Arabidopsis S6 kinase mutants display chromosome instability and altered RBR1�CE2F pathway activity

The 40S ribosomal protein S6 kinase (S6K) is a conserved component of signalling pathways controlling growth in eukaryotes. To study S6K function in plants, we isolated single‐ and double‐knockout mutations and RNA‐interference (RNAi)‐silencing lines in the linked Arabidopsis S6K1 and S6K2 genes. Hemizygous s6k1s6k2/++ mutant and S6K1 RNAi lines show high phenotypic instability with variation in size, increased trichome branching, produce non‐viable pollen and high levels of aborted seeds. Analysis of their DNA content by flow cytometry, as well as chromosome counting using DAPI staining and fluorescence in situ hybridization, revealed an increase in ploidy and aneuploidy. In agreement with this data, we found that S6K1 associates with the Retinoblastoma‐related 1 (RBR1)–E2FB complex and this is partly mediated by its N‐terminal LVxCxE motif. Moreover, the S6K1–RBR1 association regulates RBR1 nuclear localization, as well as E2F‐dependent expression of cell cycle genes. Arabidopsis cells grown under nutrient‐limiting conditions require S6K for repression of cell proliferation. The data suggest a new function for plant S6K as a repressor of cell proliferation and required for maintenance of chromosome stability and ploidy levels.     

Hit-to-lead optimization of disubstituted oxadiazoles and tetrazoles as mGluR5 NAMs

Here we report the discovery and early SAR of a series of mGluR5 negative allosteric modulators (NAMs). Starting from a moderately active HTS hit we synthesized 3,5-disubstituted-oxadiazoles and tetrazoles as mGluR5 NAMs. Based on the analysis of ligand efficiency and lipophilic efficiency metrics we identified a promising lead candidate as a starting point for further optimization.     

Optimalized transient transfection of chondrogenic primary cell cultures

We aimed to find a transfection method which provides high efficiency with minimal cytotoxic and/or apoptotic effects for gene transfer into multilayer primary chondrogenic cell cultures. The pEGFP-C1 plasmid was introduced into the cell culture and the efficiency of transformation quantified by GFP fluorescence; the resulting nucleofection was effective but resulted in severe apoptosis. Two liposomal reagents designed to allow transfection into adherent cells did not deliver the plasmids sufficiently and cartilage formation did not occur. In addition, a third liposomal compound, recommended for transfection into either adherent or suspension cell cultures, lead to acceptable transfection efficiency but no cartilage formation. When an amphiphilic reagent was used however, there was acceptable transfection efficiency as well as cartilage formation. The viability of the cells which were transfected using the amphiphilic reagent remained unaffected but proliferation was severely diminished, particularly in the presence of GFP. In addition, the amount of cartilage decreased when GFP was expressed, despite unchanged levels of mRNAs of sox9 and aggrecan core protein, factors reflecting on the efficiency of chondrogenesis. Overexpression of both the constitutively active delta and gamma isoforms of catalytic subunit of calcineurin, a protein phosphatase described as a positive regulator of chondrogenesis, decreased protein level of Sox9 and subsequent cartilage formation. In conclusion, we found that amphiphilic reagent applied prior to the adhesion of cells provides a useful means to transfer plasmids to primary differentiating chondrogenic cells.     

Evidence of the heterogeneity of mast cell population based on their biogenic amine content

The heterogeneity of the mast cells localized in various organs has been demonstrated by histochemical determination in their indole amine and histamine content. The strongest amine reaction was found in the mast cells in the mesenterium and peritoneal fluid where their maturation occurs. The reaction of the subcutaneous connective tissue is weaker; in the thyroid gland the reaction is weak as well as diffuse. In the thymus the mast cells localized along the vessels give a strong histamine reaction. The experiments support previously published data concerning the heterogeneity of mast cell populations.     

Comparison of the inhibitory effect of isothiourea and mercapto-alkylguanidine derivatives on the alternative pathways of arginine metabolism in macrophages

Novel, non-arginine based compounds have been identified as potent inhibitors of nitric oxide synthase (NOS). Members of the isothiourea and mercapto-alkylguanidine classes have generated much interest, as some members of these classes show selectivity towards the inducible isoform of NOS (iNOS), which plays a role in inflammation and shock. Here we compared the effect of a number of these compounds as well as L-arginine based NOS inhibitor reference compounds on macrophage-derived and liver arginase and macrophage iNOS activities. From the nonarginine based NOS inhibitors studied only S-aminoethyl-isothiourea (AETU) caused a slight inhibition of arginase activity. This inhibition was kinetically competitive and due to the rearrangement of AETU to mercapto-ethylguanidine (MEG). The weak inhibitory effect of non-arginine based iNOS inhibitors on arginase activity further supports the view that such compounds may be of practical use for inhibition of NO production in cells simultaneously expressing iNOS and arginase.     

The present state in the phylogeny and ontogeny of hormone receptors

Presence of signal receivers (for food, toxic, substances, "hostile" cells etc.) is essential at all levels of phylogenesis. The first encounter of a "hormone to be" with an aspecific membrane structure ("receptor to be") could result in the formation of a lasting receptor-hormone connection if it is adventageous for the cell or organism (which contains the cell), during phylogeny. At higher levels of phylogenesis receptors (ontogenetically) develop according to the differentiation program of the cell, however reinforcement (by the hormone) is necessary in a critical (neonatal) period of receptor development. This is the hormonal imprinting. In that time the receptor could be damaged by the presence of molecules analogous to the hormone. The hormonal imprinting belongs to the perinatal recognition mechanisms of organisms. The possible mechanisms of receptor development are also discussed.     

Solid-phase synthesis of an N-(phenylalkyl)cinnamide library via Horner-Wadsworth-Emmons reaction

A readily automated solid-phase approach to the synthesis of diverse N-(phenylalkyl)cinnamides, analogues of the NR2B antagonist 2, is described. The procedure utilizes polymer supported N-(phenylalkyl)amines, (diethylphosphono)acetic acid and a wide range of commercially available hydroxybenzaldehydes. The key step, a Horner-Wadsworth-Emmons reaction is achieved under mild conditions and was found to be general for a large number of benzaldehydes. A 225-member focused library was synthesized using a Tecan Combitec synthesizer.