Vol 3, No 3 (2011)

Deregulation of the expression of the genes that are involved in the control of the cell cycle impairs cellular differentiation and leads to cell death. This process can result in uncontrollable cell proliferation and, subsequently, cancer development. In this study, we examined the effect of the silencing of cancer-related genes by small interfering RNAs (siRNA) targeted at mRNA of Her2, cyclin B1 (CCNB1), and protein kinase C (PKC) on the proliferation of human cancer cells of different origins. Maximum silencing of CCNB1, Her2 (in KB-3-1, SK-N-MC, MCF-7 cells), and PKC (in MCF-7 cells) was achieved 72 h after transfection of the corresponding siR-NAs, and 12 days after the transfection, the initial levels of the target mRNAs were fully recovered. Silencing of Her2, CCNB1, and PKC differently effected the proliferation of the cell lines under study. The most pronounced antiproliferative action of the investigated siRNAs was observed in neuroblastoma SK-N-MC cells (3 - 10-fold reduction in the proliferation rate) even after the recovery of the initial levels of expression of the Her2, CCNB1, and PKC genes. The obtained data indicate that the CCNB1 and PKC genes can be used as targets in the development of drugs for neuroblastoma treatment.

The crystal structure of the human transcription factor DLX5 has been used for the screening of a library consisting of 10 6 compounds by the molecular docking technique. In vitro tests of the 14 top-rated ligands showed that compound Q12 displays the best ability to inhibit the proliferation of Dlx5 positive mouse lymphoma cells, which correlates with the down-regulation of c-myc expression. Compound Q12 has low toxicity on normal human ovarian epithelial cells and mouse lymphoma cells with absent expression of Dlx5, and can be used for further chemical optimization and for the development of novel, highly efficient cancer treatments.

The present study is devoted to the feasibility of expressing the single-domain mini-antibody (nanoantibody) selected from the library of sequences of the variable domains of special single-stranded antibodies derived from an immunized camel, a gene of which was introduced into eukaryotic cells within a recombinant adenoviral vector. A vector bearing the gene of a single-domain nanoantibody was obtained using the AdEasy Adenoviral Vector System (Stratagene). This method of delivering the nanoantibody gene facilitates efficient expression of this gene and functional activity of the nanoantibody. The results obtained can be used to produce passive immunizing tools against pathogens or new-generation immunobiological antitoxic medication.

The fibroblast growth factor receptor 3 (FGFR3) is a protein belonging to the family of receptor tyrosine kinases. FGFR3 plays an important role in human skeletal development. Mutations in this protein, including Gly380Arg or Ala391Glu substitutions in the transmembrane (TM) region, can cause different disorders in bone development. The determination of the spatial structure of the FGFR3 TM domain in a normal protein and in a protein with single Gly380Arg and Ala391Glu mutations is essential in order to understand the mechanisms that control dimerization and signal transduction by receptor tyrosine kinases. The effective system of expression of eukaryotic genes in bacteria and the purification protocol for the production of milligram amounts of both normal TM fragments of FGFR3 and those with single pathogenic mutations Gly380Arg and Ala391Glu, as well as their 15N- and [ 15N, 13C]-isotope-labelled derivatives, were described. Each peptide was produced in Escherichia coli BL21(DE3)pLysS cells as a C-terminal extension of thioredoxin A. The purification protocol involved immobilized metal affinity chromatography and cation- and anion-exchange chromatography, as well as the fusion protein cleavage with the light subunit of human enterokinase. The efficiency of the incorporation of target peptides into DPC/SDS and DPC/DPG micelles was confirmed using NMR spectroscopy. The described methodology of production of the native FGFR3 TM domain in norma and with single Gly380Arg and Ala391Glu mutations enables one to study their spatial structure using high-resolution heteronuclear NMR spectroscopy.

Astrocytes are quite interesting to study because of their role in the development of various neurodegenerative disorders. The present work describes an examination of the arrangement and mechanical properties of cytoskeleton of living astrocytes using atomic force microscopy (AFM). The experiments were performed with an organotypic culture of dorsal root ganglia (DRG) obtained from a chicken embryo. The cells were cultivated on a gelatinous substrate and showed strong adhesion. AFM allows one to observe cytoskeleton fibers, which are interpreted as actin filaments and microtubules. This assumption is supported by confocal microscopy fluorescence imaging of α-tubulin and fibrillar actin. Mapping of the local Young’s modulus of a living astrocyte showed that the stiff areas correspond to the sites where the cytoskeleton fibers are located. Thus, the data obtained indicate that AFM is a promising method to study neural cells cytoskeleton integrity and arrangement in in vitro models of neurodegeneration.