MET-dependent cancer invasion may be preprogrammed by early alterations of p53-regulated feedforward loop and triggered by stromal cell-derived HGF

MET, a receptor protein tyrosine kinase activated by hepatocyte growth factor (HGF), is a crucial determinant of metastatic progression. Recently, we have identified p53 as an important regulator of MET-dependent cell motility and invasion. This regulation occurs via feedforward loop suppressing MET expression by miR- 34-dependent and -independent mechanisms. Here, by using Dicer conditional knockout, we provide further evidence for microRNA-independent MET regulation by p53. Furthermore, we show that while MET levels increase immediately after p53 inactivation, mutant cells do not contain active phosphorylated MET and remain non-invasive for a long latency period at contrary to cell culture observations. Evaluation of mouse models of ovarian and prostate carcinogenesis indicates that formation of desmoplastic stroma, associated production of HGF by stromal cells and coinciding MET phosphorylation precede cancer invasion. Thus, initiation mutation of p53 is sufficient for preprogramming motile and invasive properties of epithelial cells, but the stromal reaction may represent a critical step for their manifestation during cancer progression.Key words: feedforward loop, HGF, invasion, MET, metastasis, motility, p53 mutation     

An improved method for the construction of decoy peptide MS/MS spectra suitable for the accurate estimation of false discovery rates

The relevance of libraries of annotated MS/MS spectra is growing with the amount of proteomic data generated in high-throughput experiments. These reference libraries provide a fast and accurate way to identify newly acquired MS/MS spectra. In the context of multiple hypotheses testing, the control of the number of false-positive identifications expected in the final result list by means of the calculation of the false discovery rate (FDR). In a classical sequence search where experimental MS/MS spectra are compared with the theoretical peptide spectra calculated from a sequence database, the FDR is estimated by searching randomized or decoy sequence databases. Despite on-going discussion on how exactly the FDR has to be calculated, this method is widely accepted in the proteomic community. Recently, similar approaches to control the FDR of spectrum library searches were discussed. We present in this paper a detailed analysis of the similarity between spectra of distinct peptides to set the basis of our own solution for decoy library creation (DeLiberator). It differs from the previously published results in some key points, mainly in implementing new methods that prevent decoy spectra from being too similar to the original library spectra while keeping important features of real MS/MS spectra. Using different proteomic data sets and library creation methods, we evaluate our approach and compare it with alternative methods.     

Diversity of wild and domestic pig populations estimated by a set of serum allotypes

Variation of serum protein allotypes serving as genetic markers of the blood has been analyzed in 29 populations of the domestic pig and subspecies of the wild boar. The population biodiversity and genetic structure have been estimated by two methods: by the frequencies of allotype combinations and with the use of a map constructed in the space of two principal components. The results obtained are the basis for determining the characteristics of the microevolution of wild boars and formation of the breeds of domestic pigs.     

Inhibition of protein synthesis during associative memory reactivation produces reversible or irreversible amnesia in the snail Helix lucorum

Effects of protein synthesis inhibitors on reactivation processes of food aversion conditioning were inverstigated in snail Helix lucorum. Protein synthesis inhibitor (PSI, anisomycin, 0.4 mg, or cycloheximede, 0.6 mg) was injected into snail body cavity 24 hours after 3-day training; then conditioned stimulus (banana) was presented and memory was tested. It was found that 2.5-3 hours after first reminding, associative food conditioning was suppressed, recovering of the conditioning was observed 4.5-5.5 hours after first reminding. In other group of snails, PSI injections were single (1.8 mg) or triple (0.6 mg with 2-hour interval). Reminding stimulus was presented after each injection. In this case, suppression of food aversion conditioning was also observed 2.5-3 hours after first reminding, while amnesia in this case lasted over 30 days. Repeated training of the group of snails recovered the food aversion conditioning only partially. In control snails (saline instead of PSI or 3 injections of PSI without reminding), foot aversion conditioning was detected 30 days after first training. Thus we found that PSI effects during reminding of food aversion conditioning produced two phases amnesia: (1) the easily suppressed by PSI transient phase lasted 2-3 hours, and (2) irreversible phase, its suppression by high doses of PSI-initiated amnesia lasting over 1 month. Second phase of amnesia was not recovered after repeated training. It was suggested that reminding induced reconsolidation of initial memory. Its suppression by protein synthesis inhibitors results in erasing of memory trace and disturbs repeated consolidation.     

Effect of zif268 early gene antisense oligonucleotide on mechanisms of synapse-specific plasticity

It was found that nociceptive sensitization was followed by long-term facilitation of synaptic responses evoked by chemical sensory stimulation of the snail "head", tactile stimulation of the snail "head" and foot in LP11 command neuron of defence behavior in snail Helix lucorum. Sensitizing stimulation during the intracellular injection of antisense olygonucleotide immediate early gene zif268 resulted in a selective suppression of synaptic facilitation in LP11 neuron responses evoked by tactile and chemical stimulation of the snail "head". At the same time, development of synaptic facilitation of responses in the LP11 neuron evoked by tactile stimulation of the foot was the same as in control sensitized snails. The results suggest that immediate early gene zif268 is selectively involved in the mechanisms of specific regulation of plasticity of the synaptic "input" of LP11 neuron from sensory receptors of the snail "head".     

Taxonomic characterization of new alkaliphilic and alkalitolerant methanotrophs from soda lakes of the Southeastern Transbaikal region and description of Methylomicrobium buryatense sp.nov

Five strains of obligate methanotrophic bacteria (4G, 5G, 6G, 7G and 5B) isolated from bottom sediments of Southeastern Transbaikal soda lakes (pH 9.5-10.5) are taxonomically described. These bacteria are aerobic, Gram-negative monotrichous rods having tightly packed cup-shaped structures on the outer cell wall surface (S-layers) and Type I intracytoplasmic membranes. All the isolates possess particulate methane monooxygenase (pMMO) and one strain (5G) also contains soluble methane monooxygenase (sMMO). They assimilate methane and methanol via the ribulose monophosphate pathway (RuMP). The isolates are alkalitolerant or facultatively alkaliphilic, able to grow at pH 10.5-11.0 and optimally at pH 8.5-9.5. These organisms are obligately dependent on the presence of sodium ions in the growth medium and tolerate up to 0.9-1.4 M NaCl or 1 M NaHCO3. Although being mesophilic, all the isolates are resistant to heating (80 degrees C, 20 min), freezing and drying. Their cellular fatty acids profiles primarily consist of C(16:1). The major phospholipids are phosphatidylethanolamine and phosphatidylglycerol. The main quinone is Q-8. The DNA G+C content ranges from 49.2-51.5 mol %. Comparative 16S rDNA sequencing showed that the newly isolated methanotrophs are related to membres of the Methylomicrobium genus. However, they differ from the known members of this genus by DNA-DNA relatedness. Based on pheno- and genotypic characteristics, we propose a new species of the genus Methylomicrobium Methylomicrobium buryatense sp. nov.     

Fast and aimed delivery of voltage-sensitive dyes to mammalian brain slices by biolistic techniques

Fast voltage-sensitive dyes (VSD) are widely used in modern neuroscience for optical recording of electrical potentials at many levels, from single cell compartment to brain areas, containing populations of many neural cells. The more lipophilic a VSD, the better signal-to-noise ratio of the optical signal, but there are no effective ways to deliver a water-insoluble dye into the membrane of live cell. Here we report a new protocol based on rapid biolistic delivery of VSDs, which is optimal for further recordings of optical signals from live neurons of rat brain slices. This protocol allows us to stain locally (150 mkm) neural somata of brain structures with a Golgi-like pattern, and a VSD propagates even to distant neurites of stained cells very quickly. This technique also can be used for rapid local delivery of any lipophilic and water-insoluble substances into live cells, further optical recording of neural activity, and analysis of potential propagation in a nerve cell.     

Dominant activation of the hedgehog signaling pathway alters development of the female reproductive tract

The role of hedgehog (HH) signaling in reproductive tract development was studied in mice in which a dominant active allele of the signal transducer smoothened (SmoM2) was conditionally expressed in the Müllerian duct and ovary. Mutant females are infertile, primarily because they fail to ovulate. Levels of mRNA for targets of HH signaling, Gli1, Ptch1, and Hhip, were elevated in reproductive tracts of 24-day-old mutant mice, confirming overactivation of HH signaling. The tracts of mutant mice developed abnormally. The uterine luminal epithelium had a simple columnar morphology in control mice, but in mutants contained stratified squamous cells typical of the cervix and vagina. In mutant mice, the number of uterine glands were reduced and the oviducts were not coiled. Expression of genes within the Hox and Wnt families that regulate patterning of the reproductive tract were altered. Hoxa13, which is normally expressed primarily in the vagina and cervix, was expressed at 12-fold higher levels in the uterus of mutant mice compared with controls. Wnt5a, which is required for development of the cervix and vagina and postnatal differentiation of the uterus, was expressed at higher levels in the oviduct and uterus of mutant mice compared with controls. Mating mutant females with fertile or vasectomized males induced a severe inflammatory response in the tract. In summary, overactivation of HH signaling causes aberrant development of the reproductive tract. The phenotype observed could be mediated by ectopic expression of Hoxa13 in the uterus and elevated levels of Wnt5a in the oviducts and uterus.