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This paper discusses the selection of mini-antibody (nanoantibody, nanobody® or single domain antibody) sequences of desired specificity by phage display-based method using a generated library of antigen-binding domains of special heavy-chain only antibodies (single-stranded antibodies) of immunized camel. A comprehensive comparison of the efficiency of parallel selection procedures was performed by using the traditional (M13KO7) and modified (with N-terminal deletion in the surface gIII protein) helper phages. These two methods are partly complementary, and by using them in parallel one can significantly improve the selection efficiency. Parallel restriction analysis (fingerprinting) of PCR-amplified cloned sequences coding for mini-antibodies (HMR-analysis) is proposed for identifying individual clones, as a replacement to sequencing (to a certain extent). Using this method, unique data were collected on the selection of mini-antibody variants with the required specificity at various stages of a multi-stage selection procedure. It has been shown that different sequences coding for mini-antibodies are selected in different ways, and that, if this feature is not taken into account, some mini-antibody variants may be lost.

Chitosan, CN(-), or H(2)O(2) caused the death of epidermal cells (EC) in the epidermis of pea leaves that was detected by monitoring the destruction of cell nuclei; chitosan induced chromatin condensation and marginalization followed by the destruction of EC nuclei and subsequent internucleosomal DNA fragmentation. Chitosan did not affect stoma guard cells (GC). Anaerobic conditions prevented the chitosan-induced destruction of EC nuclei. The antioxidants nitroblue tetrazolium or mannitol suppressed the effects of chitosan, H(2)O(2), or chitosan + H(2)O(2) on EC. H(2)O(2) formation in EC and GC mitochondria that was determined from 2',7'-dichlorofluorescein fluorescence was inhibited by CN(-) and the protonophoric uncoupler carbonyl cyanide m-chlorophenylhydrazone but was stimulated by these agents in GC chloroplasts. The alternative oxidase inhibitors propyl gallate and salicylhydroxamate prevented chitosan- but not CN(-)-induced destruction of EC nuclei; the plasma membrane NADPH oxidase inhibitors diphenylene iodonium and quinacrine abolished chitosan- but not CN(-)-induced destruction of EC nuclei. The mitochondrial protein synthesis inhibitor lincomycin removed the destructive effect of chitosan or H(2)O(2) on EC nuclei. The effect of cycloheximide, an inhibitor of protein synthesis in the cytoplasm, was insignificant; however, it was enhanced if cycloheximide was added in combination with lincomycin. The autophagy inhibitor 3-methyladenine removed the chitosan effect but exerted no influence on the effect of H(2)O(2) as an inducer of EC death. The internucleosome DNA fragmentation in conjunction with the data on the 3-methyladenine effect provides evidence that chitosan induces programmed cell death that follows a combined scenario including apoptosis and autophagy. Based on the results of an inhibitor assay, chitosan-induced EC death involves reactive oxygen species generated by the NADPH oxidase of the plasma membrane.

Current targeting strategies for genetic vectors imply the creation of a specific vector for every targeted receptor, which is time-consuming and expensive. Therefore, the development of a universal vector system whose surface can specifically bind molecules to provide efficient targeting is of particular interest. In this study, we propose a new approach in creating targeted vectors based on the genome of human adenovirus serotype 5 carrying the modified gene of the capsid protein pIX (Ad5-EGFP-pIX-ER): recombinant pseudoadenoviral nanoparticles (RPANs). The surfaces of such RPANs are able to bind properly modified chimeric nanoantibodies that specifically recognize a particular target antigen (carcinoembryonic antigen (CEA)) with high affinity. The efficient binding of nanoantibodies (aCEA-RE) to the RPAN capsid surfaces has been demonstrated by ELISA. The ability of the constructed vector to deliver target genes has been confirmed by experiments with the tumor cell lines A549 and Lim1215 expressing CEA. It has been shown that Ad5-EGFP-pIX-ER carrying aCEA-RE on its surface penetrates into the tumor cell lines A549 and Lim1215 via the CAR-independent pathway three times more efficiently than unmodified RPAN and Ad5-EGFP-pIX-ER without nanoantibodies on the capsid surface. Thus, RPAN Ad5-EGFP-pIX-ER is a universal platform that may be useful for targeted gene delivery in specific cells due to “nanoantibody–modified RPAN” binding.