PRODUCTION OF SINGLE CHAIN FRAGMENT VARIABLE (scFv) ANTIBODY AGAINST PARANOSEMA LOCUSTAE ALPHA/BETA-HYDROLASE AND IMMUNOLOCALIZATION OF MICROSPORIDIAN ENZYME IN THE INFECTED HOST CELL.

Microsporidia is a widespread group of fungi-related intracellular parasites. Direct contact of the most microsporidia species with host cytoplasm suggests that these parasites may control physiological processes of infected cells by secretion of various proteins. In previous experiments, secretion of significant amounts of microsporidia Paranosema locustae alpha/beta-hydrolase into infected cells of Locusta migratoria fat bodies was demonstrated using polyclonal antibodies against the enzyme. However, heterologous expression of microsporidian hydrolase in yeast Pichia pastoris cells was not accompanied by its secretion. In this study, we have constructed library of recombinant single chain antibodies (scFv-fragments) against proteins of fat bodies of infected locusts and isolated mini-antibody specifically recognizing the studied enzyme using phage display technology. Immunoblotting and immunofluorescent microscopy with selected scFv-fragment confirmed secretion of two different in size forms of P. locustae alpha/beta-hydrolase into infected host cell. Prospects of scFv-fragment use to explore the role of microsporidian hydrolase in host-parasite relations and mechanism of its secretion are discussed in the paper.

Microsporidia Alfvenia sibirica sp. n. and Agglomerata cladocera (Pfeiffer) 1895, from Siberian microcrustaceans and phylogenetic relationships within the "Aquatic outgroup" lineage of fresh water microsporidia.

Here we report on two microsporidia from freshwater crustaceans collected during the ongoing survey for microsporidia in the river Karasuk and adjacent waterbodies (Novosibirsk region, Western Siberia). The first species parasitized hypoderm and fat body of a cyclopid Cyclops sp. (Maxillopoda, Copedoda) and produced oval spores, measured 2.0×3.6µm (fixed) enclosed individually or in small groups in fragile sporophorous vesicles (SVs). We describe it here as Alfvenia sibirica sp. n. The second species infected the same tissues of a cladoceran Daphnia magna (Branchiopoda, Phyllopoda). Its spores were pyriform, 2.3×4.0µm (fixed), and resided in relatively persistent SVs in groups of 8-16. This species was identified as a Siberian isolate (Si) of Agglomerata cladocera (Pfeifer) because ultrastructurally it was hardly distinguishable from A. cladocera recorded from England from the same host (Larsson et al., 1996). A. cladocera (Si) shared 99% SSU rDNA sequence similarity to Binucleata daphniae from D. magna collected in Belgium (Refardt et al., 2008). The major outcome of our work is that we present molecular (SSUrDNA) characterization coupled with EM description, for representatives of two genera, Alfvenia (encompasses 3 described so far species) and Agglomerata (7 species), which allowed us to place these two genera on the phylogenetic tree. We also summarized the literature data on Alfvenia and Agglomerata spp., and provided their comparative morphological analysis. These two genera belong to so called "Aquatic outgroup" (Vossbrinck et al., 2004), a poorly resolved lineage, a sister to Amblyosporidae. This lineage probably includes majority of fresh water forms of microsporidia, most of which remain undescribed. SSUrDNA-based phylogenetic analysis and analysis of hosts suggest that diversification within the "Aquatic outgroup" could have been connected with the host switch from dipterans or copepods to cladocerans that had occurred in some ancestral Amblyospora-related lineage(s).

[THE MICROSPORIDIUM GLUGEA GASTEROSTEI VORONIN 1974 (MICROSPORIDIA: MARINOSPORIDIA) FROM THE THREE-SPINED STICKLEBACK GASTEROSTEUS ACULEATUS (ACTINOPTERYGII: GASTEROSTEIFORMES) AS AN INDEPENDENT SPECIES].

The microsporidium Glugea gasterostei from the three-spined stickleback Gasterosteus aculeatus was described as an independent species basing upon morphological and ecological traits of the parasite (Voronin, 1974), further supported by ultrastructural characters of its spores (Voronin, 1983). During the revision of microsporidia of the genus Glugea (Canning, Lom, 1986; Lom, 2002), the validity of this species was doubted and it was synonymized with G. anomala. Nevertheless, the molecular phylogenetic analysis performed in the present study showed the unique molecular haplotype of small subunit rRNA gene of G. gasterostei (Genbank accession number KM977990) and its close relatedness to G. anomala, G. atherinae and G. hertwigi (sequence similarity of 99.7 %). One of typical characters of G. gasterostei, as opposed to G. anomala, is the formation of xenomas on inner tissues and not on the surface of infected fishes. This feature is retained even after the infection of different host species. Taken together, these data confirm the validity of G. gasterostei as a separate species among closely related taxa that had diverged comparatively recently.

Optimization of the Protocol for the Isolation and Refolding of the Extracellular Domain of HER2 Expressed in Escherichia coli.

Receptor 2 of the human epidermal growth factor (HER2/neu, c-erbB2) is a 185 kDa proto-oncogene protein characterized by an overexpression in some oncological diseases, including 30% of mammary glands cancers, as well as tumors in the ovary, stomach and other organs of the human body. Since HER2- tumor status testing is the essential part of a successful cancer treatment, the expression and purification of substantial amounts of the extracellular domain (ECD) of HER2 is an important task. The production of ECD HER2 in Escherichia coli has several advantages over the use of eukaryotic expression systems, but the bulk of the recombinant product in bacteria accumulates as insoluble protein inclusion bodies. In this study, we obtained ECD HER2 in Escherichia coli as insoluble inclusion bodies and elaborated a simple, efficient, and fast protocol for the solubilization, refolding, and isolation of the protein in soluble form.