Novel inhibitors of leukocyte transendothelial migration

Leukocyte transendothelial migration is one of the most important step in launching an inflammatory immune response and chronic inflammation can lead to devastating diseases. Leukocyte migration inhibitors are considered as promising and potentially effective therapeutic agents to treat inflammatory and auto-immune disorders. In this study, based on previous trioxotetrahydropyrimidin based integrin inhibitors that suboptimally blocked leukocyte adhesion, twelve molecules with a modified scaffold were designed, synthesized, and tested in vitro for their capacity to block the transendothelial migration of immune cells. One of the molecules, namely, methyl 4-((2-(tert-butyl)-6-((2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene) methyl) phenoxy) methyl) benzoate, (compound 12), completely blocked leukocyte transendothelial migration, without any toxic effects on immune or endothelial cells (IC₅₀ = 2.4 µM). In vivo, compound 12 exhibited significant therapeutic effects in inflammatory bowel disease (IBD)/Crohn’s disease, multiple sclerosis, fatty liver disease, and rheumatoid arthritis models. A detailed acute and chronic toxicity profile of the lead compound in vivo did not reveal any toxic effects. Such a type of molecule might therefore provide a unique starting point for designing a novel class of leukocyte transmigration blocking agents with broad therapeutic applications in inflammatory and auto-immune pathologies.     

Actomyosin mediates gravisensing and early transduction events in reoriented cut snapdragon spikes

We investigated the involvement of the actomyosin network in the early events of the gravitropic response of cut snapdragon (Antirrhinum majus L.) spikes. The effects of the actin-modulating drug, cytochalasin D (CD) and/or the myosin inhibitor, 2,3-butanedione-2-monoxime (BDM) on amyloplast displacement, lateral auxin transport and consequently on stem bending were examined. The inhibitory effect on cytoskeleton integrity was studied by using indirect immunofluorescence double-labeling of actin and myosin. Our results demonstrate that no organizational changes in actin filaments occurred in cortical and endodermal cells of the stem bending zone during reorientation. These results suggest that actin depolymerization is not required for amyloplast sedimentation. Unlike the chloroplasts in the cortex, the amyloplasts in the endodermis were surrounded by actin and myosin, indicating that amyloplasts may be attached to the actin filaments via the motor protein, myosin. This suggests the involvement of myosin as part of the actomyosin complex in amyloplast movement in vertical as well as in reoriented stems. This suggestion was supported by the findings showing that: (a) BDM or CD disrupted the normal organization of actin either by altering characteristic distribution patterns of myosin-like protein in the cortex (BDM), or by causing actin fragmentation (CD); (b) both compounds inhibited the gravity-induced amyloplast displacement in the endodermis. Additionally, these compounds also inhibited lateral auxin transport across the stem and stem gravitropic bending. Our study suggests that during stem reorientation amyloplasts possibly remain attached to the actin filaments, using myosin as a motor protein. Thus, gravisensing and early transduction events in the gravitropic response of snapdragon spikes, manifested by amyloplast displacement and lateral auxin transport, are mediated by the actomyosin complex.     

Synthesis and characterization of a molecularly imprinted polymer and its application as SPE enrichment sorbent for determination of trace methimazole in pig samples using HPLC-UV

A novel molecularly imprinted polymer that could be applied as enrichment sorbent was prepared using methimazole (MMZ) as the template molecule, methacrylic acid as functional monomer, ethylene glycol dimethacrylate as cross-linker. Though evaluated by static, kinetic and competitive adsorption tests, the polymer exhibited high adsorption capacity, fast kinetics and good selective ability. A method for determination of trace MMZ was developed using this polymer as enrichment sorbent coupled with high performance liquid chromatography focusing on complex biological matrices. Under the optimum experimental conditions, the MMZ standard is linear within the concentration range studied, that is, from 0.5 μg L⁻¹ to 150 μg L⁻¹ (r² = 0.9941). Lower limits of detection (LOD, at S/N = 3) and quantification (LOQ, at S/N = 10) in pig samples were 0.63 μg kg⁻¹ and 2.10 μg kg⁻¹ for kidney, 0.51 μg kg⁻¹ and 1.70 μg kg⁻¹ for liver, 0.56 μg kg⁻¹ and 1.86 μg kg⁻¹ for muscle, respectively. Recoveries and relative standard deviation (RSD, n = 9) values for precision in the developed method were from 71.14% to 88.41% and from 2.53% to 6.18%.