The Expression of the PDZ Protein MALS-1/Velis Is Regulated by Calcium and Calcineurin in Cerebellar Granule Cells

Activity-dependent gene expression is thought to be important in shaping neuronal development and in modifying the protein content of neurons. Ca(2+) entry into neurons appears to be one of the key effectors of activity-dependent gene expression. Among the possible downstream targets of calcium, the protein phosphatase calcineurin represents a prime candidate. We hereby report that in cultured cerebellar granule cells the activation of the Ca(2+)/calcineurin pathway via either voltage- or ligand- operated Ca(2+) channels regulates MALS-1 and MALS-2 expression at the transcriptional level. These proteins are integral parts of the post-synaptic density and are also involved in receptor trafficking. MALS regulation is not at the level of mRNA stability and does not require de novo protein synthesis, thereby suggesting a direct pathway. These data suggest that Ca(2+) entry by means of calcineurin is capable of controlling the structure of the post-synaptic density by controlling the expression of key components at the transcriptional level

Calcineurin Controls the Expression of Isoform 4CII of the Plasma Membrane Ca2+ Pump in Neurons *

Abstract The expression of the CII splice variant of the plasma membrane Ca2+ ATPase 4 (PMCA4) was down-regulated in granule neurons when they were cultured under conditions of partial membrane depolarization (25 mm KCl), which are required for long term in vitro survival of the neurons. These conditions, which cause a chronic increase of the resting free Ca2+ concentration in the neurons, have recently been shown to promote up-regulation of the PMCA2, 3, and 1CII isoforms. Whereas the chronic, i.e. >3 days, Ca2+ increase was necessary for the up-regulation of the PMCA1CII, 2, and 3, the down-regulation of the PMCA4CII mRNA was already evident 1–2 h after the start of culturing in 25 mm KCl. The immunosuppressant calcineurin inhibitor FK506 inhibited the down-regulation of the PMCA4CII at both the protein and the mRNA level but did not affect the changes of the other PMCA pumps. Direct evidence for the involvement of calcineurin in the down-regulation of the PMCA4CII was obtained by overexpressing a truncated, constitutively active, and Ca2+-independent form of calcineurin; under these conditions, depolarization was not required for the down-regulation of the PMCA4CII pump. De novosynthesis of (transcription) factors was required for the down-regulation of the PMCA4CII mRNA. Calcineurin, therefore, controls the neuronal transcription of PMCA4CII, a splice variant of the pump isoforms that is found almost exclusively in brain

NAADP receptors are present and functional in the heart

AbstractAlongside the well-studied inositol 1,4,5 trisphosphate and ryanodine receptors, evidence is gathering that a new intracellular release mechanism, gated by the pyridine nucleotide nicotinic acid adenine dinucleotide phosphate (NAADP), is present in numerous organisms, ranging from plant to mammalian cells (reviewed in [1]). Most cells have been shown to express at least two Ca2+-release mechanisms controlled by different messengers, and this can lead to redundancy, convergence, or divergence of responses. One exception appears to be muscle and heart contractile tissues. Here, it is thought that the dominant intracellular channel is the ryanodine receptor, while IP3 receptors are poorly expressed and their role appears to be negligible. We now report that NAADP receptors are functional and abundant in cardiac microsomes. NAADP binds specifically and with high affinity (130 pM and 4 nM) to two sites on cardiac microsomes and releases Ca2+ with an apparent EC50 of 323 ± 14 nM. Furthermore, binding experiments show that this receptor displays both positive and negative cooperativity, a peculiarity unique among intracellular Ca2+ channels. Therefore, we show that the heart possesses multiple mechanisms to increase the complexity of Ca2+ signaling and that NAADP may be integral in the functioning of this organ

Nicotinic Acid Adenine Dinucleotide Phosphate-induced Ca2+ Release INTERACTIONS AMONG DISTINCT Ca2+ MOBILIZING MECHANISMS IN STARFISH OOCYTES

An intracellular mechanism activated by nicotinic acid adenine dinucleotide phosphate (NAADP(+)) contributes to intracellular Ca(2+) release alongside inositol 1,4,5-trisphosphate (Ins-P(3)) and ryanodine receptors. The NAADP(+)-sensitive mechanism has been shown to be operative in sea urchin eggs, ascidian eggs, and pancreatic acinar cells. Furthermore, most mammalian cell types can synthesize NAADP(+), with nicotinic acid and NADP(+) as precursors. In this contribution, NAADP(+)-induced Ca(2+) release has been investigated in starfish oocytes. Uncaging of injected NAADP(+) induced Ca(2+) mobilization in both immature oocytes and in oocytes matured by the hormone 1-methyladenine (1-MA). The role of extracellular Ca(2+) in NAADP(+)-induced Ca(2+) mobilization, which was minor in immature oocytes, was instead essential in mature oocytes. Thus, the NAADP(+)-sensitive Ca(2+) pool, which is known to be distinct from those sensitive to inositol 1,4,5-trisphosphate or cyclic ADPribose, apparently migrated closer to (or became part of) the plasma membrane during the maturation process. Inhibition of both Ins-P(3) and ryanodine receptors, but not of either alone, substantially inhibited NAADP(+)-induced Ca(2+) mobilization in both immature and mature oocytes. The data also suggest that NAADP(+)-induced Ca(2+) mobilization acted as a trigger for Ca(2+) release via Ins-P(3) and ryanodine receptors

N-arylbenzamides: extremely simple scaffolds for the development of novel estrogen receptor agonists.

The research of estrogen receptor (ER) ligands has benefited in the last decade from the implementation of combinatorial chemistry. The general pharmacophore has been identified and subsequently a multitude of compounds have been synthesized. Surprisingly, up to now simple amides have not been taken into consideration. Here we show that amides resulting from the condensation of hydroxybenzoic acids with aminophenols result in compounds retaining the pharmacophore structure of an ER ligand with a clear estrogenic activity

Celecoxib inhibits proliferation and survival of chronic myelogeous leukemia (CML) cells via AMPK-dependent regulation of β-catenin and mTORC1/2.

CML is effectively treated with tyrosine kinase inhibitors (TKIs). However, the efficacy of these drugs is confined to the chronic phase of the disease and development of resistance to TKIs remains a pressing issue. The anti-inflammatory COX2 inhibitor celecoxib has been utilized as anti-tumour drug due to its anti-proliferative activity. However, its effects in hematological malignancies, in particular CML, have not been investigated yet. Thus, we tested biological effects and mechanisms of action of celecoxib in Philadelphia-positive (Ph+) CML and ALL cells.We show here that celecoxib suppresses the growth of Ph+ cell lines by increasing G1-phase and apoptotic cells and reducing S- and G2-phase cells. These effects were independent of COX2 inhibition but required the rapid activation of AMP-activated protein kinase (AMPK) and the consequent inhibition mTORC1 and 2. Treatment with celecoxib also restored GSK3β function and led to down-regulation of β-catenin activity through transcriptional and post-translational mechanisms, two effects likely to contribute to Ph+ cell growth suppression by celecoxib.Celecoxib inhibited colony formation of TKI-resistant Ph+ cell lines including those with the T315I BCR-ABL mutation and acted synergistically with imatinib in suppressing colony formation of TKI-sensitive Ph+ cell lines. Finally, it suppressed colony formation of CD34+ cells from CML patients, while sparing most CD34+ progenitors from healthy donors, and induced apoptosis of primary Ph+ ALL cells.Together, these findings indicate that celecoxib may serve as a COX2-independent lead compound to simultaneously target the mTOR and β-catenin pathways, key players in the resistance of CML stem cells to TKIs

The Cytokine Nicotinamide Phosphoribosyltransferase (eNAMPT; PBEF; Visfatin) Acts as a Natural Antagonist of C-C Chemokine Receptor Type 5 (CCR5)

(1) Background: Extracellular nicotinamide phosphoribosyltrasferase (eNAMPT) is released by various cell types with pro-tumoral and pro-inflammatory properties. In cancer, eNAMPT regulates tumor growth through the activation of intracellular pathways, suggesting that it acts through a putative receptor, although its nature is still elusive. It has been shown, using surface plasma resonance, that eNAMPT binds to the C-C chemokine receptor type 5 (CCR5), although the physiological meaning of this finding is unknown. The aim of the present work was to characterize the pharmacodynamics of eNAMPT on CCR5. (2) Methods: HeLa CCR5-overexpressing stable cell line and B16 melanoma cells were used. We focused on some phenotypic effects of CCR5 activation, such as calcium release and migration, to evaluate eNAMPT actions on this receptor. (3) Results: eNAMPT did not induce ERK activation or cytosolic Ca2+-rises alone. Furthermore, eNAMPT prevents CCR5 internalization mediated by Rantes. eNAMPT pretreatment inhibits CCR5-mediated PKC activation and Rantes-dependent calcium signaling. The effect of eNAMPT on CCR5 was specific, as the responses to ATP and carbachol were unaffected. This was strengthened by the observation that eNAMPT inhibited Rantes-induced Ca2+-rises and Rantes-induced migration in a melanoma cell line. (4) Conclusions: Our work shows that eNAMPT binds to CCR5 and acts as a natural antagonist of this receptor

Tempi di accesso ai farmaci in Italia nel periodo 2015-2017: analisi delle tempistiche di valutazione dell'Agenzia Italiana del Farmaco

Objectives: To describe the length of time taken by the Italian Medicines Agency (AIFA) to formulate pricing and reimbursement decisions for drugs approved via centralized procedures and to evaluate possible differences between categories of drugs (innovative drugs, oncological and rare disease drugs, products negotiated with Managed Entry Agreements and/or managed via registry). Methods: Monthly meeting reports of the Technical Scientific Committee (CTS) and the Price and Reimbursement Committee (CPR) as well European and Italian Official Journals were scrutinized from January 2015 to January 2018. Results: In the scrutinized period, 85 out of 190 drugs obtained reimbursement and were included in the analysis. Overall, time-to-market (CTS assessment opening to Official Journal) was 258 days. About one third of the whole procedure was dedicated to administrative steps, without significant differences between drug categories. Conclusions: Our analysis shows that transparency makes it possible to determine the length of time taken by AIFA to conclude the Price and Reimbursement (P&R) process. It also suggests that administrative procedures may still be optimized to allow faster access to treatments for patients and provides a starting point to determine future policies in this area