SSCP screening of individual aptamers.

Aptamers are specific binding nucleic acids that emerge from in vitro selection. During the systematic evolution of ligands by exponential enrichment (SELEX) procedure, analysis of the sequences of the numerous selected individual molecules becomes an important step in the final stage of aptamer selection. The sequencing of cloned aptamers from the selected pool generally reveals groups of identical sequences and rarely occurring individual aptamers. This study demonstrates an approach similar to the single strand conformation polymorphism (SSCP) method used for mutation testing in genes. Human angiotensin I-specific aptamers have been used to show the efficiency of the SSCP method to classify selected individual sequences into identity groups, which minimizes sequencing efforts. Additionally this approach allows the rapid isolation and identification of aptamers from a mixture.

Long-term expression of foreign genes in normal human epidermal keratinocytes after transfection with lipid/DNA complexes.

Normal human epidermal keratinocytes were isolated and cultivated in serum-free medium. The expression of the integrin subunits alpha6 and beta1 indicated that a high number of keratinocytes from the stem cell system was present. These cells were transfected with complexes made of different cationic lipids and marker genes. Effectene showed a 20-fold higher transfection efficiency, compared to Lipofectin and Lipofectamine, and a similar low toxicity. The transfection protocol was optimised. A DNA/lipid ratio of 0.133 showed the highest transfection efficiency. Keratinocytes expressed the marker gene luciferase for 20 days. The maximum expression occurred after 3-4 days, where individual patches of fluorescent keratinocytes were detected. Transfected keratinocytes, cultivated at the air-liquid interface, expressed the marker gene beta-galactosidase for at least 7 weeks.

Endogenous fragment of hemoglobin, neokyotorphin, as cell growth factor.

It is shown that neokyotorphin (the alpha-globin fragment 137-141) stimulates proliferation of normal cells (murine embryonic fibroblasts, red bone marrow and spleen cells) and tumor cells (murine melanoma and transformed fibroblasts L929) in the absence or in the presence of fetal bovine serum. In contrast to serum deprivation conditions, the ability to potentiate L929 cell growth in the presence of fetal serum is strongly cell density dependent. The peptide also enhances the viability of L929 cells, murine embryonic fibroblasts and of the primary cultures of murine red bone marrow cells and splenocytes under serum-deprivation conditions for at least 72 h. The results of flow cytometry analysis suggest that the effect of neokyotorphin on survival of L929 cells in serum-free culture medium is due to maintenance of cell proliferation in the absence of growth factors. Along with cell cycle progression the peptide induces reversible reduction of L929 cell size.

Transfection of normal human epidermal keratinocytes with lipid/dna complexes in vitro.

Highly proliferative normal human epidermal keratinocytes (NHK) were isolated from human foreskin biopsies, cultivated in serum-free medium and characterized by flow cytometry. The expression of cytokeratin 19, cytokeratin 14 and vimentin indicated that the suspension contained a high percentage of undifferentiated cells of the basal epidermal layer. The NHK were transfected in vitro with lipid/DNA complexes made of Effectene or Lipofectamine and different reporter genes. The transfection efficiency of Effectene/DNA complexes was 20fold higher compared to Lipofectamine. Transfected keratinocytes continued to grow and developed within 2 weeks a cellular multilayer (3-D culture). Areas of transfected cells were detected within this layer.

Basic properties of populations generated in the frame of one-parameter discrete model of genetic diversity.

Previously, when discussing the properties of one parameter discrete model of genetic diversity (M.Yu. Shchelkanov et al, J. Biomol. Struct. Dyn. 15, 887-894 (1998)), we took into account Hamming distance distribution only between precursor and arbitrary descendant sequences. However, really there are sets of sequence populations produced during amplification process. In the presented work we have investigated Hamming distance distributions between sequences from different descendant sets produced in the frame of one parameter discrete model. Two basic descendant generation operators (so called amplifiers) are introduced: 1) the last generation amplifier, L, which produces descendants with precursor elimination; 2) all generations amplifier, G, which produces descendants without precursor elimination. Generalization of one-parameter discrete model for the case when precursor sequences do not coincide are carried out. Using this generalization we investigate the distribution of Hamming distances between L- and G-generated sequences. Basic properties of L and G operators, L/G-choice alternative problem have been discussed. Obtained results have common theoretical significance, but they are more suitable for high level genetic diversity process (for example, HIV diversity).

A peptide construct containing B-cell and T-cell epitopes from the foot-and-mouth disease viral VP1 protein induces efficient antiviral protection.

A new peptide construct Palm135-158-GGA-170-188(Acm) has been synthesized and investigated in a number of in vitro and in vivo test systems. The construct contains a virus specific T-helper epitope within the 170-188 sequence of VP1, in addition to the main antigenic 135-158 region of the foot-and-mouth disease viral VP1 protein (strain A22). The construct has higher protective, antigenic, immunogenic and T-cell proliferative activity then the previously described shorter peptide Palm(2)135-159. The 170-188 part of the construct serves as a virus specific T-epitope, responsible for the enhanced immunogenic and protective activity of the construct.

Integrin receptor-targeted transfer peptides for efficient delivery of antisense oligodeoxynucleotides.

Integrin receptor-targeted transfer of oligodeoxynucleotides (ODNs) by small synthetic peptides was used for improving delivery of antisense ODNs. An 18-mer phosphodiester bond containing ODN complementary to c-myb-encoded mRNA was complexed with several transfer peptides, containing as their parts two modules: (a) an RGD-motif as targeting sequence for integrin receptor and (b) nucleocapsid protein (NCp) 7 of HIV-1 or NCp7-derived peptides for complex formation with the ODNs. The amount of antisense ODN required for the inhibition of proliferation of human myeloid cell line HL-60 in vitro can be more than 50-fold reduced by complexing with transfer peptides. The efficiency of antisense delivery was increased by multimerization of the targeting sequence for the integrin receptor. Competition with integrin peptide abolished the effect, indicating that the integrin receptor is indeed responsible for the reaction.