Genetic Control of Biogenic-Amine Systems in Drosophila Under Normal and Stress Conditions

The contents of octopamine and its precursors (tyrosine and tyramine) were studied in adults of two lines of Drosophila virilis with contrasting stress responses. It was demonstrated that in individuals responding to stress by a hormonal stress reaction (line 101), the contents of octopamine and tyrosine are lower than in nonresponding flies (line 147). It was found that there is no difference between the lines in the level of tyramine under normal conditions. The dopamine response to stressor was also studied. Genetic analysis of these differences revealed that they are controlled by a single gene and that the gene is not sex-linked. The gene controlling the response was found to be linked to chromosome 6 of D. virilis.     

Olive Phenolics as c-Met Inhibitors: (-)-Oleocanthal Attenuates Cell Proliferation,Invasiveness, and Tumor Growth in Breast Cancer Models

Dysregulation of the Hepatocyte growth factor (HGF)/c-Met signaling axis upregulates diverse tumor cell functions, including cell proliferation, survival, scattering and motility, epithelial-to-mesenchymal transition (EMT), angiogenesis, invasion, and metastasis. (-)-Oleocanthal is a naturally occurring secoiridoid from extra-virgin olive oil, which showed antiproliferative and antimigratory activity against different cancer cell lines. The aim of this study was to characterize the intracellular mechanisms involved in mediating the anticancer effects of (-)-oleocanthal treatment and the potential involvement of c-Met receptor signaling components in breast cancer. Results showed that (-)-oleocanthal inhibits the growth of human breast cancer cell lines MDA-MB-231, MCF-7 and BT-474 while similar treatment doses were found to have no effect on normal human MCF10A cell growth. In addition, (-)-oleocanthal treatment caused a dose-dependent inhibition of HGF-induced cell migration, invasion and G1/S cell cycle progression in breast cancer cell lines. Moreover, (-)-oleocanthal treatment effects were found to be mediated via inhibition of HGF-induced c-Met activation and its downstream mitogenic signaling pathways. This growth inhibitory effect is associated with blockade of EMT and reduction in cellular motility. Further results from in vivo studies showed that (-)-oleocanthal treatment suppressed tumor cell growth in an orthotopic model of breast cancer in athymic nude mice. Collectively, the findings of this study suggest that (-)-oleocanthal is a promising dietary supplement lead with potential for therapeutic use to control malignancies with aberrant c-Met activity.     

Role of charge screening and delocalization for lipophilic cation permeability of model and mitochondrial membranes

The effects of the mitochondria-targeted lipophilic cation dodecyltriphenylphosphonium (C₁₂TPP, the charge is delocalized and screened by bulky hydrophobic residues) and those of lipophilic cations decyltriethylammonium bromide and cetyltrimethylammonium bromide (C₁₀TEA and C₁₆TMA, the charges are localized and screened by less bulky residues) on bilayer planar phospholipid membranes and tightly-coupled mitochondria from the yeast Yarrowia lipolytica have been compared. In planar membranes, C₁₂TPP was found to generate a diffusion potential as if it easily penetrates these membranes. In the presence of palmitate, C₁₂TPP induced H⁺ permeability like plastoquinonyl decyltriphenilphosphonium that facilitates transfer of fatty acid anions (Severin et al., PNAS, 2010, 107, 663–668). C₁₂TPP was shown to stimulate State 4 respiration of mitochondria and caused a mitochondrial membrane depolarization with a half-maximal effect at 6 μM. Besides, C₁₂TPP profoundly potentiated the uncoupling effect of endogenous or added fatty acids. C₁₀TEA and C₁₆TMA inhibited State 4 respiration and decreased the membrane potential, though at much higher concentrations than C₁₂TPP, and they did not promote the uncoupling action of fatty acids. These relationships were modeled by molecular dynamics. They can be explained by different membrane permeabilities for studied cations, which in turn are due to different availabilities of the positive charge in these cations to water dipoles.     

Advanced procedures for labeling of antibodies with quantum dots

Semiconductor quantum dots (QDs) are proved to be unique fluorescent labels providing excellent possibilities for high-throughput detection and diagnostics. To explore in full QDs’ advantages in brightness, photostability, large Stokes shift, and tunability by size fluorescence emission, they should be rendered stable in biological fluids and tagged with the target-specific capture molecules. Ideal QD-based nanoprobes should not exceed 15 nm in diameter and should contain on their surface multiple copies of homogeneously oriented highly active affinity molecules, for example, antibodies (Abs). Direct conjugation of QDs with the Abs through cross-linking of QDs’ amines with the sulfhydryl groups issued from the reduced Abs’ disulfide bonds is the common technique. However, this procedure often generates conjugates in which the number of functionally active Abs on the surface of QDs does not always conform to expectations and is often low. Here we have developed an advanced procedure with the optimized critical steps of Ab reduction, affinity purification, and QD–Ab conjugation. We succeeded in reducing the Abs in such a way that the reduction reaction yields highly functional, partially cleaved, 75-kDa heavy–light Ab fragments. Affinity purification of these Ab fragments followed by their tagging with the QDs generates QD–Ab conjugates with largely improved functionality compared with those produced according to the standard procedures. The developed approach can be extended to conjugation of any type of Ab with different semiconductor, noble metal, or magnetic nanocrystals.     

Retrospective molecular-cytogenetic characteristics of tetraploidy in early human embryolethality

The ploidy level of noncultivated extraembryonic tissues was studied by fluorescence in situ hybridization in 30 human I trimester spontaneous abortions with tetraploid or diploid-tetraploid karyotype after conventional cytogenetic analysis. Only thirteen embryos (43 %) were verified to be tetraploid that provides evidence for the hypothesis of placental cell polyploidization during long-term in vitro cultivation. It is shown that preferred compartmentalization of tetraploid cells in the inner cell mass derivatives is associated with blighted ovum - the most severe type of human embryo dysmorphogenesis.     

Detection of carcinoembryonic antigen using single-domain or full-size antibodies stained with quantum dot conjugates

Compact single-domain antibodies (sdAbs) are nearly 13 times smaller than full-size monoclonal antibodies (mAbs) and have a number of advantages for biotechnological applications, such as small size, high specificity, solubility, stability, and great refolding capacity. Carcinoembryonic antigen (CEA) is a tumor-associated glycoprotein expressed in a variety of cancers. Detection of CEA on the tumor cell surface may be carried out using anti-CEA antibodies and conventional fluorescent dyes. Semiconductor quantum dots (QDs) are brighter and more photostable than organic dyes; they provide the possibility for labeling of different recognition molecules with QDs of different colors but excitable with the same wavelength of excitation. In this study, the abilities for specific detection of CEA expressed by tumor cells with anti-CEA sdAbs biotinylated in vitro and in vivo, as well as with anti-CEA mAbs biotinylated in vitro, were compared using flow cytometry and the conjugates of streptavidin with QDs (SA-QDs). The results demonstrated that either in vitro or in vivo biotinylated anti-CEA sdAbs are more sensitive for cell staining compared to biotinylated anti-CEA mAbs. The data also show that simultaneous use of biotinylated sdAbs with highly fluorescent SA-QDs can considerably improve the sensitivity of detection of CEA on tumor cell surfaces.     

Interaction of tetraphenylphosphonium and dodecyltriphenylphosphonium with lipid membranes and mitochondria

The permeability of a planar lipid membrane (composed of diphytanoylphosphatidylcholine) for tetraphenylphosphonium (TPP) was investigated. The observed level of the diffusion potential generated as a function of the TPP concentration gradient differed from the theoretically expected value, possibly due to proton leakage of the membrane mediated by the traces of fatty acids in the phospholipid forming the membrane. Using the molecular dynamics approach to study movement of TPP and dodecyltriphenylphosphonium (C₁₂TPP) with different affinity to the lipid bilayer through a bilayer lipid membrane, it was found that C₁₂TPP has a greater affinity to the membrane surface than TPP. However, the two cations have the same activation energy for transmembrane transfer. Interaction of TPP and C₁₂TPP with tightly-coupled mitochondria from the yeast Yarrowia lipolytica was also investigated. At low, micromolar concentrations, both cations are “relatively weak, mild uncouplers”, do not shunt electron transfer along the respiratory chain, do not disturb (damage) the inner mitochondrial membrane, and profoundly promote the uncoupling effect of fatty acids. At higher concentrations they inhibit respiration in state 3, and at much higher concentrations they induce swelling of mitochondria, possibly due to their detergent action.     

Synthesis, biological activity and comparative analysis of DNA binding affinities and human DNA topoisomerase I inhibitory activities of novel 12-alkoxy-benzo[c]phenanthridinium salts

New antitumor 12-alkoxy-benzo[c]phenanthridinium derivatives were obtained in high yields through multistep syntheses. Analysis of DNA binding and human DNA topoisomerase I inhibitory activities demonstrates that new compounds, combining 2, 6, and 12 substitutions, interact strongly with DNA and exhibit important topoisomerase I inhibition. The cytotoxicities against solid tumor cell lines are also determined and compared with those for fagaronine and ethoxidine.     

Molecular-genetic detection of a nonpathogenic genotype of <Emphasis Type="Italic">Spironucleus barkhanus</Emphasis> (Diplomonadida: Hexamitidae) in the black Baikal grayling (<Emphasis Type="Italic">Thymallus arcticus baicalensis</Emphasis> Dybowski, 1874)

A nonpathogenic genotype of Spironucleus barkhanus (Diplomonadia: Hexamitidae) has been revealed for the first time in the Baikal grayling (Salmoniformes: Thymallidae) by molecular-genetic methods. Sequences corresponding to a fragment of the gene encoding the small ribosomal subunit RNA (SSU rRNA) have been obtained. The genotype of S. barkhanus from Baikal fish is identical to that characteristic of the parasite found in freshwater salmonid fishes.