A Kinesin Driven Enzyme Linked Immunosorbant Assay (ELISA) for Ultra Low Protein Detection Applications

<p>Gene Ontology bar charts for three different criteria: biological process (A), cell localization (B) and molecular function (C). The list of differentially expressed genes after RSV diet in neocortex were classified by Gene Ontology (GO) and drawn using Panther (<a href="https://www.pantherdb.org/" target="_blank">www.pantherdb.org/</a>)</p

Image_2_Neuronal Calcium and cAMP Cross-Talk Mediated by Cannabinoid CB1 Receptor and EF-Hand Calcium Sensor Interactions.TIF

<p>Endocannabinoids are important players in neural development and function. They act via receptors, whose activation inhibits cAMP production. The aim of the paper was to look for calcium- and cAMP-signaling cross-talk mediated by cannabinoid CB₁ receptors (CB₁R) and to assess the relevance of EF-hand CaM-like calcium sensors in this regard. Using a heterologous expression system, we demonstrated that CB₁R interacts with calneuron-1 and NCS1 but not with caldendrin. Furthermore, interaction motives were identified in both calcium binding proteins and the receptor, and we showed that the first two sensors competed for binding to the receptor in a Ca²⁺-dependent manner. Assays in neuronal primary cultures showed that, CB₁R-NCS1 complexes predominate at basal Ca²⁺ levels, whereas in the presence of ionomycin, a calcium ionophore, CB₁R-calneuron-1 complexes were more abundant. Signaling assays following forskolin-induced intracellular cAMP levels showed in mouse striatal neurons that binding of CB₁R to NCS1 is required for CB₁R-mediated signaling, while the binding of CB₁R to calneuron-1 completely blocked G_i-mediated signaling in response to a selective receptor agonist, arachidonyl-2-chloroethylamide. Calcium levels and interaction with calcium sensors may even lead to apparent Gs coupling after CB₁R agonist challenge.</p

Induction of CHOP and ASNS expression in SHSY-5Y tunicamycin ER stressed cells is reversed by PBA or PVA treatment.

<p>Cells were treated for 24 h with tunicamycin and with PBA or PVA for the times indicated. The expression of CHOP and ASNS was determined by RT-PCR and normalized to that of the corresponding 36b4 internal control. Bars represent the mean ± SEM of the relative change with respect to tunicamycin treated cells: **p<0.005 <i>vs</i> tunicamycin treated cells.</p

Primer sequences used for quantitative PCR.

<p>Primer sequences used for quantitative PCR.</p

Scheme showing the three major ER-stress sensors: PERK, ATF6 and IRE1 and the link to the targets assayed in this report.

<p>Activated PERK phosphorylates eIF2α to attenuate protein translation but allowing the expression of ATF4-dependent genes, CHOP and ASNS, involved in redox- and apoptosis-related pathways. Cleaved -active- ATF6 leads to induction of molecular chaperones (GPR78) and of the transcription factor XBP1. IRE1 activation leads to XBP1 splicing, transcriptional activation of chaperones and stimulation of protein degradation through ErdJ4, which is a component of the ER-associated degradation (ERAD) system.</p

PBA and PVA neutralize the ER stress sensors induced by tunicamycin.

<p>SH-SY5Y cells were treated as described in the Materials and Methods and the expression of GPR78 (A), spliced XBP1 (B) and ERdJ4 (C) was determined by RT-PCR. The bars represent the expression (mean ± SEM) normalized to that of the corresponding 36b4 internal control: *p<0.05; **p<0.005 <i>vs</i> tunicamycin treated cells.</p

Image_3_Neuronal Calcium and cAMP Cross-Talk Mediated by Cannabinoid CB1 Receptor and EF-Hand Calcium Sensor Interactions.TIFF

<p>Endocannabinoids are important players in neural development and function. They act via receptors, whose activation inhibits cAMP production. The aim of the paper was to look for calcium- and cAMP-signaling cross-talk mediated by cannabinoid CB₁ receptors (CB₁R) and to assess the relevance of EF-hand CaM-like calcium sensors in this regard. Using a heterologous expression system, we demonstrated that CB₁R interacts with calneuron-1 and NCS1 but not with caldendrin. Furthermore, interaction motives were identified in both calcium binding proteins and the receptor, and we showed that the first two sensors competed for binding to the receptor in a Ca²⁺-dependent manner. Assays in neuronal primary cultures showed that, CB₁R-NCS1 complexes predominate at basal Ca²⁺ levels, whereas in the presence of ionomycin, a calcium ionophore, CB₁R-calneuron-1 complexes were more abundant. Signaling assays following forskolin-induced intracellular cAMP levels showed in mouse striatal neurons that binding of CB₁R to NCS1 is required for CB₁R-mediated signaling, while the binding of CB₁R to calneuron-1 completely blocked G_i-mediated signaling in response to a selective receptor agonist, arachidonyl-2-chloroethylamide. Calcium levels and interaction with calcium sensors may even lead to apparent Gs coupling after CB₁R agonist challenge.</p

PBA and PVA protect SH-SY5Y cells against tunicamycin induced ER stress.

<p>SH-SY5Y cells were exposed to tunicamycin (500 nm) for 24 h in the absence (medium) or in the presence of PBA (panel A) or PVA (panel B). Cell viability was determined using a LDH release assay and the results are expressed as the means ± SEM. Bars represent the percentage of LDH release over that obtained in untreated cells. **p<0.005; ***p<0.0001 <i>vs</i> tunicamycin treated cells (Medium). Panel C. Immunoblot of procaspase 3 and cleaved caspase 3. A representative image is shown and the bar diagram represents the ratio of cleaved versus total protein (mean ± SEM) in the different conditions and normalizad to the ratio in absence of tunicamycin. ^##p<0.005 <i>vs</i> untreated cells; **p<0.005 <i>vs</i> tunicamycin treated cells.</p

Tunicamycin-induced ER stress in SH-SY5Y cells.

<p>A) SH-SY5Y cells were treated for 48 h with the indicated concentrations of tunicamycin and cell viability was determined using a LDH release assay. Bars represent the percentage of LDH release over that obtained in untreated cells: *p<0.05; ***p<0.0001 <i>vs</i> untreated cells. B–C) The transcriptional activity of different sensors was used to monitor the induction of ER stress by tunicamycin in SHSY-5Y (B) and HEK-293T (C) cells. Bars represent the fold change (mean ± SEM) in gene expression normalized to the control untreated cells: *p<0.05; **p<0.005; <i>vs</i> control untreated cells.</p

Number of differentially expressed genes detected by GeneSpring depending on p-value and absolute fold change (FC) values.

<p>Analysis of data, from control- and RSV-enriched diet both in quintuplicates, were performed using moderated T-test, the multiple testing correction of Benjamini-Hochberg and asymptotic p-value (adjusted) computation.</p