Cyclic AMP-Dependent Protein Kinase A Regulates the Alternative Splicing of CaMKIIδ

Ca2+/calmodulin-dependent protein kinase (CaMK) IIδ is predominantly expressed in the heart. There are three isoforms of CaMKIIδ resulting from the alternative splicing of exons 14, 15, and 16 of its pre-mRNA, which is regulated by the splicing factor SF2/ASF. Inclusion of exons 15 and 16 or of exon 14 generates δA or δB isoform. The exclusion of all three exons gives rise to δC isoform, which is selectively increased in pressure-overload-induced hypertrophy. Overexpression of either δB or δC induces hypertrophy and heart failure, suggesting their specific role in the pathogenesis of hypertrophy and heart failure. It is well known that the β-adrenergic-cyclic AMP-dependent protein kinase A (PKA) pathway is implicated in heart failure. To determine the role of PKA in the alternative splicing of CaMKIIδ, we constructed mini-CaMKIIδ genes and used these genes to investigate the regulation of the alternative splicing of CaMKIIδ by PKA in cultured cells. We found that PKA promoted the exclusion of exons 14, 15, and 16 of CaMKIIδ, resulting in an increase in δC isoform. PKA interacted with and phosphorylated SF2/ASF, and enhanced SF2/ASF's activity to promote the exclusion of exons 14, 15, and 16 of CaMKIIδ, leading to a further increase in the expression of δC isoform. These findings suggest that abnormality in β-adrenergic-PKA signaling may contribute to cardiomyopathy and heart failure through dysregulation in the alternative splicing of CaMKIIδ exons 14, 15, and 16 and up-regulation of CaMKIIδC

Extremal Bipartite Graphs with Given Parameters on the Resistance–Harary Index

Resistance distance is a concept developed from electronic networks. The calculation of resistance distance in various circuits has attracted the attention of many engineers. This report considers the resistance-based graph invariant, the Resistance−Harary index, which represents the sum of the reciprocal resistances of any vertex pair in the figure G, denoted by R H ( G ) . Vertex bipartiteness in a graph G is the minimum number of vertices removed that makes the graph G become a bipartite graph. In this study, we give the upper bound and lower bound of the R H index, and describe the corresponding extremal graphs in the bipartite graph of a given order. We also describe the graphs with maximum R H index in terms of graph parameters such as vertex bipartiteness, cut edges, and matching numbers

Ethanol extract of propolis and its constituent caffeic acid phenethyl ester inhibit breast cancer cells proliferation in inflammatory microenvironment by inhibiting TLR4 signal pathway and inducing apoptosis and autophagy

Abstract Background Propolis and its major constituent – caffeic acid phenethyl ester (CAPE) have good abilities on antitumor and anti-inflammation. However, little is known about the actions of propolis and CAPE on tumor in inflammatory microenvironment, and inflammatory responses play decisive roles at different stages of tumor development. To understand the effects and mechanisms of ethanol-extracted Chinese propolis (EECP) and its major constituent - CAPE in inflammation-stimulated tumor, we investigated their effects on Toll-like receptor 4 (TLR4) signaling pathway which plays a crucial role in breast cancer MDA-MB-231 cell line. Methods 80% confluent breast cancer MDA-MB-231 cells were stimulated with 1 μg/mL lipopolysaccaride (LPS). Then the cells were divided for treatment by CAPE (25 μg/mL) and EECP (25, 50 and 100 μg/mL), respectively. Cell viability, nitric oxide (NO) production and cell migration were measured by sulforhodamine B assay, chemical method and scratch assay. The levels of TLR4, MyD88, IRAK4, TRIF, caspase 3, PARP, LC3B and p62 were investigated through western blotting. The expression of TLR4, LC3B and nuclear factor-κB p65 (NF-κB p65) were tested by immunofluorescence microscopy assay. Results Treatment of different concentrations of EECP (25, 50 and 100 μg/mL) and CAPE (25 μg/mL) significantly inhibited LPS-stimulated MDA-MB-231 cell line proliferation, migration and NO production. Furthermore, EECP and CAPE activated caspase3 and PARP to induce cell apoptosis, and also upregulated LC3-II and decreased p62 level to induce autophagy during the process. TLR4 signaling pathway molecules such as TLR4, MyD88, IRAK4, TRIF and NF-κB p65 were all down-regulated after EECP and CAPE treatment in LPS-stimulated MDA-MB-231 cells. Conclusions These findings indicated that EECP and its major constituent - CAPE inhibited breast cancer MDA-MB-231 cells proliferation in inflammatory microenvironment through activating apoptosis, autophagy and inhibiting TLR4 signaling pathway. EECP and CAPE may hold promising prospects in treating inflammation-induced tumor

Augmentation of EPR Effect and Efficacy of Anticancer Nanomedicine by Carbon Monoxide Generating Agents

One obstacle to the successful delivery of nanodrugs into solid tumors is the heterogeneity of an enhanced permeability and retention (EPR) effect as a result of occluded or embolized tumor blood vessels. Therefore, the augmentation of the EPR effect is critical for satisfactory anticancer nanomedicine. In this study, we focused on one vascular mediator involved in the EPR effect, carbon monoxide (CO), and utilized two CO generating agents, one is an extrinsic CO donor (SMA/CORM2 micelle) and another is an inducer of endogenous CO generation via heme oxygenase-1 (HO-1) induction that is carried out using pegylated hemin. Both agents generated CO selectively in solid tumors, which resulted in an enhanced EPR effect and a two- to three-folds increased tumor accumulation of nanodrugs. An increase in drug accumulation in the normal tissue did not occur with the treatment of CO generators. In vivo imaging also clearly indicated a more intensified fluorescence of macromolecular nanoprobe in solid tumors when combined with these CO generators. Consequently, the combination of CO generators with anticancer nanodrugs resulted in an increased anticancer effect in the different transplanted solid tumor models. These findings strongly warrant the potential application of these CO generators as EPR enhancers in order to enhance tumor detection and therapy using nanodrugs

Alternative splicing of CaMKIIδ exons 14, 15, and 16 generates three splicing variants, corresponding to CaMKIIδ isoforms A, B, and C, respectively.

<p>A and B, Schematic diagram of the alternative splicing of exons 14, 15, and 16 of mini -CaMKIIδ-genes, pCI/CaMKIIδ_E12–E17 (A) and pCI/CaMKIIδ_E13–E17 (B). C and D, Three splicing variants was generated from mini-CaMKIIδ gene, pCI/CaMKIIδ_E12–E17 (C) or pCI/CaMKIIδ_E13–E17 (D), after transfection into HEK-293T or COS7 cells, respectively, for 48 hrs. The total RNA was used for measurement of the splicing products with RT-PCR.</p

PKA phosphorylates and interacts with SF2/ASF.

<p>A, Recombinant GST-SF2/ASF or GST was incubated with PKA in the presence of [γ-³²P]ATP at 30°C for 10 min, and the reaction mixture was then separated by SDS-PAGE and visualized with Coommassie blue staining (lower panel) or autoradiograph (upper panel). B, PKA-Cα was pull-down by SF2/ASF. GST-SF2/ASF or GST coupled onto glutathione-Sepharose or glutathione-Sepharose (GSH-beads) was incubated with rat brain extract. After extensively washing, the bound proteins were analyzed by Western blots developed with anti-GST or anti-PKA-Cα. C, PKA-Cα was co-immunoprecipitated by anti-HA. SF2/ASF tagged with HA were expressed in HEK-293FT cells for 48 h. The cell extracts were immunoprecipitated with anti-HA, and the immunoprecipitates were subjected to Western blots developed with anti-HA and anti-PKA-Cα. D, HeLa cells were transfected with pCEP4/SF2/ASF and treated without (Con) or with forskolin (Fors) for 30 min, followed by triple immunofluorescence staining.</p

PKA activation enhances SF2/ASF-promoted exclusion of exons 14, 15, and 16 of CaMKIIδ.

<p>A, HEK-293FT cells were transfected with pCI/CaMKIIδ_E13–E17 for 40 hrs and then treated with 10 µM forskolin or 20 µM isoproterenol for 8 hrs. The splicing products were measured with RT-PCR. Each splicing product was quantitated by densitometry and the percentage of each splicing form was calculated. The Data are presented as mean ± S.D. *<i>p</i><0.05 versus control treatment. B, Proposed mechanism by which abnormalities of β-adrenergic-PKA-pathway dysregulates the alternative splicing of exons 14, 15, and 16 of CaMKIIδ via SF2/ASF.</p