Background: The current study was directed to determine the values of both GM3 ganglioside and specific anti-GM3 antibodies in in vitro-incubated normal cells, malignant cell and mixed cultures of co-cultivated cells from both types as well. So a better understanding of the molecular mechanisms, underlining differences between various cellular types and changes during the process of cell differentiation was necessary. Methods: Total lysates from cultivated normal cells from mouse embryos, mouse malignant myeloma line and mixed of both were prepared and passed through GSHAgarose Columns. Molecules with affinity to the reduced form of Glutathione (GSH) were separated. The levels of ganglioside GM3 and of specific antibodies to it were assessed by enzyme-linked immuno-sorbent assay (ELISA) technique. Results: In the normal cells’ lysate, supplemented with molecules, possessing affinity to GSH the lowest values were assessed. These differences could be due to contention of many non-possessing such affinity molecules in equal volume of biological material, as well as with the presence of various bonded forms of GM3. Conclusions: The data obtained confirm literature data about the increased levels of GM3 as a marker for malignancy. We propose derivation of lymphoid-like cells from the embryonic progenitors. Another hypothesis could be the production of immunoglobulins (IgG antibodies) from non-lymphoid cells in appropriate conditions.

The potential of viruses as appropriate vectors for the development of new therapeutic strategies, as well as for the design of molecular (DNA, RNA, and/or protein) vaccines via substitution of nucleotide sequences, has been proven. Among the most appropriate DNA and/or RNA fragments, members belonging to families Parvoviridae (particularly adeno-associated virus, AAV) and Poxviridae have frequently been suggested for this purpose. In previous studies, the vaccine avipoxvirus strains FK (fowl) and Dessau (pigeon) have been proven able to infect mammalian cells (as well as avian cells), and to replicate productively in a small number of them; thus, we may be able to adapt them using incubation, and in these conditions. Additionally, we have previously proved, based on AAV recombinant DNA vectors, that it is possible to transfer appropriate genes of interest via mouse embryonic stem cells (mESCs). In the current study, we develop methods for the application of the same vaccine avipoxviral strains, based on the AAV DNA genome recombinant constructs, to be used for gene transfer in cells, for the transfer of DNA and/or RNA fragments (for the suppression of unwanted viral and/or cellular genes), and for the production of molecular (DNA, RNA, and/or protein) anti-cancer and anti-viral vaccines. To this end, sub-populations of embryonic mammalian cells infected with the two forms of both vaccine avipoxviral strains were frozen in the presence of cryo-protector dimethylsulfoxide (DMSO), subsequently thawed, and re-incubated. In most cases, the titers of the intra-cellular forms of the two strains were higher than those of their extra-cellular forms. These data were explained by the probable existence of the intra-cellular forms as different sub-forms, including those integrated in the cellular genome proviruses at a given stage of the cellular infection, and suggest the possibility of transferring nucleotide (DNA and/or RNA) fragments between cellular and viral genomes; this is due to the influence of activated fusion processes on DMSO, as well as drastic temperature variations.

Sialidases/neuraminidases remove terminal sialic acid residues from glycoproteins, glycolipids, and oligosaccharides. Our previous research has revealed the distribution of sialidase in non-clinical fungal isolates from different ecological niches, including Antarctica. Fungi adapted to extremely low temperatures possess defense mechanisms necessary for their survival such as the response against oxidative stress. The relationship between oxidative stress and sialidase synthesis has been studied extremely sparsely. The aim of the present study was to investigate the involvement of sialidase in the cell response of the Antarctic strain P. griseofulvum P29 against oxidative stress induced by long- and short-term exposure to low temperatures. The changes in growth temperatures for 120 h (long-term stress) affected biomass accumulation, glucose consumption, sialidase synthesis, and the activity of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT). The short-term temperature downshift (6 h) caused oxidative stress, evidenced by changes in the levels of biomarkers, including lipid peroxidation, oxidatively damaged proteins, and the accumulation of reserve carbohydrates. Simultaneously, a sharp increase in SOD and CAT activity was found, which coincided with a significant increase in sialidase activity. This study marks the first demonstration of increased sialidase activity in filamentous fungi isolated from extreme cold environments as a response to oxidative stress.

Sialidases (neuraminidases) catalyze the removal of terminal sialic acid residues from glycoproteins. Novel enzymes from non-clinical isolates are of increasing interest regarding their application in the food and pharmaceutical industry. The present study aimed to evaluate the participation of carbon catabolite repression (CCR) in the regulation of cold-active sialidase biosynthesis by the psychrotolerant fungal strain Penicillium griseofulvum P29, isolated from Antarctica. The presence of glucose inhibited sialidase activity in growing and non-growing fungal mycelia in a dose- and time-dependent manner. The same response was demonstrated with maltose and sucrose. The replacement of glucose with glucose-6-phosphate also exerted CCR. The addition of cAMP resulted in the partial de-repression of sialidase synthesis. The CCR in the psychrotolerant strain P. griseofulvum P29 did not depend on temperature. Sialidase might be subject to glucose repression by both at 10 and 25 °C. The fluorescent assay using 4MU-Neu5Ac for enzyme activity determination under increasing glucose concentrations evidenced that CCR may have a regulatory role in sialidase production. The real-time RT-PCR experiments revealed that the sialidase gene was subject to glucose repression. To our knowledge, this is the first report that has studied the effect of CCR on cold-active sialidase, produced by an Antarctic strain.

Background: Changes in biomolecules under the influence of chemical and physical factors on cells, tissues, and whole organisms are investigated. Material and methods: In vitro-incubated bovine embryonic cells were inoculated with low titers (high dilutions of viral suspensions) of vaccine avipoxviral strains. Mouse embryonic fibroblasts were co-cultivated with mouse malignant myeloma cells (P3-X63-Ag8) transfected by recombinant DNA plasmid or preincubated in culture fluid from prior incubation of the same cells. Sub-populations of virus-inoculated, co-cultivated, and preincubated cell cultures were frozen in the presence of the cryoprotectant dimethylsulfoxide (DMSO), subsequently thawed, and re-incubated. Newly formed cell monolayers were inoculated with extracellular and intracellular forms of each viral strain, both before and after exposure to DMSO and drastic temperature changes. Extracellular forms were derived from the cultural fluids of inoculated cell cultures, while intracellular forms were obtained from suspensions of mechanically scraped virus-inoculated cells. Results: Exchange of nucleotide (DNA and/or RNA) fragments between cellular and viral genomes, as well as between genomes of separate cells, was suggested. These changes were explained by activated fusion induced by the organic detergent (DMSO) combined with drastic temperature changes. Such processes could provide vectors for gene-engineering manipulations and the development of molecular (DNA-based, RNA-based, and/or protein-based) antiviral and antimalignant vaccines. Production of immune molecules by nonimmune cell types under appropriate conditions, such as the presence of immunomodulators, was also proposed. Conclusions: The results suggest the possibility of nucleotide (DNA and/or RNA) fragment exchange between separate cells, as well as between cells and virions. Nonimmune cells demonstrated the capacity to produce immune molecules under appropriate conditions.