Integrating proteomic and functional genomic technologies in discovery-driven translational breast cancer research

The application of state-of-the-art proteomics and functional genomics technologies to the study of cancer is rapidly shifting toward the analysis of clinically relevant samples derived from patients, as the ultimate aim of translational research is to bring basic discoveries closer to the bedside. Here we describe the essence of a long-term initiative undertaken by The Danish Centre for Translational Breast Cancer Research and currently underway for cancer biomarker discovery using fresh tissue biopsies and bio-fluids. The Centre is a virtual hub that brings together scientists working in various areas of basic cancer research such as cell cycle control, invasion and micro-environmental alterations, apoptosis, cell signaling, and immunology, with clinicians (oncologists, surgeons), pathologists, and epidemiologists, with the aim of understanding the molecular mechanisms underlying breast cancer progression and ultimately of improving patient survival and quality of life. The unifying concept behind our approach is the use of various experimental paradigms for the prospective analysis of clinically relevant samples obtained from the same patient, along with the systematic integration of the biological and clinical data.     

Study of the association between constitutional exogenous obesity and polymorphism of the apolipoprotein B gene

An attempt was made to associate the insertion-deletion (Ins/Del) polymorphism of the apolipoprotein B gene (apoB) with obesity and to identify alleles and genotypes predisposing to this disorder. The apoB Ins/Del allele frequencies observed in the Russian population were similar to those in West European populations and significantly differed from frequencies reported for Asian populations. Patients with obesity did not differ from healthy individuals in allele and genotype frequencies regardless of whether total or sex-stratified samples were compared. Estimation of relative risk for individuals with genotype Ins/Ins did not reveal a significant association between obesity and this genotype. Thus, constitutional exogenous obesity did not prove to be associated with the Ins/Del polymorphism of the apoB gene in the Russian population.     

Changes in the relative number of bipolar-like cells in the retina of Pleurodeles waltl as a function of age and as a result of light exposure

The study of the population of bipolar-like cells (displaced bipolars) was continued in order to establish their role in development and regeneration of the retina in lower vertebrates. The size of the population of these cells was estimated on serial semithin sections in the retina of normal eyes in adult and young newt Pleurodeles waltl, as well as in adult newts subjected to long-term bright illumination. The population of displaced bipolars was significantly increased with reference to all cells of the outer nuclear layer. In young and illuminated animals, their numbers were approximately 1.3 and 1.4 times that in the adult animals not exposed to constant light. The results obtained favor the earlier suggestion of the involvement of the displaced bipolars in growth and restoration of the outer nuclear layer in the retina of newts during development and after trauma.     

Interaction of spin-labeled methacyne analog with butyrylcholinesterase

Interaction between spin-labeled methacyne (I) and butyrylcholinesterase (BChE) was studied by ESR and enzyme kinetic methods. The compound (I) was shown to be a competitive reversible inhibitor, the value of Ki appeared to be 1.3 X 10(-5) M. Insertion of nitroxyl fragment in the methacyne molecule results in a two-fold increase of its inhibitory activity. The ESR spectrum of the enzyme-inhibitor complex was registered. This complex dissociates under the action of eserine, tetramethylammonium and hexamethonium. Scatchard plot reveals two different types of binding sites with Kdiss values 1.5 X 10(-5) M and 2.6 X 10(-4) M. One type of binding sites is identified as the enzyme active centre. The restricted motion of (I) in complex with BChE proves the assumption that the enzyme active centre is located in the split of macromolecule surface.     

Binding of reversible spin-labeled inhibitors with an butyrylcholinesterase active center

The interaction of spin-labeled metacyn, procaine, carbolin and bivalent cations (Ca2+, Co2+, Ni2+) with butyrylcholinesterase (BChE) was studied by ESR and enzyme kinetic methods. The effect of pH, ionic strength and organic solvent was analysed. Spin-labeled metacyn binds at the anionic site of BChE active centre. This complex is stabilized both with coulombic and hydrophobic interactions, ionizing group of active centre with pK 6-7 also affects the binding. Spin-labeled procaine appeared to be enzyme competitive inhibitor (Ki = 4 X 10(-5) M) and is located, most probably, at the same site. Activating effect of Ca2+ ions on BChE was confirmed. Simultaneous application of spin labels and paramagnetic ions demonstrates that cations Co2+ and Ni2+ bind with BChE in the close vicinity of spin-labeled inhibitor site. Paramagnetic cations are located more closely to the cationic part of the inhibitor molecule than to the hydrophobic one, and can be displaced by surplus of Ca2+ ions. The experimental data testify the model of anionic centre which consists of bivalent metal ions and aminoalcyl cationic group subsites and is located in a hydrophobic pocket of the enzyme surface.     

Optimization of the schemes for using interferon inducers

The results of study on 2 Soviet interferon inductors, i.e. the synthetic polyguacyl polynucleotide and natural double-strand phage RNA or dsRNA were studied. It was shown that the time course of accumulation and period of circulation of interferon depended on the route of the inductor administration. The antiviral activity of polyguacyl and dsRNA in experimental influenza and tick-borne encephalitis is described. The maximum protective effect with respect to experimental influenza was observed with intranasal administration of the drugs 4 hours before inoculation. A pronounced protective effect with respect to tick-borne encephalitis was observed with intraperitoneal administration of the inductors or their use in the form of aerosols. Direct correlation between interferon production and the final protective effect was found.     

Motor reactions and vestibular reflexes in cats and monkey in weightlessness

Close morpho-functional relationships of the cerebellum and vestibular system at all stages of phylogenesis of vertebrates suggest that cerebellum can be regarded as an important center of gravireceptive function. Direct examination of electrical activity of the labyrinth in cats during transient (1-2 sec) state of weightlessness produced by free fall has shown that there was an almost two fold increase in both the rate and amplitude of electrical activity in the vestibular ganglion. It is commonly accepted at present time that the conditions of orbital flight around Earth closely connect with weightlessness that usually manifests itself as undesirable factor of flight. It is known, that vestibular, proprioceptive, visual and other sensory modalities are converted on the cerebellum, which would indicate that this information is used for motor coordination and spatial orientation. Undoubtedly, origin of many vestibulo-motor disturbances during flight and in postflight period to a considerable degree depends on weightlessness. On the whole the visual illusions, motor discoordination, and space sickness, including vomiting are referred to the "space adaptation syndrome." But nature of these disturbances still is not well understood. This investigation was dedicated to study of vestibular and motor reactions of cats and monkey in short-term microgravity.