Identification of novel PTEN-binding partners: PTEN interaction with fatty acid binding protein FABP4

PTEN is a tumor suppressor with dual protein and lipid–phosphatase activity, which is frequently deleted or mutated in many human advanced cancers. Recent studies have also demonstrated that PTEN is a promising target in type II diabetes and obesity treatment. Using C-terminal PTEN sequence in pEG202–NLS as bait, yeast two-hybrid screening on Mouse Embryo, Colon Cancer, and HeLa cDNA libraries was carried out. Isolated positive clones were validated by mating assay and identified through automated DNA sequencing and BLAST database searches. Sequence analysis revealed a number of PTEN-binding proteins linking this phosphatase to a number of different signaling cascades, suggesting that PTEN may perform other functions besides tumor-suppressing activity in different cell types. In particular, the interplay between PTEN function and adipocyte-specific fatty-acid-binding protein FABP4 is of notable interest. The demonstrable tautology of PTEN to FABP4 suggested a role for this phosphatase in the regulation of lipid metabolism and adipocyte differentiation. This interaction was further studied using coimmunoprecipitation and gel-filtration assays. Finally, based on Biacore assay, we have calculated the K _D of PTEN–FABP4 complex, which is around 2.8 μM.     

Antiviral activity of 2,3-secotriterpenic hydrazones of the lupane and 19β,28-epoxy-18α-oleanane types

Novel hydrazones of the lupane and 19β,28-epoxy-18α-oleanane types have been synthesized via the interaction of 2,3-secotriterpenic aldehydonitriles with substituted hydrazines. As a result of the investigation of the antiviral activity of 2,3-secotriterpenic hydrazones against the Indiana strain of the vesicular stomatitis virus on two models of mammalian cell line infection, the acetylhydrazone of 1-cyano-2,3-seco-19β,28-epoxy-18α-olean-3-al has been found to have a high prophylactic activity of 0.00016 μg/ml to the vesicular stomatitis virus and to inhibit virus reproduction in primarily infected cells in a 0.21-μg/ml concentration.     

Experimental models of transgenic plants promising for the modern technologies

Transgenic popato plants have been created which express recombinant proteins, analogues of spidroin 1, the protein of the cobweb skeleton thread. Expression of the hybrid spidroin 1 genes possessing some repeated sequences retains both in the model test-tube-growing plants and in the crops. Expression level of the synthetic spidroin 1 genes and the level of accumulation of their products in plants depend on the type of promoter, number of repeats, organ specificity and plant species but not on the duration of plant material storage. The results show that the strategy based on constuction and expression of hybrid proteins which include the reporter protein makes it easier to select and analyse expression of hybrid proteins in transgenic organisms.     

Molecular Diagnostics of the Involvement of Visually Normal Mucosa in the Malignancy Process in Urothelial Bladder Cancer

Molecular Biology - Immunofluorescent method by flow cytometry was used to quantify the expression of the tumor-associated protein βIII-tubulin (TUBB3) in the tissue of urothelial bladder...     

Study of Multiple Enzymatic Incorporation of Modified Nucleotides of Purine and Pyrimidine Nature in the Growing DNA Chain

Molecular Biology - The substrate properties of nitrogen-base modified derivatives of purine and pyrimidine deoxynucleoside triphosphates during their simultaneous pairwise insertion into the...     

FRET-based biosensor for oleic acid in nanomolar range with quantum dots as an energy donor

A self-assembling sensor for oleic acid has been developed. The sensor consists of a self-assembling fluorescent dye labeled BSA and quantum dots CdSe/ZnS capped with 3-mercaptopropionic acid. The detection limit of the new sensor is 10-1000 nM. The influence of the quantum dot size on the FRET efficiency in the course of the interaction of the sensor system with the analyte has been studied. The pH dependence, aggregation stability. and electrophoretic properties of the sensor have been examined. The data suggest a new approach for the development of nanoscale FRET-based sensors operating effectively due to unique fluorescent properties of quantum dots as well as due to selective protein-ligand interactions.     

Phosphorylation of proteasomes and alpha-RNP from rat liver cells

In eukaryotic cells the population of proteasomes is heterogeneous. Here we have shown that proteasomes from nuclei and cytoplasm of rat liver cells differ in their subunit patterns. The subunit pattern of alpha-RNP differs from that of proteasomes, however, alpha-RNP particles contain the number of 26S proteasome subunits. Moreover, the proteasomes contain subunits of alpha-RNP. We have shown for the first time that nuclear proteasomes and alpha-RNP are hyperphosphorylated on threonine residues. Differences in phosphorylation state of subunits of nuclear and cytoplasmic proteasomes and alpha-RNP on threonine and tyrosine residues have been revealed. A suggestion is put forward that hyperphosphorylation of subunits may determine nuclear localization of these complexes in rat liver cells. The results obtained suggest that a highly specialized system of protein kinases and phosphatases may be involved in the regulation of phosphorylation state of different populations of proteasomes and alpha-RNP in rat liver cells.     

Phorbol 12-myristate 13-acetate inhibits apoptosis in erythroleukemia K562 cells induced by some nucleosides

We studied the ability of phorbol 12-myristate 13-acetat to prevent erythroid differentiation and apoptosis in erythroleukemic K562 cells induced by cytidine, thymidine, and guanosine. The exposure of cancer cells to combinations of phorbol 12-myrsitate 13-acetate (100 nM) nucleosides for two days led to a loss of hemoglobin production (marker of erythroid differentiation) in cells and increased expression of monocyte-macrophage lineage associated surface antigen CD14. The treatment of K562 cells with nucleosides only was accompanied by the activation of caspase-3 and caspase-9, rather than caspase-6, increased fluorescence of ethidium bromide and DAPI upon binding to DNA, and apoptosis. Intracellular activation of caspase-6, inhibition of caspase-9, a markedly decreased activity of caspase-3 and of fluorescence of DNA-binding dyes, and inhibition of apoptosis were observed when the cells were treated with phorbol 12-myeristet 13-acetate combined with nucleosides.     

Modification of the Sunflower Defensin SD2 Gene Sequence and Its Expression in Bacterial and Yeast Cells

To achieve broader range of the defensin antimicrobial activity, based on the sd2 gene sequence, the modified gene, sd2mod, was constructed. Hybrid genes, sd2-licBM2, licBM2-sd2, licBM2-sd2mod, and sd2mod-licBM2, in which the wild-type and modified gene sequences were fused in frame with the reporter gene encoding thermostable lichenase, were constructed. Expression of the wild-type, modified, and hybrid genes was examined in the cells of pro- and eukaryotes. It was demonstrated that these genes were efficiently expressed in the cells of lower eukaryotes, the yeast. Inhibiting effect of the SD2 and SDmod proteins as the components of the hybrid proteins, SD2-LicBM2 and SD2mod-LicBM2, on the growth of the Fusarium culmorum hyphae was similar to that of the wild-type and modified proteins. It was shown that the presence of lichenase in the hybrid proteins facilitated selection and analysis of the hybrid proteins expression in transgenic organisms.