Anti-Inflammatory Activity of a CB2 Selective Cannabinoid Receptor Agonist: Signaling and Cytokines Release in Blood Mononuclear Cells

The endocannabinoid system (ECS) exerts immunosuppressive effects, which are mostly mediated by cannabinoid receptor 2 (CBR2), whose expression on leukocytes is higher than CBR1, mainly localized in the brain. Targeted CBR2 activation could limit inflammation, avoiding CBR1-related psychoactive effects. Herein, we evaluated in vitro the biological activity of a novel, selective and high-affinity CBR2 agonist, called JT11, studying its potential CBR2-mediated anti-inflammatory effect. Trypan Blue and MTT assays were used to test the cytotoxic and anti-proliferative effect of JT11 in Jurkat cells. Its pro-apoptotic activity was investigated analyzing both cell cycle and poly PARP cleavage. Finally, we evaluated its impact on LPS-induced ERK1/2 and NF-kB-p65 activation, TNF-α, IL-1β, IL-6 and IL-8 release in peripheral blood mononuclear cells (PBMCs) from healthy donors. Selective CB2R antagonist SR144528 and CBR2 knockdown were used to further verify the selectivity of JT11. We confirmed selective CBR2 activation by JT11. JT11 regulated cell viability and proliferation through a CBR2-dependent mechanism in Jurkat cells, exhibiting a mild pro-apoptotic activity. Finally, it reduced LPS-induced ERK1/2 and NF-kB-p65 phosphorylation and pro-inflammatory cytokines release in human PBMCs, proving to possess in vitro anti-inflammatory properties. JT11 as CBR2 ligands could enhance ECS immunoregulatory activity and our results support the view that therapeutic strategies targeting CBR2 signaling could be promising for the treatment of chronic inflammatory diseases.     

Thermal Shift Assay as a Tool to Evaluate the Release of Breakdown Peptides from Cowpea β-Vignin during Seed Germination

The present work aimed to characterize the molecular relationships between structure and function of the seed storage protein β-vignin, the vicilin storage protein of cowpea (Vigna unguiculata, l. Walp) seeds. The molecular characterization of β-vignin was carried out firstly by assessing its thermal stability, under different conditions of pH and ionic strength, by thermal shift assay (TSA) using SYPRO Orange fluorescent dye. Secondly, its aggregation propensity was evaluated using a combination of chromatographic and electrophoretic techniques. Two forms of β-vignin were considered: the native form purified from mature quiescent seeds, and a stable breakdown intermediate of 27 kDa produced while seeds germinate. TSA is a useful tool for determining and following over time the structural changes that occur to the protein during germination. The main result was the molecular characterization of the 27 kDa intermediate breakdown polypeptide, which, to the best of our knowledge, has never been described before. β-vignin seems to retain its trimeric conformation despite the evident degradation of its polypeptides.     

Electrochemical behaviour of a Cu/CuSe microelectrode and its application in detecting temporal and spatial localisation of copper(II) fluxes along Olea europaea roots

The electrochemical behaviour of a Cu/CuSe electrode was studied in order to define its selectivity towards cupric ions, Nerstian response, limit of detection and response time. The chalcogenide electrode was prepared by cathodic deposition of Se and subsequent formation of a thin layer of CuSe on a copper substrate. A Cu/CuSe microelectrode was prepared using copper wire 75 μm in diameter. The dimensions and response time (<0.5 s) allowed use of this electrode in the “vibrating probe method” with the aim of measuring net influxes as well as effluxes of copper(II) ions in Olea europaea roots. The electrode potential was measured along the root at a distance of 5 μm from the surface for 5 s, and then again for 5 s at a distance of 55 μm, moving the microelectrode with respect to the root surface by steps with a frequency of 0.1 Hz. The potentials measured at the two extremes of vibration were then converted to copper(II) concentrations. Substitution of these values in Fick's law yields the flux, assuming the diffusion constant D for copper ions in aqueous solutions. The results enabled us to detect copper(II) fluxes as small as 0.05 pmol cm⁻² s⁻¹. Copper(II) influx showed marked spatial and temporal features: it was highest at about 1.5 mm from the root apex and exhibited an oscillatory pattern in time. Received: 29 September 1999 / Accepted: 11 January 2000     

Development of an LC–DAD–MS-Based Method for the Analysis of Hydroxyanthracene Derivatives in Food Supplements and Plant Materials

Products based on plants containing hydroxyanthracene derivatives (HADs)—such as Rheum, Cassia, and Aloe species—are widely used in food supplements or nutraceuticals due to their laxative effects. A more restricted control of HAD contents in food supplements has been implemented by EU Regulation 2021/468, in order to increase the safety of these preparations. Due to their toxicity, aloin A, aloin B, aloe emodin, emodin, and the synthetic derivative danthron have been listed as prohibited substances in food supplements, being tolerated in amounts < 1 mg kg⁻¹ in marketed products. In this work, we report the development of a sensitive and fast LC–DAD–MS-based procedure for the determination of these five compounds in food supplements and plant materials or extracts. The entire procedure includes a simple sample preparation step, where target analytes are concentrated by means of solvent extraction and evaporative concentration (solid samples), or by lyophilisation (liquid samples). The average LOQ of 0.10 mg/L, LOD of 0.03 mg/L, accuracy, and precision with CVs below 12.72 were obtained for the studied analytes. This method is suitable for assessing the compliance of commercial products and raw materials with EU Regulation 2021/468. Furthermore, the proposed method can represent a starting point for the development of a unique and standardised analytical approach for the determination of other HADs under the attention of EU authorities.     

Direct optimization of orbitals for multiconfiguration self-consistent field theory by unitary transformations

In this paper the orbital variations which make stationary the MCSCF energy are generated by unitary transformations worked out directly from the null-gradient condition. A number of iterative procedures are given, compared with other recent proposals and tested numerically on LiH for SCF and geminal theory. A satisfactory convergence rate is found, particularly for the MCSCF case.