Julia Kazmierczak-Baranska

Specific Silencing of L392V PSEN1 Mutant Allele by RNA Interference.

RNA interference (RNAi) technology provides a powerful molecular tool to reduce an expression of selected genes in eukaryotic cells. Short interfering RNAs (siRNAs) are the effector molecules that trigger RNAi. Here, we describe siRNAs that discriminate between the wild type and mutant (1174 C→G) alleles of human Presenilin1 gene (PSEN1). This mutation, resulting in L392V PSEN1 variant, contributes to early onset familial Alzheimers disease. Using the dual fluorescence assay, flow cytometry and fluorescent microscopy we identified positions 8th–11th, within the central part of the antisense strand, as the most sensitive to mismatches. 2-Thiouridine chemical modification introduced at the 3′-end of the antisense strand improved the allele discrimination, but wobble base pairing adjacent to the mutation site abolished the siRNA activity. Our data indicate that siRNAs can be designed to discriminate between the wild type and mutant alleles of genes that differ by just a single nucleotide.

5-Ethynyl-1-β-D-ribofuranosyl-1H-[1,2,3]triazole-4-carboxylic acid amide (ETCAR) and its analogues: synthesis and cytotoxic properties.

Efficient Pd(0)-catalysed synthesis of 5-alkynyl-1-β-D-ribofuranosyl-1H-[1,2,3]triazole-4-carboxylic acid amide depends on the presence of different protecting groups of the ribose moiety. Peracetylated 5-iodo substrate (15) couples with terminal alkynes or trimethyl-[(tributylstannyl)ethynyl]silane in 50-71% and 72% yield (ETCAR), respectively, although its hydrodehalogenation to 19 is noticeable. On the other hand, hydrodehalogenation of acetonide (16) predominates over coupling with terminal alkyne and slightly decreases a yield of cross-coupling reaction with trimethyl[(tributylstannyl)ethynyl]silane. Alternative conditions of reaction with terminal alkynes, to exclude so far identified hydride sources to produce hydridopalladium species, have been established for acetonide 16 and allowed to achieve 72% of coupling. Fluoromethyl derivative (42) was prepared from its 5-hydroxymethyl precursor by fluorination with DAST. Additionally, X-ray structural analysis of 42 was performed. All 1,2,3-triazolonucleosides and two synthesized cycloSal-pronucleotides were evaluated for cytotoxic activity against K562, HeLa and HUVEC cells.

High Cytotoxic Activity of Phosphonium Salts and Their Complementary Selectivity towards HeLa and K562 Cancer Cells: Identification of Tri-n-butyl-n-hexadecylphosphonium bromide as a Highly Potent Anti-HeLa Phosphonium Salt.

Quaternary ammonium and phosphonium salts have been screened for their toxic effect on HeLa and K562 cancer cell lines, as well as on normal HUVEC cells. Tri-n-butyl-n-hexadecylphosphonium bromide, the first phosphonium salt with a halogen anion tested against HeLa cells, was 12 times more potent (IC50 <5 μm after 24 and 48 h) than the clinically used reference compound cisplatin and 17 times more potent than tri-n-hexyltetradecylphosphonium bis(trifluoromethylsulfonyl)imide, which, to the best of our knowledge, is the first phosphonium salt to be evaluated in HeLa cells. However, it was inactive against K562 cells (24 and 48 h). According to a caspase-3/7 assay, its toxicity has not been connected with the induction of apoptosis. In contrast, triphenylalkylphosphonium iodides with shorter C1–5 alkyl chains were inactive against HeLa cells but very active against K562 cells (IC50=6–10 μm after 48 h). Phosphonium cations with halide counterions proved to be more potent than those with (CF3SO2)2N− as the anion, as in the anticancer agent NSC 747251, or other anions in molecules with similar alkyl chain lengths. On the other hand, a series of ammonium salts containing a short methylthiomethyl or methoxymethyl side chain revealed low cytotoxicity (IC50 >500 μm after 24 and 48 h) against both HeLa and K562 cancer cell lines as well as normal HUVEC cells, showing that the nontoxic N+CH2YMe (Y=S, O) structural motif in ammonium salts could be suitable for further optimization and development, especially in transfection experiments.

Synthesis of a Fluorescent Cationic Phosphorus Dendrimer and Preliminary Biological Studies of Its Interaction with DNA

The synthesis of a water-soluble phosphorus-containing dendrimer possessing a fluorophore (maleimide-type) linked to the core is described. This dendrimer is found brightly fluorescent in CH2Cl2, but poorly fluorescent in water. The cytotoxicity of this compound is relatively low towards HeLa and A549 cells, and less toxic after 48 hours than after 24 hours. Association of this dendrimer with plasmid DNA (BACE-GFP) analyzed with circular dichroism (CD) indicates a possible disturbing of the helical B-type structure of DNA. The strength of this association (a “dendriplex”) with BACE-GFP (also with HygEGFP) was measured by electrophoresis.

Phytotoxicity and Cytotoxicity of Imidazolium Ionic Liquids Containing Sulfur Atom

Abstract Ionic liquids have attracted considerable interest in various areas as new, nonvolatile and nonflammable organic solvents, catalysts, reaction additives, ligands, drugs, and other dedicated materials. Ionic liquids exhibit solubility both in organic solvents and in water. They also constitute very good solvents for many inorganic, organic, and organometallic compounds. Yet, the most important feature of ionic liquids is that they provide the possibility of obtaining substances of optimal properties for a given process through modification of the cation structure and selection of the appropriate anion. Therefore, the term designer solvents has been invented for these salts. The above characteristics and relatively low production cost may soon lead to the situation, in which huge amounts of these chemicals will be brought into environment via industry, agriculture, and commercial trading. Their general use, sometimes in bulky quantities, requires determination of their potential ecotoxicity on selected plant and animal organisms. Other biological tests, are also very important. GRAPHICAL ABSTRACT

Evaluation of BACE1 Silencing in Cellular Models

Beta-secretase (BACE1) is the major enzyme participating in generation of toxic amyloid-beta (Aβ) peptides, identified in amyloid plaques of Alzheimers disease (AD) brains. Its downregulation results in decreasing secretion of Aβ. Thus, BACE1 silencing by RNAi represents possible strategy for antiamyloid therapy in the treatment of AD. In this study, a series of newly designed sequences of synthetic and vector-encoded siRNAs (pSilencer, pcPURhU6, and lentivirus) were tested against overexpressed and endogenous BACE1 in several cell lines and in adult neural progenitor cells, derived from rat hippocampus. SiRNAs active in human, mouse, and rat cell models were shown to diminish the level of BACE1. In HCN A94 cells, two BACE1-specific siRNAs did not alter the expression of genes of BACE2 and several selected genes involved in neurogenesis (Synapsin I, βIII-Tubulin, Calbidin, NeuroD1, GluR2, CREB, MeCP2, PKR), however, remarkable lowering of SCG10 mRNA, coding protein of stathmin family, important in the development of nervous system, was observed.

RNA interference in silencing of genes of Alzheimer's disease in cellular and rat brain models

Accumulation of insoluble aggregates of beta-amyloid peptide, a cleavage product of amyloid precursor protein, is thought to be a central step in the pathogenesis of Alzheimers disease. The major enzymes required for the generation of toxic amyloid-beta peptide are beta-(BACE1) and gamma-secretases. Here, we present the rational design and the application of synthetic and lentivirus vector-encoded siRNAs for specific and efficient knockdown of overexpressed and endogenous BACE1, both in dividing and neural stem cells and in a rat brain. We also tested an approach to anti-amyloid therapy by the use of the allele-specific siRNAs to silence the mutant presenilin 1 (L392V PS-1), the main component of gamma-secretase, responsible for development of Familial Alzheimers disease. Reducing the level of beta-amyloid accumulation in the brain could be beneficial for metabolic studies as well as potential therapeutic approach for prevention and treatment of Alzheimers disease.