Ribonuclease binase apoptotic signature in leukemic Kasumi-1 cells

Cytotoxic exogenous RNases triggering apoptotic response in malignant cells have potential as anticancer drugs; surprisingly, detailed characterization of the RNase-induced apoptosis has not been conducted so far. Here we show that a cytotoxic RNase from Bacillus intermedius (binase) induces extrinsic and intrinsic apoptotic pathways in leukemic Kasumi-1 cells. The experiments were performed using TaqMan Array Human Apoptosis 96-well Plate for gene expression analysis, and flow cytometry. Cytometric studies demonstrated dissipation of the mitochondrial membrane potential, opening of mitochondrial permeability transition pores, activation of caspases, increase of intracellular Ca²⁺ and decrease of reactive oxygen species levels. We found that expression of 62 apoptotic genes is up-regulated, including 16 genes that are highly up-regulated, and only one gene was found to be down-regulated. The highest, 16 fold increase of the expression level was observed for TNF gene. Highly up-regulated genes also include the non-canonical NF-κB signaling pathway and inflammatory caspases 1,4. The obtained results suggest that binase induces evolutionary acquired cellular response to a microbial agent and triggers unusual apoptosis pathway.     

An Adiabatic Calorimetry Method to Determine the Thermodynamic Characteristics of Cryoprotectants

Biophysics - Abstract—The efficiency of cryoprotectants used to protect cells from damage is usually evaluated by the changes in vital cell parameters after a freezing–thawing cycle....     

Erratum: “Regulation of antimicrobial peptide synthesis in larvae of <Emphasis Type="Italic">Calliphora vicina</Emphasis> (Diptera,Calliphoridae): a dose-dependent effect of ecdysteroids”

On page 297, left column, the final line of the ACKNOWLEDGMENTS should read: This research was supported by the grant of the Russian Scientific Foundation (project no. 1.53.392.2016).     

Targeting of adenovirus via genetic modification of the viral capsid combined with a protein bridge

A potential barrier to the development of genetically targeted adenovirus (Ad) vectors for cell-specific delivery of gene therapeutics lies in the fact that several types of targeting protein ligands require posttranslational modifications, such as the formation of disulfide bonds, which are not available to Ad capsid proteins due to their nuclear localization during assembly of the virion. To overcome this problem, we developed a new targeting strategy, which combines genetic modifications of the Ad capsid with a protein bridge approach, resulting in a vector-ligand targeting complex. The components of the complex associate by virtue of genetic modifications to both the Ad capsid and the targeting ligand. One component of this mechanism of association, the Fc-binding domain of Staphylococcus aureus protein A, is genetically incorporated into the Ad fiber protein. The ligand is comprised of a targeting component fused with the Fc domain of immunoglobulin, which serves as a docking moiety to bind to these genetically modified fibers during the formation of the Ad-ligand complex. The modular design of the ligand solves the problem of structural and biosynthetic compatibility with the Ad and thus facilitates targeting of the vector to a variety of cellular receptors. Our study shows that targeting ligands incorporating the Fc domain and either an anti-CD40 single-chain antibody or CD40L form stable complexes with protein A-modified Ad vectors, resulting in significant augmentation of gene delivery to CD40-positive target cells. Since this gene transfer is independent of the expression of the native Ad5 receptor by the target cells, this strategy results in the derivation of truly targeted Ad vectors suitable for tissue-specific gene therapy.     

The hybridization of tetraploid wheat with the octoploid wheat-wild rye amphiploid Elytricum fertile and the chromosome behavior in meiotic MI

The hybridization and chromosome behaviour regularities at meiotic MI of reciprocal F1 hybrids of tetraploid wheats T. durum and T. turgidum with 56-chromosome incomplete wheat-Elymus amphiploid (IWEA) Elytricum fertile, were investigated. Variation of main indices of the hybridization has been revealed to be determined by variance of such factors as cross direction, genotype peculiarities of wheat strains and their interaction. Elymus sibiricus genome, contained in IWEA karyotype, carries several genes, that can suppress wheat diploidization system. Reduction of homologous chromosome synapsis is not determined by these genes, but is connected with influence of other Elymus sibiricus chromosome factors.     

Production of transgenic pea (Pisum sativum L.) plants resistant to the herbicide pursuit

Transgenic pea (Pisum sativum L.) plants containing mutant ahas/als gene were obtained using Agrobacterium-mediated genetic transformation. Transformation has been carried out using cocultivation of pea explants with Agrobacterium tumefaciens strain lBA4404 carrying genetic vectors pCB004, pCB006 and pCB007 containing ahas/als and nptII genes. The presence of transferred genes in the genomes of transgenic plants has been confirmed by PCR analysis.     

Expression of the FLT3-ITD oncogene sensitizes murine progenitor B-cell line BAF3 to cytotoxic action of binase

Acute myeloid leukemia makes up about 30% of all leukemia cases in adults. Mutations in the genes of the receptor tyrosine kinases KIT and FLT3, along with chromosomal translocations, are frequently found in leukemic cells. In the current work, we show that the transgenic B-cells BAF3/FLT3-ITD are significantly more sensitive to cytotoxic action of the ribonuclease binase than original BAF3 cells. BAF3/FLT3-ITD cells differ from BAF3 in expression of the FLT3-ITD oncogene, which results in the alteration of normal signaling pathways. We observed a similar effect previously when studying binase cytotoxic action in cells Kasumi-1 and FDC-P1-N822K, in which the activated oncogene KIT-N822K was expressed. An elevated cytotoxicity of binase to the cells that express the FLT3-ITD oncogene indicates that, as in case of the FDC-P1 cells transduced by the KIT oncogene, the expression of an activated oncogene determines the cell’s sensitivity to the binase action.     

Neurogenic and septic inductions of synthesis of peptide antibiotics in larvae of <Emphasis Type="Italic">Calliphora vicina</Emphasis> R.-D. (Diptera: Calliphoridae)

Synthesis of antimicrobial peptides in diapausing larvae of the blowfly Calliphora vicina can be induced by two different pathways. One pathway is well known in insects and includes recognition of microbial particles by the pattern-recognizing receptors. The other pathway includes perception and transduction of stress signal to immunocompetent cells by neuroendocrine system. This phenomenon consists in stimulation of synthesis of defensins, cecropins, and diptericins under effect of chromic stimulation of mechanoreceptors with ligature applied on the larva head end. Formation of immune response in brain is established to need less than 30 s, after which isolation of the neuroendocrine complex does not eliminate activation of immune response. As judging from rate of the neurogenic induction, transduction of the stimulating signal from brain to the immune system cells can be connected with release into hemolymph of biogenic amines or other neurohormones stored preliminarily in the neurohemal organ. The nature of this inductor at present remains unknown, as analysis of role of octopamine, dopamine, and adipokinetic hormone did not reveal stimulating effect on synthesis of bactericidal peptides. Physiological mechanism of this phenomenon is not finally understood, its key links seem to be CNS, hormonal factor of cardiac bodies, and system of antimicrobial peptides. Synthesis of antimicrobial peptides is directly regulated by the neuroendocrine system that can produce both stimulating and stress action by reminding in this aspect the known immunoneuroendocrine interrelations in vertebrates. The existence of similar integrating mechanisms in such polar animal kingdom groups as insects and vertebrates indicate that they are more ancient than this was considered earlier.     

Alkaline serine proteinase and lectin isolation from the culture fluid of Bacillus subtilis

Bacillus subtilis strain 316 M was found to produce extracellular alkaline serine proteinase and lectin. The characteristics of proteinase and lectin accumulation during the growth of the producer organism were found to be similar. The maxima of proteolytic and lectin activities were close and observed at 16 h and 14 h of B. subtilis 316 M batch cultivation, respectively. Alkaline serine proteinase was purified by ion exchange chromatography directly from the culture fluid. Proteinase (eluate) purified 40-fold possessed 60–90 units/ml of caseinolytic activity and 240–320 units/ml of elastolytic activity. Eluate obtained after enzyme sorption on the ion exchanger was used for lectin isolation followed by ammonium sulphate precipiration. Lectin purified 12.3-fold was shown to have a high carbohydrate specificity to N-glycolylneuraminic, N-acetylneuraminic, N-acetylmuramic and d-galacturonic acids with minimal inhibiting concentrations of 2.5–7.5 mm. *** DIRECT SUPPORT *** AG903053 00002     

High-level expression of human interferon alpha-2b in transgenic carrot (Daucus carota L.) plants

In this study, we report the obtaining of carrot plants expressing human interferon alpha-2b via Agrobacterium-mediated transformation using two vector constructs containing the sequence coding for interferon gene fused with Nicotiana plumbagenifolia calreticulin apoplast targeting signal driven by 35S CaMV promoter and root-specific Mll promoter. The human interferon alpha-2b gene was correctly translated in carrot plants according to Western blot analysis. The recombinant protein exhibited antiviral activity in vitro by inhibition of vesicular stomatitis virus replication in established piglet testicular cells. The results demonstrated the higher activity of interferon accumulated in carrot plants for young leaves (up to 50.7 × 10³ IU/g FW) compared to the mature ones probably due to the degradation-susceptible nature of this protein. The taproot-expressing system could have also provided the sufficient protein amounts (up to 16.5 × 10³ IU/g FW) and could possibly be used for generating interferon alpha-2b protein in planta for preventing and curing infectious diseases.