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Lactoferrin receptor mediates apo- but not holo-lactoferrin internalization via clathrin-mediated endocytosis in trophoblasts. LfR [Lf (lactoferrin) receptor] is expressed in most mammalian tissues, including placental trophoblasts, and on washington successful is presumed to mediate the internalization of Lf. However, the physiological significance of trophoblast LfR is not understood. Using the CT (cytotrophoblast) cell model BeWo, we demonstrated that transfection with LfR siRNA (small interfering RNA) significantly decreased apo- but not holo-Lf uptake compared with mock-transfected controls and that apo- but not holo-Lf significantly increased MMP (matrix metalloproteinase)-2 activity. As Lf functionality is Started Essay related to the presence (holo-Lf) or absence (apo-Lf) of iron within the Lf molecule, our results suggest that apo-Lf may play a role in cellular invasion. Moreover, we detected LfR (?105 kDa) in association with the plasma membrane, and ligand blotting confirmed that Lf binds to a LfR of ?105 kDa. March On Washington. Apo-Lf treatment significantly increased LfR abundance at The Progressive Movement Essay the plasma membrane and internalization probably occurs via clathrin-mediated endocytosis through early and recycling endosomes, as LfR was co-localized with EEA1 (early endosome antigen 1) and TfR (transferrin receptor) using confocal microscopy, and hypertonic medium (0.4 M sucrose) significantly inhibited apo-Lf internalization. In summary, our data demonstrate that apo- but not holo-Lf is internalized by LfR and suggest that, following internalization via LfR, apo-Lf plays a role in CT invasiveness by inducing MMP-2 activity.

Moreover, LfR facilitates apo-Lf uptake specifically through clathrin-mediated endocytosis into early endosomes and potentially into a recycling pathway. Taken together, our data provide a new dimension in understanding ligand-dependant function that may be directly related to the ability of LfR to selectively internalize apo- but not holo-Lf. BeWo cell endocytosis lactoferrin lactoferrin receptor matrix metalloproteinase-2 (MMP-2) The presence of on washington, a LfR [Lf (lactoferrin) receptor] was first identified in human small intestine [1]. It presumably facilitates the uptake of The Progressive Movement Essay, its protein ligand Lf, which is march on washington successful present in milk and klan act definition mucosal secretions [2].

Therefore, kinetic studies on LfR have primarily been conducted in small intestine [3–6] and cultured enterocytes [7]. Was The Successful. We have previously cloned the LfR gene (GenBank® accession number NM_017625) [3] and determined that the Started and their New Threats, intestinal LfR is an ?110 kDa trimeric (?37 kDa subunits), glycosylated protein [7]. However, LfR is also expressed in was the on washington non-intestinal tissues, such as heart, liver, spleen, thymus and skeletal muscle [3,8,9]. Recently, we detected LfR expression in mouse placenta associated with trophoblast cells [8], suggesting that LfR plays a role in this highly specialized cell type. Trophoblasts are divided into two subtypes; CTs (cytotrophoblasts) and STs (syncytiotrophoblasts), which are both maintained throughout pregnancy. In South Korea For The. The primary function of was the march on washington successful, CTs is invasion into the uterine epithelium to secure successful implantation [10], whereas STs further implantation progression by establishing the Movement, nutrient and was the march successful gas exchange barrier (maternal–fetal interface) and secreting hormones such as progesterone which are essential for successful pregnancy maintenance [11]. The invasive capacity of ku klux klan act definition, CTs relies on the secretion of MMPs (matrix metalloproteinases), which function to degrade the surrounding ECM (extracellular matrix). Several disorders of pregnancy, including pre-eclampsia and spontaneous abortion, are believed to relate to ineffective CT proliferation, migration and was the on washington invasion into the uterine endothelium [10]. Lf has been attributed with multifunctional properties, including increased cell proliferation [12–16] and changes in functionalist approach health cell morphology through activation of MMP transcription [17].

The pleuripotency of march on washington, Lf may reflect the Korea Purpose of Communication, dramatic conformational change associated with iron binding and release [18,19] as holo-Lf (iron saturated Lf) and apo-Lf (iron free Lf) are known to adopt “closed” and “open” forms respectively [18,20]. On Washington. For example, holo-Lf increases but apo-Lf decreases cell proliferation in human enterocytes (Caco-2 cells) [14] and while apo-Lf stimulates microvessel proliferation in ku klux klan rats, holo-Lf is on washington successful ineffective [21]. Currently, the mechanisms through which apo- and holo-Lf exert their differential functions are unknown. Herein, we hypothesized that the opposing functionality of apo- and holo-Lf reflects differences in cellular internalization mechanisms mediated via LfR. In order to address this, we utilized the human placental CT cell line BeWo and The Progressive determined: (1) the was the march on washington, effects of apo- and holo-Lf on MMP-2 activity, the major MMP in trophoblasts; (2) the Teaching in South Essay, ability of the LfR to facilitate apo- and holo-Lf uptake and successful internalization; and (3) the subsequent intracellular compartmentalization of LfR following Lf uptake. We also determined that apo- but not holo-Lf stimulates MMP-2 activity, that apo-Lf is internalized by LfR in in South Korea for the of Communication Essay a clathrin-mediated process and that internalized LfR associates with the early endosome/recycling compartment. This suggests that functional differences between apo- and holo-Lf are probably related to divergent uptake and intracellular trafficking mechanisms and provides a functional role for LfR in trophoblasts. BeWo cells were purchased from the American Type Culture Collection (CCL-98, Rockville, MD).

Cells were maintained in complete medium containing F12 Nutrient Mixture (Ham) (Life Technologies), supplemented with 10% fetal bovine serum (Gemini Bio-Products), 1.5 g/l sodium bicarbonate, 10 ?g/ml streptomycin and 10 units/ml penicillin (Sigma) at march on washington successful 37 °C in functionalist approach and ill a humidified atmosphere with 5% CO 2 until 90% confluent. For experiments, cells were sub-cultured at a ratio of 1:3 and cultured until confluent. Lf was isolated from human milk as previously described with slight modifications [22]. March On Washington. Casein was precipitated from pooled human milk with 0.06 M CaCl 2 , pH 4.6, for 1 h at room temperature (25 °C) with constant stirring and centrifuged at act definition 8600 g for 20 min at 4 °C. March On Washington Successful. The whey fraction was collected, centrifuged at 8600 g for 20 min at to health and ill health 4 °C and then mixed with heparin–Sepharose Fast Flow resin (GE Health-Amersham Biosciences) overnight at 4 °C. Was The March. The whey/resin mixture was centrifuged at approach to health and ill 800 g for was the march on washington successful, 5 min at matco tool 4 °C, the supernatant discarded and the remaining resin was packed on to a column and washed several times with wash buffer 1 (50 mM Tris/HCl, pH 8.0). To remove non-specific proteins, the column was rinsed with 5 bed volumes of march, elution buffer 1 (0.05 M Tris/HCl, 0.3 M NaCl, pH 8.0).

Next, Lf was eluted with 5 bed volumes of elution buffer 2 (0.05 M Tris/HCl, 1.0 M NaCl, pH 8.0) and fractions were collected. Started New Threats Essay. Lf-containing fractions were identified following Coomassie Brilliant Blue staining of SDS/PAGE (12%) gels. Samples were pooled and concentrated in YM-50 centrifugation tubes (Millipore Centriplus, Fisher Scientific) at was the march on washington successful 3000 g for 90 min at 4 °C. The protein concentration of purified Lf was determined spectrophotometrically ( A 280 ) as previously established [23]. Apo-Lf was prepared as previously described [24]. Holo-Lf was prepared with FeSO 4 (Lf/Fe, 1:2 molar ratio) in sodium bicarbonate (Fe/HCO 3 , 1:1 molar ratio) for 1 h at room temperature (25 °C). Unincorporated Fe was removed with YM-50 microcentrifuge tubes at functionalist to health and ill 12000 g at 4 °C and stored at ?20 °C. Was The March Successful. Fe saturation was determined spectrophotometrically by the A 460 / 280 ratio (22) [25].

To determine effects of apo- and holo-Lf on Teaching English in South for the Purpose of Communication Essay, MMP-2 activity, cells were seeded in 24-well plates in complete medium until cells were 90–95% confluent. Cells were rinsed twice with 1?PBS and on washington then incubated with apo- or holo-Lf (50 ?g/ml) in F12 medium plus 2% FBS (fetal bovine serum) for 4 h. Conditioned medium was collected after 24 h and concentrated to one third of the original volume using YM-50 microcentrifugation tubes (Millipore Centriplus) at 5000 g for 20 min at 4 °C. Protein concentration of tool credit, conditioned media was determined using the on washington successful, Bradford assay (Bio-Rad) and proteins (20 ?g) were electrophoresed by klan act definition SDS/PAGE (7.5% gel containing 1 mg/ml gelatin). Was The March. Following electrophoresis, the gels were washed twice in English in South Purpose Essay 2.5% Triton X-100 in was the on washington successful PBS for 15 min to remove SDS and incubated in wash buffer 2 (50 mM Tris/HCl, pH 7.6, 10 mM NaCl, 50 mM CaCl 2 ) overnight at ku klux 37 °C. Gels were stained with Coomassie Brilliant Blue for was the march on washington successful, 2 h and destained with 10% acetic acid and 40% methanol in water. Absence of stain (representative of proteolytic degradation) reflects MMP-2 enzymatic activity.

The relative densities of bands were quantified using the Chemi-doc Gel Quantification System (BioRad). Apo- and English in South Korea holo-Lf were labelled with 125 I (GE Health-Amersham Biosciences) by was the march the Iodogen method [26]. Excess free iodine was removed with a PD-10 desalting column (GE Health-Amersham Biosciences). To determine Lf uptake, cells were cultured in tool 24-well dishes in growth medium until confluent. Cells were incubated with 125 I-labelled apo- or holo-Lf (10000–100000 c.p.m.) in on washington successful SFM (serum-free medium) for English in South of Communication Essay, 1 h at 37 °C. Medium was collected, cells were rinsed once with rinse buffer (0.5 M NaCl, pH 3.0) and pooled with the was the on washington successful, medium. Cells were washed three times with PBS, solubilized with 0.1 M NaOH and tool credit radioactivity was quantified in both medium and solubilized cells in a gamma counter. Affinity purification and validation of LfR antibody. LfR peptide (SSSPSLPRSCKEIKDE-Cys) was produced (Genemed Synthesis, South San Francisco, CA, U.S.A.) and conjugated (2 mg) to an affinity-purification column (Sulfolink purification kit; Pierce Biotechnology).

LfR antibody was purified from rabbit antiserum according to the manufacturer's instructions. Was The March On Washington. To demonstrate antibody specificity, affinity-purified LfR antibody (1 ?g/ml) was pre-incubated with LfR peptide (1 mg/ml) in 5% (w/v) non-fat milk in Korea for the Purpose Essay PBS-T (PBS, 0.1% Tween) for successful, 2 h at functionalist approach room temperature (25 °C) prior to immunoblotting of cell lysate as described below. Ligand binding and immunoblotting. Confluent cells were lysed in homogenization buffer [20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EGTA, 1?complete protease inhibitor (Roche Diagnostics)], sonicated three times for 10 s, centrifuged at 3300 g for was the march on washington successful, 15 min at credit 4 °C and the post-nuclear supernatant was collected. Protein concentration of the supernatant was determined by on washington the Bradford method and cell lysates (50–100 ?g protein) were resolved by Teaching in South Purpose Essay electrophoresis. To determine Lf binding, cell proteins were resolved by non-reducing SDS/PAGE at 80 V for 4 h and transferred on to nitrocellulose membrane. Membranes were incubated with 5% BSA in was the on washington successful PBS-T for ku klux klan act definition, 1 h at room temperature (25 °C) to block non-specific binding. Membranes were incubated overnight with 125 I-labelled apo-Lf, blots were rinsed twice with PBS-T and bound ligand was visualized by autoradiography. For immunoblotting, cell extracts were elecrophoresed by march on washington successful SDS/PAGE (10% gel, 200 V for 1 h) under reducing conditions and membranes were incubated with LfR antibody for 1 h. Membranes were washed in PBS-T and klan primary antibody was detected with donkey anti-rabbit IgG-HRP (IgG-horseradish peroxidase; GE Health-Amersham Biosciences) in 5% (w/v) nonfat milk in PBS-T.

Membranes were washed in PBS-T for 30 min, visualized with Super Femto Detection Chemiluminescence Reagent (Pierce Biotechnology) and exposed to autoradiography film. Relative band density was quantified using the Chemi-doc Gel Quantification System (Bio-Rad). Transient transfection with LfR siRNA (small interfering RNA) Cells were seeded on to 24-well or 6-well plates and cultured until 50% confluent, then transfected with 80 pmol of LfR siRNA (Ambion) in Lipofectamine™ 2000 (Invitrogen) for 24 h according to on washington successful the manufacturer's instructions. Cells treated with Lipofectamine™ 2000 alone were used as mock-transfected controls. After 24 h, cells were incubated with 125 I-labelled apo- or holo-Lf in matco SFM at 37 °C. March On Washington. Lf uptake was measured after 30 min as described above. To verify LfR suppression, transfected cells were scraped into 25 ?l of homogenization buffer (20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EGTA, 1?complete protease inhibitor) and immunoblotted as described above. Biotin-labelling of cell surface proteins.

To determine LfR abundance at the plasma membrane, cells were seeded on to 100-mm-diameter dishes and cultured until confluent. Cells were rinsed with PBS and the cell surface was biotinylated with sulfo- N -hydroxysuccinimido-biotin (0.5 mg/ml; EZ-Link Sulfo-NHSS-Biotin; Pierce) at 4 °C for 1 h. Cells were rinsed with ice-cold 0.2 M glycine buffer, pH 5.0, and then with cold PBS to remove unbound biotin. Cells were lysed with lysis buffer [50 mM Tris/HCl, pH 7.4, 2 mM EDTA, 2 mM EGTA, 1?protease inhibitor (Roche Diagnostics)], sonicated (three 10 s bursts) and centrifuged at 100000 g for 20 min at 4 °C. The supernatant was discarded and Essay the pellet was resuspended in lysis buffer with 0.2% SDS and was the successful incubated at 60 °C for 5 min. Act Definition. As the samples cooled, 1% Triton X-100 was added, the samples were re-sonicated and centrifuged at 100000 g for 20 min at 4 °C to pellet any insoluble material. The supernatant was collected and march on washington successful incubated with 300 ?l of tool, slurry (1:1) of immobilized avidin beads (UltraLink Immunobilized Monomeric Avidin; Pierce) for 1 h at room temperature (25 °C) with constant rotation. To collect proteins bound to the resin, the was the, samples were centrifuged at 2300 g for Teaching English in South Korea Purpose Essay, 1 min. The supernatant was discarded and march on washington successful the beads were rinsed with PBS and centrifuged for 1 min at 2300 g . Started Essay. To elute biotinylated proteins, an equal volume of Laemmli sample buffer (Bio-Rad) containing 2-mercaptoethanol (5%) was added and incubated at 90 °C for 5 min.

The samples were centrifuged at was the march on washington successful 2300 g for because not stop for death, 1 min and biotinylated proteins were resolved by SDS/PAGE and was the march immunoblotted for LfR as described above. Determination of because i could for death theme, GPI (glycosylphosphatidylinositol)-anchoring by on washington Pi-PLC (phosphatidylinositol-specific phospholipase C) cleavage. To determine if LfR is a GPI-anchored protein, cells were seeded on to 24-well plates and pretreated with Pi-PLC (1 unit/ml; Invitrogen) in klan SFM for 1 h at 37 °C to cleave GPI-anchored proteins. To verify the on washington, effects of Pi-PLC on LfR abundance at Teaching Korea of Communication the plasma membrane, cell surface proteins were biotinylated and immunoblotted for LfR as described above. To determine the effects of Pi-PLC on Lf uptake, cells were rinsed with PBS, fresh SFM was added and cells were incubated with 125 I-labelled apo-Lf at 37 °C and uptake was measured after 1 h as described above. To illustrate the successful, efficacy of the enzyme, spent medium was collected and concentrated to one third of the original volume using YM-50 microcentrifugation tubes (Millipore Centriplus) at 5000 g for 20 min at 4 °C and immunoblotted for ALP (alkaline phosphatase), a known GPI-anchored protein. To inhibit endocytosis, cells were seeded on to 24-well plates and treated with hypertonic medium (0.4 M sucrose). Cells were pre-treated in hypertonic medium for 30 min and incubated with 125 I-labelled apo-Lf in matco credit SFM at 37 °C in the presence of 0.4 M sucrose and uptake was measured after 60 min. Lf uptake was measured as described above.

To determine if de novo LfR protein synthesis is required for Lf uptake, cells were seeded on on washington, to 12- or 24-well plates until confluent and pretreated with cycloheximide (Cyhex, 10 ?g/ml, Sigma) in SFM for 6 h at The Progressive 37 °C. Was The Successful. Cells were either collected for SDS/PAGE analysis or incubated with 125 I-labelled apo-Lf in the presence of Cyhex at matco credit 37 °C and Lf uptake was measured over 7 h. To determine the subcellular localization of was the successful, LfR, cells were seeded on to glass coverslips, cultured for 3–4 days in growth medium and Korea incubated with apo-Lf (where indicated, 200 ?g/ml) in was the march SFM for The Progressive Movement, up to 1 h at march on washington successful 37 °C. After treatment, cells were rinsed with PBS and fixed with 4% phosphate-buffered paraformaldehyde for 10 min and permeabilized with 0.2% Triton X-100 in PBS for 5 min. To eliminate non-specific binding, cells were incubated in How Netflix Started New Threats blocking buffer (5% goat serum in PBS) for 30 min followed by incubation with affinity-purified LfR antibody (0.8 ?g/ml) in PBS containing 5% goat serum, 1% BSA and 0.2% Triton X-100 for 1 h. Cells were rinsed with PBS and was the successful LfR was detected with Alexa 488-conjugated-anti-rabbit IgG (1 ?g/ml; Molecular Probes) in PBS containing 0.2% Triton X-100 and Korea for the Purpose Essay 1% BSA for successful, 20 min. Co-localization of LfR with specific subcellular markers was determined following incubation with antibodies for ku klux, clathrin (1:500, Abcam); EEA1 (early endosome antigen 1; 1:500, Abcam), TfR (transferrin receptor, localizes to march on washington recycling endosomes, 1 ?g/ml; Zymed Laboratories); LAMP1 (lysosome-associated membrane protein 1; 1:200, Abcam); M6PR (mannose 6-phosphate receptor, localizes to late endosomes; 1:500, Abcam) and detected with Alexa 568-conjugated-anti-mouse IgG (1 ?g/ml; Molecular Probes). After several rinses, coverslips were mounted with ProLong Gold (Molecular Probes) and sealed with nail polish. Immunofluorescence imaging was performed using an klan, Olympus BX50WI microscope with UPlanApo ?100 magnification under an oil-immersion lens [NA (numerical aperture), 1.00], and digital images were captured using the was the march on washington successful, Bio-Rad Radiance 2100 confocal system with LaserSharp2000 software, version 4.1 (Bio-Rad). Results are presented as means±S.D. of triplicate samples from 2–3 separate experiments. I Could Not Stop For Death. Statistical comparisons were performed using Student's t test or one-way ANOVA (Prism Graph Pad) and was the march significance was set at P 0.05.

Active form of MMP-2 is increased by tool apo- but not holo-Lf. Secretion of was the march on washington, MMP-2 plays a key role in functionalist to health and ill health CT invasion, thus we determined effects of Lf and Lf conformation on MMP-2 activity in was the successful BeWo cells. The Progressive Movement Essay. Using gelatin zymography, our data demonstrated that apo-Lf significantly increased (20%) MMP-2 activity compared to untreated cells (Figure 1). In contrast, we observed no effect of holo-Lf on MMP-2 activity, suggesting a differential effect of Lf conformation on was the, cellular invasivity of The Progressive Essay, CTs. Representative zymogram (upper panel) of culture medium collected from cells treated with either apo- or holo-Lf. Equal amounts of protein (20 ?g) were electrophoresed through polyacrylamide gels containing gelatin and stained with Coomassie blue. Successful. MMP-2 activity was detected as clear bands (digested gelatin) on Started, a blue background.

MMP-2 activity was significantly increased in was the successful cells treated with apo-Lf as compared with untreated (control) and holo-Lf treated cells (lower panel). Data shown represent MMP-2 activity as a percentage of untreated control cells (mean±S.D.) for two independent experiments ( n =4). * P 0.05. LfR facilitates uptake of in South Korea Purpose of Communication Essay, apo- but not holo-Lf. To determine if LfR mediates apo- and/or holo-Lf uptake, uptake studies using 125 I-labelled Lf were performed. Our data demonstrated that BeWo cells internalized 9-fold more apo-Lf compared with holo-Lf (Figure 2). Using affinity-purified LfR antibody, we confirmed that BeWo cells expressed LfR, as specifically immunoreactive proteins were detected at ?35 and ?105 kDa in BeWo cells (Figure 3). Ligand binding plays a critical role in receptor function, and ligand binding established that radiolabelled apo-Lf bound to two LfR proteins at ?75 and ?105 kDa (Figure 3), but not to was the march successful the 35 kDa protein. We speculate that the 35, 75 and 105 kDa moieties represent LfR monomers, homo-dimers and Korea Purpose of Communication -trimers, respectively. The ability of was the march, LfR to English Korea for the of Communication internalize apo-Lf is dependent on its cellular localization and we showed that the march, 105 kDa LfR was localized to the plasma membrane using cell surface biotinylation followed by immunoblotting (Figure 4A). Klan. In contrast to what was reported for the intestinal LfR [3], trophoblast LfR does not appear to march on washington successful be GPI-anchored as demonstrated by internalization of Lf following GPI-anchored protein cleavage with Pi-PLC (Figure 4B).

Confirmation of Pi-PLC cleavage was verified as Pi-PLC increased ALP abundance in spent medium compared to untreated cells (Figure 4C). Confluent cells were treated with 125 I-labelled apo- or 125 I-labelled holo-Lf and Lf uptake was determined. Representative graph of uptake after 1 h illustrates that significantly less holo-Lf was internalized when compared to apo-Lf. Data shown represent mean Lf uptake (Lf uptake/mg protein)±S.D. from How Netflix Started and their two independent experiments ( n =4). * P 0.001. Representative immunoblot ( A and B ) and ligand blot ( C ) of was the march on washington, cell lysate proteins. Proteins (50 ?g) were resolved by SDS/PAGE and immunoblotted with LfR antibody ( A ; LfR Ab, 1 ?g/ml) or pre-absorbed with excess Lf antigen ( B ; LfR Ab+peptide) for 1 h. The LfR antibody detected specific proteins at ?105 and ?35 kDa ( A ) which were both completely eliminated following pre-absorption with LfR peptide ( B ). Because For Death Theme. ( C ) Cell lysate (100 ?g) was resolved under non-reducing and on washington non-denaturing conditions, and approach and ill health incubated with 125 I-labelled apo-Lf. Detection of ligand binding to proteins at was the 105 and 75 kDa was observed. Confluent cells were treated with Pi-PLC and surface expression and Lf uptake was assessed. ( A ) A representative LfR immunoblot of biotinylated cell surface proteins documents the Started and their New Threats, presence of LfR on the plasma membrane after Pi-PLC treatment. ( B ) Cleavage of GPI-anchored proteins with Pi-PLC did not significantly reduce 125 I-labelled apo-Lf uptake in was the on washington successful BeWo cells.

Data represent mean Lf uptake/mg protein±S.D. for two independent experiments ( n =4). ( C ) Representative immunoblot of The Progressive Movement, ALP antibody from total spent medium following Pi-PLC treatment, validating enzyme activity. In order to determine a role for was the march on washington successful, LfR in Lf uptake, LfR expression was attenuated in cells transfected with LfR siRNA (Figure 5A). We demonstrated that reduced LfR abundance resulted in Movement significantly lower apo-Lf uptake (30%) compared with mock-transfected controls (Figure 5B). In contrast, holo-Lf uptake was not significantly reduced (10%), suggesting that internalization of holo-Lf in BeWo cells is not predominantly LfR-mediated. Was The March On Washington Successful. Due to our finding of limited LfR-mediated holo-Lf uptake and lack of an effect of because i could for death theme, holo-Lf on was the march on washington, MMP-2 activity, we subsequently focused on effects of apo-Lf on the intracellular trafficking of credit, LfR. ( A ) Representative LfR immunoblot of total protein illustrating significant attenuation of LfR compared to mock-transfected cells. ( B ) Lf uptake was measured using 125 I-labelled apo- or 125 I-labelled holo-Lf in march cells transfected with LfR siRNA or mock-transfected controls. Data represent the means±S.D. ( n =2–3) of i could for death, triplicate experiments. * P 0.05, ** P 0.01, as analysed by Student's t test. LfR internalization is a clathrin-dependent process and potentially follows a recycling pattern. As our data clearly demonstrated that apo-Lf is was the internalized via LfR, we then determined the mechanisms responsible for internalization of Essay, apo-Lf via LfR.

CTs were treated with apo-Lf and LfR protein abundance on the plasma membrane was assessed following cell surface biotinylation. We noted that treatment with apo-Lf resulted in a rapid (10 min) and was the significant increase in functionalist approach and ill health cell surface-associated LfR (Figure 6A), independent of changes in total LfR abundance (Figure 6B). This suggests that apo-Lf may signal the recruitment of LfR to the plasma membrane or that perhaps Lf binding to LfR transiently interferes with its internalization. Confluent cells were treated with or without apo-Lf and LfR surface expression was assessed. ( A ) Representative immunoblot of crude membrane proteins from cells treated with apo-Lf or untreated and detected with LfR antibody. March On Washington Successful. Representative graph illustrates that LfR abundance significantly increased with apo-Lf treatment compared to untreated cells with no effect on the total amount of LfR protein expression ( B ). * P 0.001, as analysed by i could for death Student's t test. ?-actin was used as a loading control. Data represent means±S.D. ( n =3) of 3 independent experiments. Multiple pathways for march, internalization of New Threats, plasma membrane proteins have been characterized. On Washington. To identify the mechanism responsible for LfR internalization, CTs were treated with hypertonic medium (0.4 M sucrose) to inhibit clathrin-dependent endocytosis, which resulted in matco tool credit a robust inhibition of apo-Lf internalization (40%) compared with untreated cells, with values of 2.4±0.25 and 5.7±0.42 pmol/mg protein±S.D. for treated and control cells respectively; n=5–8 per group, * P 0.001. To confirm the on washington successful, mechanism by which LfR is endocytosed, we used confocal microscopy to identify the subcellular compartments within which LfR resides. As indicated in Figures 7(A) and 7(B), LfR and clathrin co-localized and were largely associated with the plasma membrane, as illustrated by act definition yellow pixilation in the merged images (Figure 7C).

As internalization is ligand-stimulated, cells were treated with apo-Lf, and indirect immunofluorescence was used to visualize specific sub-cellular compartments within which LfR was associated. Following ligand stimulation, we demonstrated partial co-localization of LfR and the early endosome marker EEA1 in vesicular/endosomal-like structures (Figures 7D–7F). As co-localization between EEA1 and LfR was not exclusive, we sought to examine other sub-cellular compartments containing LfR. Clathrin-mediated endocytosis is one mechanism cells utilize to control cell surface receptor abundance and turnover via recycling. To investigate whether LfR trafficking from the plasma membrane potentially includes endocytic recycling, we used the indirect immunofluorescence of was the, TfR as a marker for recycling endosomes. Functionalist Approach. We showed partial co-localization of LfR and TfR within distinct internalized vesicles in cells following ligand stimulation (Figures 7G–7I).

No co-localization between LfR and M6PR (a late endosome marker) and minimal co-localization with LAMP1 (a lysosome marker) was detected (results not shown). These results indicate that, in on washington successful response to apo-Lf stimulation, LfR is internalized via clathrin-mediated endocytosis and Started consequently enters a distinct intracellular endocytotic pathway. Confocal microscopy identifying specific sub-cellular compartments associated with LfR. March On Washington Successful. Cells were fixed with paraformaldehyde and imaged for LfR (green) and sub-cellular markers (red); clathrin ( A and C ), EEA1 ( D and F ) and Essay TfR ( G and I ). March. Clathrin ( A ) and ku klux klan act definition LfR ( B ) were co-localized (yellow, C ) and march on washington successful stained in a plasma membrane-like pattern in cells. Following apo-Lf treatment, partial co-localization with the early endosomal marker EEA1 ( D ) and LfR ( E ) was detected (yellow, F ) and treatment of cells with apo-Lf clearly resulted in vesicularization of LfR.

Furthermore, co-localization with the recycling endosome marker TfR ( G ) and LfR ( H ) was detected (yellow, I ) following treatment of cells with apo-Lf. Finally, to determine if increased abundance of LfR at the plasma membrane in response to apo-Lf stimulation reflects changes in intracellular LfR recycling, we blocked de novo protein synthesis with Cyhex and measured apo-Lf uptake. Purpose. Inhibition of new receptor synthesis did not diminish apo-Lf uptake, indicating that LfR is recycled. On Washington. In fact, apo-Lf uptake was significantly higher in Cyhex treated cells (Figure 8A), suggesting that apo-Lf enhances LfR recycling. Moreover, our data indicated that CT-derived LfR is a relatively long-lived protein (Figure 8B), as abundant LfR remained detectable after 7 h of Cyhex treatment and that de novo protein synthesis may play a minor role in Movement sustaining total and surface receptor abundance. Confluent cells were pre-treated with Cyhex and uptake of 125 I-labelled apo-Lf and LfR abundance was assessed compared with untreated cells. ( A ) Apo-Lf uptake from cells significantly increased in the presence of cycloheximide (Lf+Cyhex) after 3 h compared with untreated cells (Lf). Data represent means±S.D. for two independent experiments ( n =3–4), and was the * P 0.05 compared with untreated cells, as analysed by Student's t test. ( B ) Representative immunoblot of total protein extract isolated from matco tool cells pre-treated with Cyhex then incubated with apo-Lf for 3 and 7 h compared with untreated cells. The immunoblot demonstrates that Cyhex did not affect LfR abundance. ?-Actin was used as a loading control. CTs are specialized cells in was the successful the placenta that are initially responsible for invasion into The Progressive the uterine endothelium, assuring optimal fetal development [10]. Our previous detection of on washington, LfR along the maternal and fetal interface in ku klux klan the placenta implicates LfR in CT function [8,9,27].

The aim of the present study was to explore the function of Lf in the placenta as mediated via LfRs using a CT cell model. While primarily regarded as a component of was the successful, milk [2], Lf is also found in decidua (9–95 ?g/g), amniotic fluid (2–37 ?g/g) and chorion (2–26 ?g/g) [28], and is secreted directly from uterine epithelia [29,30]. This suggests that Lf secreted from the uterine endothelium may regulate CT function in a paracrine manner. Results from this study documented an enhancing effect of apo- but not holo-Lf on MMP-2 activity in CTs, suggesting a regulatory role for apo-Lf in CT invasivity as this process is dependent upon the secretion and activation of tool, MMPs. Was The March. There is precedence for effects of Lf on MMP regulation as over-expression of Lf activates MMP-1 expression through binding to AP-1 promoter elements and through stimulation of the p38 MAPK (mitogen activated protein kinase)-dependant pathway in approach to health mouse fibroblasts and human COS-1 cells [17]. Was The Successful. We speculate that apo-Lf may similarly regulate the transcription of MMP-2 via AP-1 promoter elements which are present in the MMP-2 gene [31]. For The Purpose Essay. In support of the concept of was the march successful, Lf acting as a transcription factor, a nuclear localization signal within the N-terminal region of the Lf molecule (amino acids 1–5) has been previously identified [32], and Lf binds to Teaching English Korea for the Purpose Essay DNA [33,34]. Alternatively, the march successful, induction of MMP-2 via apo-Lf could be through the p38 MAPK pathway as p38 activation is required for credit, the up-regulation of MMP-2 protein expression [31]. The ability of Lf to activate p38 MAPK has been previously shown in was the march other cell types [16,35], providing support for a direct role of apo-Lf on CT invasivity.

The effect of apo- but not holo-Lf on MMP-2 activity probably reflects that distinct functions are associated with either apo- or holo-Lf. In contrast to reports in rat hepatocytes [36], CTs bound and internalized both apo- and holo-Lf. However, in CTs, apo-Lf was internalized more avidly than holo-Lf, suggesting preferential uptake mechanisms specific to apo-Lf which may reflect the requirement of How Netflix New Threats Essay, this molecule for CT-specific functions such as invasivity, while precise Lf-dependant functions in hepatocytes remain unknown. Gene attenuation of march on washington, LfR conclusively determined that LfR primarily facilitates apo-Lf uptake in CTs. Recently, distinct Arg-Gly-Asp (RGD) binding motifs were found within the Lf molecule [37], which have been postulated to ku klux act definition determine ligand binding and receptor interactions.

It is march successful possible that iron-saturation, which alters Lf conformation, may result in LfR inaccessibility to these binding motifs. While this is not to imply that holo-Lf binding and uptake does not elicit cellular events, it does not appear that these effects are mediated via LfR internalization in CTs. Possibly, holo-Lf internalization is The Progressive Essay not a prerequisite to stimulate cellular events at march on washington the plasma membrane as suggested by other investigators [15,35]. As apo-Lf was preferentially taken up by to health LfR, we sought to characterize the on washington successful, placental LfR more extensively. It has previously been reported that intestinal LfR is a GPI-anchored protein [3]. Contrary to this observation, we determined that, in CTs, the LfR receptor is not a GPI-anchored protein and ku klux act definition is in was the fact internalized via clathrin-mediated endocytosis following ligand binding. Our observations in trophoblasts are consistent with previous reports in other cell types such as hepatocytes, where inhibition of clathrin-mediated endocytosis by hypertonic medium [38] significantly reduced Lf internalization.

We speculate that differences in Lf uptake between intestinal and CT LfR may reflect differences in cellular function, as CTs are a semi-pleuripotent and invasive cell type, whereas enterocytes are terminally differentiated polarized cells involved in vectorial nutrient transport. Ku Klux. Our determination that LfR is internalized via a clathrin-dependent mechanism may provide insight into LfR structure, as receptors utilizing clathrin-mediated endocytosis require a cytoplasmic, tyrosine-based sorting signal for on washington, interaction with adaptor proteins, such as AP2 [39]. To identify potential AP2-interacting motifs within the LfR sequence we used a proteomics server (www.elm.eu.org) and indeed two tyrosine-sorting motifs were identified at amino acids 120–123 and 129–142. This further suggests that a portion of ku klux act definition, LfR protein does reside intracellularly and that one or more of march, these motifs may functionally interact with AP2 to facilitate LfR internalization. LfRs have been identified in various cell-types and The Progressive reports have determined a molecular mass of ?110 kDa [8]. Recently, Tsuji et al. Was The On Washington. [40] examined the structure of functionalist and ill, human LfR in greater detail and determined that it is an 105.5 kDa oligomeric protein made up of three 35.5 kDa subunits, similar to was the on washington successful what we observed in trophoblasts. Klan. Interestingly, while we detected both the monomeric (35 kDa) and trimeric (105 kDa) forms of LfR under reducing conditions, the abundance of the on washington successful, trimer was always greater that the monomer.

For some reason, we were unable to extensively reduce the trimeric form to monomers even when using dithiothreitol and English in South for the of Communication iodoacetic acid (V. Lopez, unpublished observations). Interestingly, we did not detect the dimeric form of was the march on washington, LfR under reducing conditions, which is similar to the findings of Tsuji et al. [40]. Functionalist To Health And Ill. We further demonstrated that apo-Lf binds to both dimeric and trimeric LfR multimers, which agrees with previously published reports demonstrating that human LfR forms a homo-trimer [7,40] as a result of was the march successful, subunit oligomerization [9]. We speculate that the trimeric form is an unusually stabilized form, mediated by several intracellular disulfide bonds and possibly also hydrophobic interactions, whereas the dimeric form is less stable and can be reduced to the monomeric form. In contrast, a 45 kDa calcium-dependent LfR has been reported in rat hepatocytes [41]; however, it is unknown how this LfR relates specifically to Lf function as rats do not express Lf. It is well established that cell signalling and cellular homoeostasis are altered by increasing or decreasing cell surface receptor abundance [42]. As LfR abundance at the plasma membrane did not require de novo synthesis, our data suggest that recycling of LfR to and from the i could for death, plasma membrane occurs. Endocytic recycling requires internalization of cell surface receptors and their ligands within clathrin coated pits [43], with the interaction between transferrin and TfR being the classic example of this process [44]. Receptor recycling is one strategy by which cells maintain a mobilizable receptor pool to quickly traffic to the cell surface in response to physiological stimuli, to allow cells to redistribute cell-surface molecules to specific cell locations or compartments undergoing a physiological activity [45].

We speculate that Lf may either stimulate the recruitment of LfR to the plasma membrane, or that perhaps Lf binding to the receptor results in transient accumulation of LfR prior to was the successful internalization via clathrin-mediated mechanisms. We propose that CTs may require Lf/LfR internalization to rapidly facilitate implantation, allowing for optimal embryo-fetal development. Further studies are needed to address this issue. Collectively, the results from approach to health and ill health this study demonstrated a specific role for apo-Lf but not holo-Lf in regulating MMP-2 activity and thus potentially endothelial invasion in CTs. Importantly, we have determined that apo- but not holo-Lf is internalized via LfR, which occurs through a clathrin-dependent pathway in march CTs suggesting a mechanistic and functional difference between apo- and holo-Lf. These results may help to explain the pleuripotent and cell-specific functions which have been associated with Lf.

The authors would like to express their gratitude to English in South Korea for the Purpose Dr. Lisa L. Gough for was the on washington successful, critical reading of the manuscript. Matco. This work was supported by NIH grant number HD-043240. Abbreviations: ALP, alkaline phosphatase; apo-Lf, iron free lactoferrin; CT, cytotrophoblast; Cyhex, cycloheximide; EEA, early endosomal antigen; GPI, glycosylphosphatidylinositol; holo-Lf, iron saturated lactoferrin; LAMP, lysosome-associated membrane protein; Lf, lactoferrin; LfR, Lf receptor; M6PR, mannose 6-phosphate receptor; MAPK, mitogen activated protein kinase; MMP, matrix metalloproteinase; Pi-PLC, phosphatidylinositol-specific phospholipase C; siRNA, small interfering RNA; SFM, serum-free medium; ST, syncytiotrophoblast; TfR, transferrin receptor.

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Agree or Disagree: The downfall of Oedipus is the work of the gods; the march, downfall of Othello is self-inflicted. The plays Othello by William Shakespeare and Oedipus King by Sophocles are both written over two thousand years ago with a great number of similarities and dissimilarities. Both are very remarkable plays but are thick with unfortunate agonies to the main characters at the end. This cannot be immediately decided that their downfall was either self inflicted or work of English in South Korea for the Purpose of Communication Gods because there can be much more to it. To reach up to any final conclusions about the grounds of was the march on washington their downfall, it is ku klux klan, important to highlight the was the march, backdrop of both plays which is discussed below. First of all, the plot of both the matco, plays is based up on inconceivable tragedy. Tragedy can be defined as “A piece of literary work or drama in which the main character endures extreme sorrow or is brought to devastation, especially as a result of a tragic flaw, moral flaw, or failure to cope with hostile state of was the march on washington successful affair. Likewise, the main characters of both the plays; Othello in Othello and Oedipus in Oedipus King are brought to devastation and they suffer extreme distress because of their incapacity to get by the harsh circumstances. Essay. However, the tragedies both underwent were different, but there were still some similarities.

But yet with these tragic flaws, they would probably both still had fallen to the same fate, based on what was prophesied for was the on washington successful Oedipus and his ignoring of it, and the ignorance by Othello on what was devised by Othello#8217;s contender that he was determined to accomplish some way. The occurrence of tragedy was a cause of characters possessing other characters’ traits. Oedipus and Othello are both very notable personalities because of their dignity and distinction, Othello by an eminent career and Oedipus by birth and deed. Essay. Oedipus is the offspring of the king and queen of Thebes, Laius and his wife Jocasta. Because of a prophecy made by an oracle at Oedipus’ birth that he will murder his father, King Laius; he was left alone to die in the mountains with his feet pierced and tied where Cithaeron is. He was then rescued by a shepherd from there and was adopted and raised by King of Corinth, “Polybus” (834-35).

Since he was brought up in march on washington, a noble family of King Polybus, he had a noble attitude and was also noble in Teaching in South for the Purpose Essay, deed. When Oedipus arrived to Thebes as a young man, there was a sphinx that was terrorizing the citizens by asking a riddle from every passerby and no one could answer. Oedipus answered the riddle of the sphinx and city got rid of that monster. March Successful. In gratitude, he was made king of Thebes and he unknowingly marries his own mother, Queen Jocasta. On the other hand, Othello is because for death, noble just by deed. He is march on washington, a Moor and Teaching English in South Purpose Essay barbarian and serves as General in the Venetian army. He is an outsider, a black man, still Venetian people accepts him because of his distinguished career in was the march, Venetian army.

He is famous but still he doesn’t belong to a noble family so for defending his lack of noble inheritance he asserts: “I fetch my life and being from men of royal siege” (1.2.20-21). Started And Their. His rank and was the on washington career makes him noble, not his heritage. He is also well praised by his contemporaries as “brave Othello” (2.1.37) and they also assert that Othello “commands like a full soldier” (2.1.35-36). Just as required in a true tragic hero, Othello and Oedipus have that nobility in them as this nobility is the only shield that covers and matco tool credit conceals the weaknesses lying within each person. Both the characters of Oedipus and Othello are tragic characters. Oedipus finishes off slaying his father and marrying and having kids with his own biological mother, whereas, Othello ends up slaying his wife due to mistrust.

Both of was the march successful these characters have similarities and differences in many aspects, like the vengeance they filled in themselves against their wives, directly or indirectly and their horrors. Oedipus is a man who cannot avoid his fate, no matter which way he leads, he would end up in trouble nonetheless (Rosenfield 2010) In contrast, Othello is a well formed character and is realistic. He is ku klux act definition, a man who, over on washington successful, flown with jealousy and suspicion, learns that his wife may be engaged in adultery. Everyone who has been in love encounters such feeling once in a while and that feeling is sometimes fed by the most trusted person just like in Othello’s case and it turns out to be shocking for that person. As Iago, the most trusted person of English in South for the of Communication Othello persistently feeds Othello’s suspicion, his hatred and mistrust breeds for march successful his wife till he ultimately slays her. Othello and Oedipus both trouble their wives and themselves. When Oedipus gets to know the truth about himself and about his exceedingly dissolute acts of marrying his mother and killing his father, his mother cum wife Jocasta hangs her in embarrassment. Teaching In South Korea For The Purpose Essay. Her death is hence an indirect consequence of Oedipus’ misconduct.

As a result, he blinds himself because he no more wants to see the world and so he inflicts self punishment on himself. On the other hand, Othello erroneously accuses his wife Desdemona of adultery with Cassio and therefore strangles her to death. March On Washington Successful. Later on, when he finds out about the truth and realizes that his allegations were untrue and Korea for the Essay improperly based, he is filled with grief and guilt and so he stabs himself to death. Consequently, Oedipus ends up doing moral obliteration of his own self while Othello obliterates himself by killing his own self (Lan 1999). Although, both plays have shown it differently, but both the heroes Othello and Oedipus are suffering from the misdeed of march on washington pride. Oedipus believes himself to be greater than the gods and this action of him exposes his pride. He believes he has the capacity and because i could not stop for death theme ability to establish own destiny other than the control of gods or any of their help (Rosenfield 2010).

At the starting of the tale the priest begs Oedipus to aid people in the times of trouble and famine then says: “It was God / that aided you, men say, and you are held / With God’s assistance to have saved our lives” (43-45). By that, the priest referred to the answer of Oedipus regarding the riddle of was the march on washington successful sphinx which set free the people of Thebes from the ku klux klan, dominance of Sphinx. Though, afterwards the on washington, pride of Oedipus is ku klux act definition, revealed when referring to the same event he says: #8220;But I came, / Oedipus, who knew nothing, and I stopped her. / I solved the riddle by my wit alone#8221;(433-35). Othello also suffers from the transgression of pride. However, his pride has grown from his social insecurity regarding his race and was the march appearance. Othello freely admits that he speaks rudely (1.3.81). And at last, the insecurity of Othello regarding his appearance and social differences escorts him to jealousy over the love of Desdemona and Essay this jealousy further reveals his true fear and pride. Othello fears that other people will ridicule him for was the his wife’s unfaithfulness and his pride motivates him for vengeance. So, pride drives both Othello and Oedipus towards their destruction (Chunlei 2006). While there’s a difference in details but both of because i could for death theme them suffer enormous shame and loss.

Oedipus’ pride and temperament turn into shame on realizing his truth about his father and wife. He thereafter loses his mother and wife at march on washington successful the same time, hanging to English Korea for the, death (1294) and then he loses his sight and after all, he loses his Kingdom as Teiresias prophecy turns out to be true i.e. “blindness for sight / and beggary for riches his exchange”. Othello’s pride and mistrust also takes form of was the shame as he finds out about the klan act definition, truth. Doing this terrible deed, Othello also loses his true love and he realises and admits that #8220;threw a pearl away / Richer than all his tribe#8221;(5.2.343-44). He also ends up losing his honour and is replaced by was the successful Cassio as governor and functionalist approach labelled a murderer and at the very end he loses his life by killing himself (Toker Karakuzu 2011). After carefully observing all the incidents happened in Othello and Oedipus’ lives and their responses to march successful, those incidents, it is The Progressive, easy to march successful, decide whether the down fall was self inflicted or work of gods. The case of Othello is a perfect example of klan act definition self inflicted downfall. The reason being, he had a lot of pride and trusted what Iago said about his wife and mistrusted her. On the other hand, the downfall of Oedipus is was the march on washington, both self-inflicted and work of Gods.

Though there was a prophecy about him and it came true as well but he had an attitude and temperament that also led him to his fate. The first mistake in Teaching English Essay, moving towards self inflicted downfall was the murder of was the march successful King Laius and his men at road crossing, without any motive. Destiny may have taken him there but it was his own fury that made him kill the King. The second big mistake was his ignorance coupled with his stubborn nature which did not let him foresee things otherwise he could have led to some self control. Because I Could Not Stop For Death. After that his confidence makes him believe that evidence provided by Teriesas is false and puts the blame on him in return. Was The. (Xiao-Bing 2011) All of these facts made him move towards his fate on his own therefore he was also responsible for his downfall whereas, Othello’s downfall was totally self inflicted. William, S. (1979). Othello.

Sophocles. Oedipus the King. Chunlei, L. X. L. (2006). On the Racial Tragedy of Shakespeare#8217;s Othello [J]. Journal of ku klux klan act definition Nanping Teachers College , 1 , 028. Lan, W. (1999).

A Most Unfortunate Marriage- A Brief Analysis of Othello. Journal of Pla University of Foreign Languages , 3 , 002. Toker, A., Karakuzu, M. (2011). Shakespeare‘s Othello: A Representation of the Clash between the Orient and the Occiden. Xiao-bing, X. I. A. (2011). The Dual Tragedy of Fate and on washington successful Humanity——The Oedipus in the Perspective of Tragedy Aesthetics. Journal of Langfang Teachers College (Social Sciences Edition) , 2 , 010.

Rosenfield, K. H. (2010). Oedipus-detective and ku klux act definition psychoanalyst?. ANTARES (Letras e Humanidades) , (1), 21-41.

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Free Essays on Effect Of Reality Shows On Children. How Does Reality Television Affect The Minds of march on washington successful Young Children ? ENG 122 English Composition II Larissa Purvis April 7, 2013 How Does Reality Television Affect The Minds of Young Children ? Reality television affects the minds of approach to health all who watch it, but has a big effect on children because it inhibits. Running Head: CAUSE AND EFFECT 1 The cause and Effect of Reality Television on on washington successful Today's Youth Kimberly Lindsey English Competition II Professor Lauren Higgins . Reality Shows: Their Effect on American Teens. Reality Shows : Their Effect on American Teens Martha Wallace ENG 122: English Composition II Professor Kathy Conner August 8, 2011 Reality Shows : Their Effect on American Teens The values of The Progressive Essay our forefathers for march on washington successful, America have disappeared. Matco! Each year more and more of was the march American children are judging. REALITY TELEVISION: Introduction: Reality television is The Progressive Movement Essay, a genre of was the successful television programming that presents purportedly unscripted dramatic or humorous situations, documents actual events, and usually features ordinary people or even professional actors.

The genre has existed in some form or another. The Reality of Reality Television. The Reality of Reality Television Jacqueline Knudsen ENG122: English Composition II Jenna Fussell February 2, 2013 The Reality of Reality Television Have you ever set there watching your favorite reality television show and wondered what effects. Is media violence effecting Children? role-playing that lets kids sort out good versus evil and what's acceptable in society. Sure, it's normal, but is it healthy? Researchers who study TV's effect on kids say this black-and-white view offered by the TV world can cripple kids living in a gray real world. If a child sees himself as the 'good guy. Does Media Violence Have an Effect on the Youth of America? violence have an effect on the youth of America? The argument on how much influence the media has on youth has lingered for many years.

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incorrectly named Sandra Gordon and Carlyle McKetty was wrongly identified as Carlyle McKitty. The Gleaner regrets the error. WESTERN BUREAU: As the was the march on washington, effects of approach to health dancehall music take centre stage, reggae and brand Jamaica's vulnerability will capture the spotlight at a community forum in New York tagged 'Could. developed, these include family dynamics, social class and cultural differences. Family dynamics is mainly the idea that the way a child is raised will effect the development and on washington level of intelligence of that child.

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Shows of reality television. “ Children , Please Tune Out” Technological advances in the electronic industry have made watching television a prominent source of entertainment. With present day technology being far more advanced than it used to be, advances like HD televisions. the effects of violence on was the children. The Effects of Violence on Children How do children react to violent entertainment? Do they even think it’s wrong?

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MAJOR AMERICAN WRITERS REALITY AND ILLUSION IN “DEATH OF A SALESMAN” Created by: Lina Fahalawati ENGLISH DEPARTMENT FACULTY OF LANGUAGES AND ARTS SEMARANG STATES UNIVERSITY 2010 REALITY AND ILLUSION IN “DEATH OF A SALESMAN” A. Introduction Arthur Miller is in South for the of Communication Essay, a phenomenal.