71 research outputs found
Management of bank’s marketing communications
The understanding of bank’s marketing communications (BMC) and their management is deepened in the dissertation; the major development trends of global and domestic markets of marketing communications and banking services are identified; the
principles of systematic approach implementation to the BMC management are developed; approach to assessment of BMC’s usage effect is worked out; motivation tools
for bank front managers involved in the process of personal selling are developed; bank optimal cost-sharing framework is improved: 1) between mass and individual BMC instruments, 2) between individual instruments of mass BMCУ дисертаційній роботі поглиблено розуміння сутності маркетингових ко-мунікацій банку (МКБ) та управління ними; виявлено основні тенденції розвитку світового та вітчизняного ринків маркетингових комунікацій та банківських послуг; розвинуто засади застосування системного підходу до управління МКБ; за-пропоновано підхід до оцінювання ефекту від застосування інструмента МКБ; розроблено інструментарій мотивації фронт менеджерів банку, задіяних у процесі персональних продажів; удосконалено засади оптимального розподілу витрат банку: 1) між інструментами масових та індивідуальних МКБ; 2) між окремими інструментами масових МКБ
Characterization of Differential Protein Tethering at the Plasma Membrane in Response to Epidermal Growth Factor Signaling
Physical tethering of membrane proteins to the cortical
actin cytoskeleton
provides functional organization to the plasma membrane and contributes
to diverse cellular processes including cell signaling, vesicular
trafficking, endocytosis, and migration. For these processes to occur,
membrane protein tethering must be dynamically regulated in response
to environmental cues. In this study, we describe a novel biochemical
scheme for isolating the complement of plasma membrane proteins that
are physically tethered to the actin cytoskeleton. We utilized this
method in combination with tandem liquid chromatography/mass spectrometry
(LC–MS/MS) to demonstrate that cytoskeletal tethering of membrane
proteins is acutely regulated by epidermal growth factor (EGF) in
normal human kidney (HK2) cells. Our results indicate that several
proteins known to be involved in EGF signaling, as well as other proteins
not traditionally associated with this pathway, are tethered to the
cytoskeleton in dynamic fashion. Further analysis of one hit from
our proteomic survey, the receptor phosphotyrosine phosphatase PTPRS,
revealed a correlation between cytoskeletal tethering and endosomal
trafficking in response to EGF. This finding parallels previous indications
that PTPRS is involved in the desensitization of EGFR and provides
a potential mechanism to coordinate localization of these two membrane
proteins in the same compartment upon EGFR activation
Characterization of Differential Protein Tethering at the Plasma Membrane in Response to Epidermal Growth Factor Signaling
Physical tethering of membrane proteins to the cortical
actin cytoskeleton
provides functional organization to the plasma membrane and contributes
to diverse cellular processes including cell signaling, vesicular
trafficking, endocytosis, and migration. For these processes to occur,
membrane protein tethering must be dynamically regulated in response
to environmental cues. In this study, we describe a novel biochemical
scheme for isolating the complement of plasma membrane proteins that
are physically tethered to the actin cytoskeleton. We utilized this
method in combination with tandem liquid chromatography/mass spectrometry
(LC–MS/MS) to demonstrate that cytoskeletal tethering of membrane
proteins is acutely regulated by epidermal growth factor (EGF) in
normal human kidney (HK2) cells. Our results indicate that several
proteins known to be involved in EGF signaling, as well as other proteins
not traditionally associated with this pathway, are tethered to the
cytoskeleton in dynamic fashion. Further analysis of one hit from
our proteomic survey, the receptor phosphotyrosine phosphatase PTPRS,
revealed a correlation between cytoskeletal tethering and endosomal
trafficking in response to EGF. This finding parallels previous indications
that PTPRS is involved in the desensitization of EGFR and provides
a potential mechanism to coordinate localization of these two membrane
proteins in the same compartment upon EGFR activation
Characterization of Differential Protein Tethering at the Plasma Membrane in Response to Epidermal Growth Factor Signaling
Physical tethering of membrane proteins to the cortical
actin cytoskeleton
provides functional organization to the plasma membrane and contributes
to diverse cellular processes including cell signaling, vesicular
trafficking, endocytosis, and migration. For these processes to occur,
membrane protein tethering must be dynamically regulated in response
to environmental cues. In this study, we describe a novel biochemical
scheme for isolating the complement of plasma membrane proteins that
are physically tethered to the actin cytoskeleton. We utilized this
method in combination with tandem liquid chromatography/mass spectrometry
(LC–MS/MS) to demonstrate that cytoskeletal tethering of membrane
proteins is acutely regulated by epidermal growth factor (EGF) in
normal human kidney (HK2) cells. Our results indicate that several
proteins known to be involved in EGF signaling, as well as other proteins
not traditionally associated with this pathway, are tethered to the
cytoskeleton in dynamic fashion. Further analysis of one hit from
our proteomic survey, the receptor phosphotyrosine phosphatase PTPRS,
revealed a correlation between cytoskeletal tethering and endosomal
trafficking in response to EGF. This finding parallels previous indications
that PTPRS is involved in the desensitization of EGFR and provides
a potential mechanism to coordinate localization of these two membrane
proteins in the same compartment upon EGFR activation
CRLF1 is required for differentiation-induced resistance to 6-OHDA independent of the CNTF receptor.
<p>A, Relative expression of CRLF1 was determined by quantitative RT-PCR in in stably selected SH-SH5Y cell lines containing control (non-targeting shRNA, NT-sh) and CRLF1-targeted shRNAs. Expression was normalized relative to basal expression in undifferentiated control cells. Error bars indicate standard deviation in replicate (n = 3) samples. <b>B</b>, Survival of the indicated stable shRNA cell lines in response to increasing doses of 6-OHDA. Dose-response curves were generated to identify the LD50 (gray dashed line) of each cellular state. LD50 values ± SE are indicated in the table below the graph along with significance scores (<i>p</i>-value) comparing CRLF1-shRNA expressing lines to NT-sh. <b>C</b>, Different concentrations of 6-OHDA diluted in conditioned media harvested from RA/TPA differentiated cells of each of the indicated stable shRNA lines were used to treated naïve SH-SY5Y cells. Dose-response curves were generated to identify the LD50 (gray dashed line) of cells treated with each media condition. LD50 values ± SE are indicated in the table below the graph along with <i>p</i>-values comparing CRLF1-shRNA expressing lines to NT-sh. <b>D</b>, Immunoblot analysis of SH-SY5Y cells treated for 15 minutes with 5 ng/mL CLC/CLF ± the indicated pre-treatment (1 hour) dose of 6-OHDA indicates that STAT3 is phosphorylated in response to the ligand independent of 6-OHDA concentration. Total STAT3 and tubulin were used as controls to demonstrate equal protein loading. <b>E</b>, Survival of the indicated stable shRNA cell lines in response to increasing doses of 6-OHDA in media ±10 ng/mL recombinant CLC/CLF ligand. LD50 values ± SE are indicated in the table below the graph along with <i>p</i>-values comparing CLC/CLF-treated cells to untreated NT-sh cells. <b>F</b>, The indicated SH-SY5Y stable shRNA lines were plated at equal densities and differentiated over six days in NBA media ±10 ng/mL recombinant CLC/CLF. Error bars indicate standard deviation in replicate (n = 6) samples.</p
Cytokine Receptor-Like Factor 1 (<i>CRLF1</i>) Protects against 6-Hydroxydopamine Toxicity Independent of the gp130/JAK Signaling Pathway
<div><p>Oxidative stress is an important cause of cellular toxicity in the central nervous system and contributes to the pathology associated with neurodegenerative disorders including Parkinson’s disease. As such, elucidation of cellular mechanisms that enhance neuronal resistance to oxidative stress may provide new avenues for therapy. In this study we employed a simple two-state cellular model to identify genes that are associated with resistance to oxidative stress induced by 6-hydroxydopamine (6-OHDA). In this model, undifferentiated neuroblastoma cells display higher sensitivity to 6-OHDA than differentiated cells. By comparing the gene expression between these two states, we identified several genes whose expression is altered concomitant with changes in 6-OHDA sensitivity. This gene set includes cytokine receptor-like factor 1 (<i>CRLF1</i>), which is up-regulated during the differentiation process and has been previously implicated in neuroprotection. We show that the product of this gene is both necessary and sufficient for increased resistance to 6-OHDA in differentiated neuroblastoma cells, and that CRLF1 serves its protective role by a cell autonomous mechanism that is independent from its known role as a co-ligand for the ciliary neurotrophic factor receptor. These data provide an additional role for CRLF1 that could potentially explain its broad expression pattern and effects on cells lacking expression of this receptor.</p></div
Changes in gene expression in response to RA/TPA-induced differentiation.
<p><b>A</b>, Global gene expression in undifferentiated and differentiated SH-SY5Y and SK-N-SH cells was determined using Agilent 44 K gene expression arrays. Changes in gene expression were determined by subtracting the normalized log fold expression value of undifferentiated cells from that of differentiated cells for each line. Positive values indicate genes whose expression increase (blue), while negative values indicate genes whose expression decrease (red) in response to differentiation. The change in gene expression in SH-SY5Y cells was plotted against that of SK-N-SH cells to identify genes that were coordinately altered in both lines. <b>B</b>, Heat map illustration of genes whose expression were either significantly increased or decreased (≥1.5 fold) upon differentiation. Scale corresponds to log fold change in gene expression relative to expression control. <b>C</b>, Coordinate changes in gene expression in SH-SY5Y and SK-N-SH cells identified by expression array were validated by quantitative RT-PCR for the indicated genes. Error bars indicate standard deviation in replicate wells (n = 3).</p
Ectopic expression of CRLF1 protects differentiated SH-SY5Y cells from 6-OHDA toxicity independent of gp130 signaling.
<p><b>A</b>, Full-length CRLF1 (FL) and a 34 amino acid N-terminal deletion mutant (ΔN) were stably expressed from lentiviral vectors in SH-SY5Y cells or transiently expressed in 293FT cells. Expression of each protein was established by immunoblot analysis of cell lysates and conditioned media. The Δ34N mutant also contains a V5 epitope upstream of the CRLF1 sequence. <b>B</b>, Expression of CRLF1 and CLCF1 in conditioned media harvested from stable SH-SY5Y lines was determined by direct ELISA. Endogenous CRLF1 was below the limit of detection (n.d.) in vector and n-terminal truncation mutant (Δ34N) cell lines. <b>C,D</b>, Proteins precipitated from conditioned media of the indicated stable cell lines were separated by non-reducing (C) or reducing (D) SDS-PAGE, and then analyzed for CRLF1 or CLCF1 expression by immunoblot. <b>E</b>, The effect of stably expressing CRLF1-FL or CRLF1-ΔN on SH-SY5Y differentiation-induced cell cycle arrest was determined by analyzing the relative cell number of each line after six days culture in NBA media containing 10% FBS or 10 µM RA, or after three days in RA followed by three days in 100 nM TPA. Relative cell number was analyzed by cellular DNA content and normalized to undifferentiated conditions. Error bars indicate standard deviation in replicate (n = 6) samples. <b>F</b>, Survival of undifferentiated stable cell lines in response to increasing doses of 6-OHDA. Relative cell number was normalized to untreated cells and plotted according to 6-OHDA concentration. Dose-response curves were generated to identify the LD50 (gray dashed line) of each cellular state. LD50 values ± SE are indicated in the table below the graph along with significance scores (<i>p</i>-value) transgenic cells to vector controls. <b>G</b>, Survival of RA/TPA differentiated stable isogenic cell lines in response to increasing doses of 6-OHDA was determined as in F. <b>H</b>, Survival of RA/TPA differentiated stable isogenic cell lines in response to increasing doses of 6-OHDA was determined in the presence of gp130 neutralizing (α-gp130) or control (IgG) antibodies. Data was analyzed as in F.</p
Inhibition of gp130/JAK signaling fails to affect the response of SH-SY5Y cells to 6-OHDA treatment.
<p><b>A</b>, CLCF1/CRLF1 heterodimers are ligands for the ciliary neurotrophic factor receptor (CNTFR) complex, which includes the co-receptor gp130. This complex activates JAK family tyrosine kinases, prominently JAK2, which subsequently phosphorylate an array of targets including STAT family proteins STAT1 and STAT3. STAT proteins are dimeric transcription factors that translocate to the nucleus and regulate gene expression upon phosphorylation. <b>B</b>, Immunoblot analysis demonstrates that inhibition of the CNTFR/JAK/STAT signaling pathway with gp130 neutralizing antibodies (α-gp130) or the JAK1/2 specific inhibitor ruxolitinib (1 µM) blocks activation of STAT1 and STAT3 in both undifferentiated and RA/TPA differentiated SH-SY5Y cells compared to untreated control (con) cells of the same condition. Note that these inhibitors are highly specific for this pathway, and fail to impact growth/survival signaling by other key pathways including the MAPK (pERK), PI(3)K/AKT (pAKT) and mTOR (pS6) pathways. Control blots tyrosine hydroxylase (TH) are included to demonstrate differentiation, whereas blots for ERK1/2, AKT, S6 and tubulin are included to demonstrate equal protein loading. <b>C</b>, Undifferentiated SH-SY5Y cells cultured in media containing 10% FBS and α-gp130 (1 µg/mL), ruxolitinib (1 µM) or vehicle control (1 µg/mL non-specific mouse IgG or 0.1% DMSO) were concomitantly treated with different doses of 6-OHDA for 24 hours. Relative cell number was normalized to untreated cells and plotted according to 6-OHDA concentration. Dose-response curves were generated to identify the LD50 (gray dashed line) of each cellular state. LD50 values ± SE are indicated in the table below the graph along with significance scores (<i>p</i>-value) comparing antibody or drug-treated cells to controls. <b>D</b>, RA/TPA differentiated SH-SY5Y cells were treated with pathways inhibitors and 6-OHDA, and cell survival was analyzed as in C. LD50 values ± SE are indicated in the table below the graph along with <i>p</i>-values comparing antibody or drug-treated cells to controls. Note that neither inhibitor has a significant effect on the survival of cells in response to 6-OHDA.</p
Differentiation of neuroblastoma cells promotes resistance to 6-OHDA toxicity.
<p><b>A</b>, SH-SY5Y cells were plated and allowed to adhere overnight, then differentiated with 10 µM retinoic acid only (six days) or with retinoic acid and 100 nM TPA (three days each). Cells were subsequently treated for 24 hours with different doses of 6-OHDA diluted in serum-free NBA/B27 media and analyzed for cellular viability. Relative cell number was normalized to untreated cells and plotted according to 6-OHDA concentration. Dose-response curves were generated to identify the LD50 (gray dashed line) of each cellular state. LD50 values ± SE are indicated in the table below the graph along with significance scores (<i>p</i>-value) comparing treated samples to untreated controls. <b>B</b>, SK-N-SH cells were treated and assayed as in A. LD50 values ± SE are indicated in the table below the graph along with <i>p</i>-values comparing treated samples to untreated controls. <b>C</b>, SH-SY5Y cells were plated and allowed to adhere overnight. They were subsequently treated with the indicated doses of 6-OHDA diluted either in fresh NBA media (alone or containing 10% FBS, 10 µM RA or 100 nM TPA), or in conditioned media harvested from SH-SY5Y cells that had been cultured in 10% FBS or differentiation media (RA only, TPA only or RA/TPA) for six days. After 24 hours the cells were analyzed for viability and relative cell number was determined relative to untreated controls. <b>D</b>, SK-N-SH cells were treated and assayed as in C. <b>E</b>, LD50 values ± SE for naïve SH-SY5Y and SK-N-SH cells cultured in fresh or conditioned media from each treatment paradigm. For all experiments, cells were treated and assayed as in C. Significance scores (<i>p</i>-value) are shown for comparisons between fresh and conditioned media (CM) for each condition. <b>F</b>, The ratio of LD50 values in conditioned versus fresh media for each media condition was calculated to determine whether media from conditioned cells contains factors that protect naïve/undifferentiated cells from 6-OHDA toxicity. Values >1 indicate a survival advantage in conditioned media, whereas values ≤1 indicate a lack of survival advantage or disadvantage in conditioned media.</p
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