armando Benedetti, decide quedarse en su propio movimiento, Colombia Siempre.

Por otro lado la senadora Claudia Blum, cuya fórmula en Bogotá fue Gina Parody tampoco se les une, quedándose en Cambio Radical. Por último los Senadores Habib Merheg y Dieb Maloof tampoco ingresan a la nueva adhesión, quedándose en el partido por el que resultaron electos, Colombia Viva. El no lograr unir la fuerza uribista en un solo partido, manteniéndose fraccionada en varios movimientos, hace que el primer desafío electoral del Nuevo Partido las elecciones locales de año 2003, no tengan tan buenos resultados como los esperados.

Plataforma ideológica

La Declaración Programática era la plataforma ideológica oficial del partido:

 La defensa y protección de la cristiandad, la familia y sus tradiciones.

 Buscar acabar los actos de corrupción y clientelismo a lo ancho y largo de Colombia.

 Hacer énfasis en el desarrollo educacional de niños y adolescentes en toda Colombia.

 Llevar el mensaje de la seguridad democrática como única bandera a todas las regiones del país.

 Apoyar la autonomía de las regiones del país por medio de la descentralización económica en Colombia

Resultados Electorales

Elecciones legislativas

Elecciones regionales

Referencias

Partidos políticos colombianos de centroderecha
Partidos conservadores de Colombia
Partidos políticos de Colombia


Est is situated on George St above Establishment and is he headed up by Peter Doyle it currently has two SMH chef hats although in the past it has held three.
The menu for lunch is $69 , $95 or $118 for two , three or four courses. The service is impeccable when the food comes out the plates are both placed presented on the table at the same time in what seems like choreographed movements. All the staff are so knowledgeable about the dishes not just the ingredients but how its prepared and cooked.
The last time a ate at Est was a few years ago I remember bring very impressed by it, it was also one of the first posts on this blog. Fast forward to toady I was equally as impressed the presentation & taste of the food together with the amazing service made for a very enjoyable lunch at Est.
Salad of spanner crab, almond cream, baby zucchini, heirloom tomato jelly, basil such a beautiful dish and it tastes as good as it looks! As it was described in the menu as a salad I was not expecting something like this to come out.
wow each dish is beautiful esp that salad!
Yes the salad dish was amazing both to eat and look at!
The plating is artwork! Good to see Est is still going strong.
Love the plating of every dish! So fresh and pretty.


Are you at a point in your life when you're asking yourself, "What's next?" You've finished one phase of your life and are moving into a new phase, much of which is unknown.
The good folks at AARP have developed a program called Life Reimagined which can serve as a guide from working life to retirement life. You can log into the Life Reimagined website or check out the book of the same title, co-written by Richard J. Leider and Alan M. Webber.
Reflect – look deeply into your life to connect with key benchmarks.
Connect – request feedback from friends and mentors to build your own information and support team.
Explore – test different opportunities.
Choose – narrow down the possibilities.
Repack – decide what's essential for the road ahead.
Act – take small steps to make possibilities real.
Of course, the process is more complex than this brief outline. For more information, don't hesitate to contact me.