Separation of mesenchymal stem cells with magnetic nanosorbents carrying CD105 and CD73 antibodies in flow-through and batch systems

The aim of this study is to develop magnetically loaded nanosorbents carrying specific monoclonal antibodies (namely CD105 and CD73) for separation of mesenchymal stem cells from cell suspensions. Super-paramagnetic magnetite (Fe3O4) nanoparticles were produced and then coated with a polymer layer containing carboxylic acid functional groups (average diameter: 153 nm and polydispersity index: 0.229). In order to obtain the nanosorbents, the monoclonal antibodies were immobilized via these functional groups with quite high coupling efficiencies up to 80%. These nanosorbents and also a commercially available one (i.e., microbeads carrying CD105 antibodies from Miltenyi Biotec., Germany) were used for separation of CD105+ and CD73+ mesenchymal stem cells from model cell suspension composed of peripheral blood (97.6%), human bone marrow cells (1.2%) and fibroblastic cells (1.2%). The initial concentrations of the CD105+ and CD73+ cells in this suspension were measured as 5.86% and 6.56%, respectively. A flow-through separation system and a very simple homemade batch separator unit were used. We were able to increase the concentration of CD105+ cells up to about 86% in the flow-through separation system with the nanosorbents produced in this study, which was even significantly better than the commercial one. The separation efficiencies were also very high, especially for the CD73+ cells (reached to about 64%) with the very simple and inexpensive homemade batch unit.     

Premature cytokinesis in pollen mother cells of transgenic tobacco plants (<Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L.)

For the majority of dicotyledonous plants, cytokinesis in PMC is staged only once, i.e., after the completion of two cycles of caryokinesis. In the article, a cytological picture and the frequency characteristics of anomalies are shown, in which the cytokinesis in the PMCs of transgenic tobacco plants was already initiated after the first meiotic division. It is shown that, in such cells, the basic processes of cytoskeletal reorganization, which is typical for the simultaneous type of cytokinesis, remained unmodified. However, in most cases, premature division of cytoplasm took place with abnormalities, e.g., with the formation of a membranous “tunnel” or “gash.” It has been detected that the initialization of an additional round of cytokinesis is not an obstacle to the performance of the nuclear cycle or cytokinesis after the second meiotic division. Thus, in the presence of this anomaly, there is a change in the type of cytoplasm division, i.e., of simultaneous to successive.     

S-nitrosoglutathione induced nitrosative stress in yeast: modifying role of catalases

Possible role of catalases in modification of yeast Saccharomyces cerevisiae response to nitrosative stress was studied in the work. Yeast cell of a wild strain and catalase-defective strains were treated with nitric oxide donor S-nitrosoglutathione, then the cell survival rate, and the levels of protein carbonyls and oxidized glutathione were measured. It was shown that S-nitrosoglutathione decreased cells viability of wild and catalase-defective strains. Unexpected, yeast cells defective by both catalases survived successfully as compared with the cells of the wild strain. The intensity of stress was evaluated by measures of oxidative protein modifications and glutathione oxidation. Treatment with S-nitrosoglutathione did not affect the level of protein carbonyls and was lower by about 14 i 22 % in the cells of double mutant strains after treatments with S-nitrosoglutathione in concentrations of 10 and 20 mM. S-nitrosoglutathione induced a strong increase of the level of oxidized glutathione in yeast cells of the wild strain. This stress slightly increased the level of oxidized glutathione in the yeast cells defective by peroxisomal or both catalases. It is interesting, that an increase of oxidized glutathione level was not observed in the yeast cells defective by cytosolic catalase. The obtained results prove that catalases can modify yeast cell response to the nitrosative stress.     

Mutational pressure in genomes of alphaherpesviruses infecting human

Genomes of the herpes simplex viruses are extremely enriched with GC. Elevated G+C level in genomes of the simplex viruses is a result of their long-term evolution under the influence of the mutational pressure. We counted the rates of nucleotide substitutions from gene coding major capsid protein (MCP) (G+C = 0.68, 3GC = 0.89) of human simplex virus 1 (HSV-1) to the MCP gene (G+C = 0.70, 3GC = 0.91) of HSV-2 (the first pair of genes) and from the same MCP gene of HSV-1 to the homologous gene (G+C = 0.73, 3GC = 0.99) from cercopithecine herpes virus 16 (the second pair of genes). The rates of transitions from A-T to G-C base pairs increases 2.17-, 3.09-, and 1.27-fold in the first, second, and third codon positions, respectively, if compared those rates between the second and first pair of genes (the growth of GC-richness is only 3%). This effect is due to an approximately 90% GC-richness of the third codon positions in all those genes. Transitions caused by the strong mutational pressure (from A-T to G-C base pairs) have a low probability to occur in the third positions, but high probability to occur in the first and second positions. For MCP gene of human herpes 3, the probability of the occurrence of transition caused by mutational pressure in the third codon position is 2.36 times higher than in MCP gene of HSV1, and 3 times higher than in MCP gene of HSV2. These data could provide an explanation of rarely occurring relapses of herpes Zoster infection and frequently occurring relapses of herpes simplex infection.     

Disorders in lipid composition and properties of plasma membrane in epithelial cells of the placental villous chorion in chlamydial infection

Lipid composition of plasma membranes of placental epithelial cells of villous chorion of healthy women and those with chlamydiosis has been studied. Lipid composition of plasma membranes of ill women differs from that of healthy women by reduction of total phospholipids quantity, by the increase of the level of free cholesterol and free fatty acids. A disturbance in the ratio between certain lipid fractions and increasing quantity of lysophospholipids is observed. Two-fold oppression of plasma membranes Na+, K+ -ATPase activity of placental epithelial cells of villous chorion in ill women has been detected but Mg2+, Ca2+ -ATPase activity has not changed. Thus chlamydial infection causes significant disorders in lipid composition and functioning of epithelial cells membranes of chorion.     

Effect of pentosan polysulphate (SP-54) on the level of prion physiological form in rat tissues

It is established that except for already known influence of pentosan polysulphate (SP-54) on the expression of pathological prion, this preparation has an inhibiting effect in respect of physiological prion. Moreover, the reduction of concentration of physiologycal prion is registered in the central and peripheral organs of the prion-replicating system. It was also shown that inhibition of the studied protein leads to the growth in copper and zinc concentration in the proper organs and tissues, but at the same time activity of Cu/Zn-dependent superoxide dismutase does not change.     

Proteolytic enzymes and trypsin inhibitors of higher plants under stress conditions

The response of the components of a protease-inhibitor system of legume and cereal crops to stress factors was studied. It was found that salinization, heavy metal ions, and phytopathogenic flora inhibit the activity of neutral, acidic, and alkaline proteases at early stages of seed germination, the degree of the inhibition of the endoprotease activity being dependent on the index of tolerance of legume and cereal crops. It was shown that, in response to unfavorable conditions, accumulation of trypsin inhibitors occurs, which is accompanied by the appearance of new protein components, as indicated by electrophoresis. The results confirm the presumption that serine protease inhibitors are involved in the response of plants to stress factors.     

Cryopreservation of wheat (Triticum aestivum L.) egg cells by vitrification

A procedure has been developed for the cryopreservation of wheat female gametes. The procedure involves loading the cells with 25% concentrated vitrification solution consisting of 30% glycerol, 10% sucrose, 120 mM ascorbic acid (AA) and 5% propylene glycol (PG), dehydration in 80% concentrated vitrification solution, droplet vitrification and storage in liquid nitrogen, unloading and rehydration of the cells by gradual addition of isolation solution. Supplementation with AA significantly increased the proportion of viable egg cells after de- and rehydration. During the early phase of rehydration AA reduced the probability of membrane damage caused by rapid water uptake. Maintaining the temperature of the cells at 0°C during the de- and rehydration processes increased cell survival. Microscopic examination of the semi-thin sections of untreated and viable cryopreserved cells revealed that the vitrification process might cause changes in cell structure.