Aim . To analyze the effect of growing liver biological set on viability and proliferative activity of various cellular cultures in vitro. Material and Methods. The biological combination is an extract from the growing liver obtained by using of original technique. We have assessed extract’s effect on the following cellular lines: hepatic carcinoma Huh7, L-fibroblasts of mice, murine bone marrow mesenchymal stem cells. Viability and proliferative activity were assessed by staining the cells with trypan blue and visual counting of cells under phase contrast microscopy. Result. Biological combination from neonatal piglet’s growing liver dose-dependently protects hepatocyte-like cells from deprivation of fetal serum and stimulates cellular growth in presence of serum. High concentrations of HRS do not lead to growth arrest of the Huh7 cells. At the same time, it is a cytostatic (or cytotoxic) for murine L- fibroblasts. Limited protective effect of the combination on the deprivation of serum when exposed to bone marrow stem cells was revealed. Conclusion. Our data show that the extract may be considered as an important regulator of reparative regeneration of liver with protective and/or stimulating effect on mesenchymal stem and hepatic-like cells and cytostatic effect on fibroblasts. So, further trials are necessary.

Tankyrase, one of the NAD+ ADP-ribosyltransferases, is a target for drugs developed for their anticancer and other pharmacological activities. We designed an assay for estimation of the inhibition or activation of the enzyme in preclinical studies. In mice, the highest specific activity of tankyrase was observed in thymus, spleen, pancreas, and bone marrow. In murine liver, tankyrase is active in ontogenesis and during reparative regeneration; however, the basal activity is hardly detectable in normal liver and most of other organs of adult animals. We suggest that tankyrase is a part of the tissue growth and repair machinery, while its age-dependent inhibition, when an organism stops growing, turns on phenoptosis.

Mannose-binding lectin was identified as a substrate of tankyrase 2, an enzyme that catalyzes poly(ADP-ribosyl)ation. The endogenous tankyrase 2 was isolated out of cytoplasm of human embryonic kidney cells. It was bound to a soluble complex of at least two other proteins; they were identified using specific antibodies and other approaches as keratin 1 and mannose-binding lectin. Using immunoblot analysis and radioactive labeling, we detected tankyrase-2-dependent poly(ADP-ribosyl)ation of mannose-binding lectin. In the presence of NAD(+), the complex of keratin 1 and lectin was dissociated, what was recorded during elution of its separate components out of affinity columns and by decrease of their apparent molecular masses during gel-filtration. Tankyrase 2 also inhibited the carbohydrate-binding function of the lectin. The latter effect was observed using mannose-binding lectin out of human serum, which is free from keratin 1. As a result of tankyrase-2 activity, the lectin lost its affinity to mannan-agarose. The discovery of this new biochemical mechanism justifies further analysis of its physiological and medical significance.

We have isolated and purified endogenous cytosolic tankyrase from human embryonic kidney cells of line 293. Our data confirm a model of De Rycker and Price who consider that tankyrase is a master scaffolding protein capable of regulating assembly of large protein complexes. We have also studied kinetic characteristics of tankyrase in the complex, pH dependence of the enzyme activity, and its physicochemical properties.

Chromosome telomeres of humans and many model organisms contain a structure called a t-loop, which is maintained by TERF, TINF2, Pot1, and other proteins. Increase in TERF1 concentration prevents telomere elongation by telomerase. Decrease in TERF2 concentration (preventing t-loop formation) is accompanied by blockade of proliferation and appearance of other signs of cellular senescence in experiments. Natural regulation of TERF1 involves tankyrase, ATM protein kinase, and fluctuations of the protein level across a cell cycle. The telomere nucleoprotein complex also interacts with various polypeptide macromolecules (e.g., Sir2, PinX1, Rap1, Ku, Rad50/Mre11/Nbs1) responsible for heterochromatin formation, modulation of telomerase activity, DNA repair, and signaling to other cell compartments about telomere state. Study of structure and functioning of telomere nucleoprotein complex may contribute to elucidation of poorly understood mechanisms of aging and processes of tumor transformation of cells.

We studied the subcellular localization of tankyrase in primary and immortalized human cell cultures. In embryonic kidney cell line 293 the enzyme was excluded from the nuclei and distributed in fractions of soluble cytosolic proteins and low-density microsomes. Newly revealed cytosolic tankyrase in its poly(ADP-ribosyl)ated form was passed through a Sepharose 2B column and eluted as an apparently monomeric protein. The cytosolic localization of the enzyme correlated with its relatively high activity in the 293 cell line in comparison to eight other studied cell types.

Three forms of baker's yeast transketolase have been revealed. These forms differed in thermal stability and elution profiles during chromatography on a phosphocellulose column and migrated with identical rates during electrophoresis in the presence of sodium dodecyl sulfate. The same forms in yeast, pig and rat liver and in different organs and tissues of the rabbit were found to be similar in their thermal stability and chromatographic properties. The relative amounts of the forms appeared to depend on the physiological state of the organism. Crystals of the three pure forms were grown using ammonium sulfate as the precipitating agent. These crystals differed morphologically and by stability upon storage. The possibility of interconversion of the transketolase forms is discussed.