Effects of Anionic Liposome Delivery of All–Trans–Retinoic Acid on Neuroblastoma Cell Differentiation

All–trans–retinoic acid (ATRA) has long been known to affect cell growth and differentiation. To improve ATRA’s therapeutic efficacy and pharmacodynamics, several delivery systems have been used. In this study, free ATRA and anionic–liposome–encapsulated ATRA were compared for their effects on SK–N–SH human neuroblastoma cell growth and differentiation. Anionic liposomes made of L–α –phosphatidylcholine (PC) and L–α –phosphatidic acid (PA), empty (PC–PA) and loaded with ATRA (PC–PA–ATRA), were characterized by dynamic light scattering (DLS) and electrophoretic mobility measurements, and drug entrapment efficiency (EE%) was measured to evaluate the applicability of the new colloidal formulation. The results of brightfield microscopy and cell growth curves indicated that ATRA, whether free or encapsulated, reduced growth and induced differentiation, resulting in SK–N–SH cells changing from epithelioid to neuronal–like morphologies, and producing a significant increase in neurite growth. To further characterize the neuro-differentiation of SK–N–SH cells, the expression of βIII–Tubulin and synaptophysin and mitochondria localization were analyzed via immunofluorescence. Increased expression of neuronal markers and a peculiar localization of mitochondria in the neuritic extensions were apparent both in ATRA– and PC–PA–ATRA–differentiated cells. As a whole, our results strongly indicate that ATRA treatment, by any means, can induce the differentiation of parent SK–N–SH, and they highlight that its encapsulation in anionic liposomes increases its differentiation ability in terms of the percentage of neurite–bearing cells. Interestingly, our data also suggest an unexpected differentiation capability of anionic liposomes per se. This work highlights the importance of developing and carefully testing novel delivery nanocarriers, which are a necessary first “step” in the development of new therapeutic settings

Insights into Mechanical Behavior and Biological Properties of Chia Seed Mucilage Hydrogels

In this contribution we report insights on the rheological properties of chia (Salvia hispanica) seed mucilage hydrogels. Specifically, we studied the influence of temperature and polymer concentration on the viscoelastic properties of resulting networks. Creep experiments performed in steady-state conditions allowed calculating Newtonian viscosities for chia hydrogels at different polymer concentrations, pointing at inter-chain interactions as the main responsible for the different behavior toward network slipping under constant stress. The combination of oscillatory frequency and stress sweep tests highlighted a moderate effect of temperature in influencing hydrogel mechanics. The latter results prompted us to investigate potential biological functions for this set of biomaterials. Lactate Dehydrogenase assay proved lack of cytotoxicity of chia suspensions toward Human Mesenchymal Stem Cells from adipose tissue here used as cell model. Differentiation experiments were finally undertaken to verify the influence of chia samples on osteo-induction triggered by chemical differentiation factors. Alkaline Phosphatase enzyme activity assay and Alizarin red staining demonstrated that chia mucilage did not alter in vitro stem cell differentiation. Collectively, this set of experiments revealed an almost inert role associated with chia suspensions, indicating a possible application of chia-based networks as scaffold models to study osteogenesis in vitro

Extracellular calcium acts as a “third messenger” to regulate enzyme and alkaline secretion

It is generally assumed that the functional consequences of stimulation with Ca2+-mobilizing agonists are derived exclusively from the second messenger action of intracellular Ca2+, acting on targets inside the cells. However, during Ca2+ signaling events, Ca2+ moves in and out of the cell, causing changes not only in intracellular Ca2+, but also in local extracellular Ca2+. The fact that numerous cell types possess an extracellular Ca2+ “sensor” raises the question of whether these dynamic changes in external [Ca2+] may serve some sort of messenger function. We found that in intact gastric mucosa, the changes in extracellular [Ca2+] secondary to carbachol-induced increases in intracellular [Ca2+] were sufficient and necessary to elicit alkaline secretion and pepsinogen secretion, independent of intracellular [Ca2+] changes. These findings suggest that extracellular Ca2+ can act as a “third messenger” via Ca2+ sensor(s) to regulate specific subsets of tissue function previously assumed to be under the direct control of intracellular Ca2+

A Reassessment of the Effects of Luminal [Ca2+] on Inositol 1,4,5-Trisphosphate-induced Ca2+ Release from Internal Stores

Inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ release from intracellular stores displays complex kinetic behavior. While it well established that cytosolic [Ca2+] can modulate release by acting on the InsP3 receptor directly, the role of the filling state of internal Ca2+stores in modulating Ca2+ release remains unclear. Here we have reevaluated this topic using a technique that permits rapid and reversible changes in free [Ca2+] in internal stores of living intact cells without altering cytoplasmic [Ca2+], InsP3 receptors, or sarcoendoplasmic reticulum Ca2+ ATPases (SERCAs). N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylene diamine (TPEN), a membrane-permeant, low affinity Ca2+ chelator was used to manipulate [Ca2+] in intracellular stores, while [Ca2+] changes within the store were monitored directly with the low-affinity Ca2+ indicator, mag-fura-2, in intact BHK-21 cells. 200 microM TPEN caused a rapid drop in luminal free [Ca2+] and significantly reduced the extent of the response to stimulation with 100 nm bradykinin, a calcium-mobilizing agonist. The same effect was observed when intact cells were pretreated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid(acetoxymethyl ester) (BAPTA-AM) to buffer cytoplasmic [Ca2+] changes. Although inhibition of Ca2+ uptake using the SERCA inhibitor tBHQ permitted significantly larger release of Ca2+ from stores, TPEN still attenuated the release in the presence of tBHQ in BAPTA-AM-loaded cells. These results demonstrate that the filling state of stores modulates the magnitude of InsP3-induced Ca2+release by additional mechanism(s) that are independent of regulation by cytoplasmic [Ca2+] or effects on SERCA pumps

Copernicus Ocean State Report, issue 6

The 6th issue of the Copernicus OSR incorporates a large range of topics for the blue, white and green ocean for all European regional seas, and the global ocean over 1993–2020 with a special focus on 2020

The Different Facets of Extracellular Calcium Sensors: Old and New Concepts in Calcium-Sensing Receptor Signalling and Pharmacology

The current interest of the scientific community for research in the field of calcium sensing in general and on the calcium-sensing Receptor (CaR) in particular is demonstrated by the still increasing number of papers published on this topic. The extracellular calcium-sensing receptor is the best-known G-protein-coupled receptor (GPCR) able to sense external Ca2+ changes. Widely recognized as a fundamental player in systemic Ca2+ homeostasis, the CaR is ubiquitously expressed in the human body where it activates multiple signalling pathways. In this review, old and new notions regarding the mechanisms by which extracellular Ca2+ microdomains are created and the tools available to measure them are analyzed. After a survey of the main signalling pathways triggered by the CaR, a special attention is reserved for the emerging concepts regarding CaR function in the heart, CaR trafficking and pharmacology. Finally, an overview on other Ca2+ sensors is provided

Palmitic acid is associated with halorhodopsin as a free fatty acid. Radiolabeling of halorhodopsin with 3H-palmitic acid and chemical analyses of the reaction products of purofied halorhodopsin with thiols and NaBH4

Halorhodopsin, isolated from Halobacterium, salinarium cells incubated with tritiated palmitic acid, co-elutes with labeled palmitate in phenylsepharose CL-4B chromatography. Halorhodopsin-bound H-3-palmitate is not readily displaced by prolonged exposure to a large excess of detergents and by re-chromatography of radiolabeled halorhodopsin on phenylsepharose. On other hand, the association of labeled palmitate with purified halorhodopsin is not resistant to denaturation induced either by isopropanol/hexane or by SDS gel electrophoresis. We have tested the hypothesis that tightly associated palmitate is bound to halorhodopsin through a thioester bond, which is unstable in denaturing conditions. Using GC/MS, we have analysed the reaction products of native halorhodopsin with specific thioester reagents, thiols and NaBH4, which are inactive on free fatty acids. The results of this analytical approach indicate that there is no thioester bond between halorhodopsin and palmitic acid and that palmitic acid is associated with halorhodopsin as a free fatty acid. (C) 1998 Elsevier Science B.V

Recent advances in understanding the extracellular calcium-sensing receptor

The extracellular calcium-sensing receptor (CaR), a ubiquitous class C G-protein-coupled receptor (GPCR), is responsible for the control of calcium homeostasis in body fluids. It integrates information about external Ca 2+ and a surfeit of other endogenous ligands into multiple intracellular signals, but how is this achieved? This review will focus on some of the exciting concepts in CaR signaling and pharmacology that have emerged in the last few years

The role of Store-Operated Cyclic AMP Signalling (SOcAMPS) in cardiac physiology and pathology: an in vitro study on neonatal rat cardiomyocytes.

Background and Aims: Store-Operated Cyclic AMP Signaling (SOcAMPS) represents a recently identified mechanism of cross-talk between Ca2+ and cAMP signals. In this process, depletion of Ca2+ in the endoplasmic reticulum (ER) leads to increases in cAMP levels, independently of cytosolic Ca2+ changes. Expression and functionality of STIM1 (Stromal Interaction Molecule 1), a transmembrane ER Ca2+ sensor protein, is necessary for SOcAMPS to occur. Interestingly, recent reports have demonstrated a critical role for STIM1 in the development of cardiac hypertrophy, a process notoriously controlled both by Ca2+ and cAMP signaling. Here we aimed to evaluate whether SOcAMPS was manifest in neonatal rat cardiomyocytes and its potential role in cardiac cell hypertrophy. Methods: To monitor changes in cAMP levels, real time imaging experiments were performed on neonatal rat cardiomyocytes transiently transfected with an EPAC-based fluorescent probe for [cAMP], EPAC H30. Fura-2 and Fluo-4 were used to monitor cytosolic Ca2+ levels and an ER/SR targeted probe, D1ERcameleon, was used to measure ER [Ca2+]. Long term incubation (48h) of cardiomyocytes with angiotensin II (1 μM) and aldosterone (1 μM) was used to induce "in vitro" cell hypertrophy. Increases in cell size and/or sarcomere alignment were monitored microscopically after labeling with phalloidin-TRITC. Results: To verify the existence of SOcAMPS in neonatal rat cardiomyocytes, cells were stimulated in Ca2+-free Ringer's solutions with the low affinity membrane permeant Ca2+ chelator TPEN (1mM), able to induce a reduction of SR Ca2+ levels ([Ca2+]SR) without affecting cytosolic [Ca2+]. SR Ca2+ measurements demonstrated that under these experimental conditions, 1 mM TPEN led to a reduction in intraluminal [Ca2+] that was 50,5±2,4% (8 exp, 11 cells, p<0.001) of the maximal store depletion. Parallel experiments performed with the EPAC H30 cAMP sensor showed increases in [cAMP] that were 26,5±3% (13 exp, 13 cells, p<0.001) of the maximum delta ratio. In the presence of 5 μM Forskolin (FRSK) the TPEN-induced cAMP augmentation resulted 63,7±3,9% of the maximal response (16 exp, 19 cells, p<0.001). Also depletion of SR by the Ca2+ ionophore ionomycin (10 μM) was found to induce significant cAMP increases both in the absence and presence of FRSK. The participation of STIM1 in the observed phenomenon was proven by the 47 % reduction of the TPEN+FRSK induced [cAMP] signal after transfection of cells with a shRNA against STIM1 (6 exp, p<0,01). To evaluate the putative role of SOcAMPS in cardiac hypertrophy, cAMP measurements were performed on angio+aldo treated cells and compared to control cardiomyocytes. Under these experimental conditions a 20% increase of the TPEN+FRSK induced response was observed in hypertrophic myocytes (16 exp, p<0,01). Conclusions: These data straightforwardly establish, for the first time, the existence of SOcAMPS in the neonatal cardiomyocyte cell model. Also, a significantly increased SOcAMP signalling was shown to exist in hypertrophic cardiomyocytes. Further experiments to ascertain whether a causeand- effect relationship exists between SOcAMPS and cardiac cell hypertrophy are in progress