Mechanism of inactivation and mutagenesis induced by ethyleneimine in Drosophila germ cells. V. Relation of complete and mosaic mutations during the supplementary action of high temperature]

Supplementary effect of high temperature (37 degrees C) an hour after the treatment of mature sperms of Drosophila with ethylene imine resulted in an increased level of the inactivation (the frequency of dominant lethal mutation). The frequency of complete mutations (recessive sex-linked lethal mutations) increased by the supplementary effect of high temperature at low doses of E1, and it did not change under a comparatively high dose of the mutagen. The frequency of mosaic mutations decreased under the effect of high temperature at both doses of E1. No effect of high temperature was observed in 4 and 24 h after the E1 treatment. The results obtained are discussed in connection with the proportion of inactivation and mutagenesis under the effect of chemical mutagens on Drosophila germ cells.     

Characteristics of ceruloplasmin synthesis in mammalian embryogenesis. 2. The yolk sac as the site of the primary expression of the ceruloplasmin gene in rats

It was shown that the omphaloid placenta and, first of all, visceral wall of yolk sac is the site of primary synthesis of ceruloplasmin (CP), whereas the activation of CP synthesis in the liver cells is secondary and is revealed from the 12th day of embryo-genesis. The CP synthesis in the yolk sac cells proved by selective CP localization in the cells of the yolk sac visceral wall and, first of all, in the cells of visceral endoderm on sections stained by the method of indirect immunofluorescence and using the reaction of soluble peroxidase-antiperoxidase complex. A specific CP-mRNA has been revealed in the yolk sac cells which is actively translated in the polyribosomes isolated from the yolk sac and in the cell-free translation system from the rabbit reticulocytes. on the 14th day of embryogenesis CP amounts to ca. 4% of all polypeptides secreted by the yolk sac cells. As the embryogenesis proceeds, the relative rate of CP synthesis progressively decreases in the yolk sac and increases in the liver cells. CP synthesized by the yolk sac cells has a molecular mass of ca. 122 kD. Possible causes of differences between the "embryonic" and "adult" rat CPs are discussed. A suggestion has been put forward that the time of activation of CP synthesis coincides with the yolk sac formation (8-9th days of embryogenesis) and the cells of visceral endoderm are the site of primary expression of the CP gene.     

Expression of ceruloplasmin gene in mammalian organs from the data of hybridization analysis with complementary DNA probes

Expression of ceruloplasmin (Cp)-coding gene in rat and human liver and brain tissues was studied by Northern blot hybridization and by in situ hybridization with cloned species-specific cDNA probes. In rat brain structures, different levels of Cp mRNA were detected, the maximal one was found in cerebellum. The steady-state level of Cp mRNA in rat and human brain was several times lower than in parenchymatous liver cells. The size heterogeneity of Cp mRNA was found. Polyadenylated RNA prepared from human liver contains two equally abundant Cp mRNAs differing in their chain length (3.6 and 4.5 kb) while brain polyadenylated RNA contains a single Cp mRNA (4.5 kb).     

Polymorphism of nucleotide sequences of human genomic DNA linked to a mucoviscidosis locus

Polymorphism in the restriction fragments length of human DNA sequences linked to mucoviscidosis locus was studied in the healthy control group and in the families affected by mucoviscidosis. The plasmid clones metH, pJ3.11,XV-2c and pKM.19 were used as hybridization probes. The allelic frequencies of the polymorphic loci were determined for total population and for affected families. The linkage disequilibrium between the disease locus and linked polymorphic loci detectable with XV-2c (TaqI endonuclease) and pKM.19 (PstI endonuclease) was demonstrated. The high informational value of DNA-diagnosis of mucoviscidosis in the family studies with the use of four DNA probes combination has been demonstrated.     

Analysis of mutation frequency following continuous and fractionated application of ethyleneimine and ethyl methanesulfonate to male sex cells of Drosophila melanogaster]

The increase in the frequency of recessive lethal sex-linked mutations induced by fractionated effect of ethylene imine (EI) an ethylmethanesulphonate (EMS) on mature sperm of Drosophila melanogaster was observed and compared uith prolonged treatment (8h) and with the additive effect. This effect of dose fractionation was observed in the case of the treatment of sperms in male gonads and in female spermathecas. The increase of the mutation frequency was noted by brood-pattern method after fractionated treatment of spermatocytes and spermatogonia only with EMS. This increase was not observed under the effect of EI on spermatocytes and spermatogonia because of the high sierilization activity of EI. Possible mechanisms of the effects observed are discussed.     

Immunogenic properties of neomycin conjugates with macromolecular carriers]

To obtain diagnostic antibodies to neomycin, immunogenic properties of the neomycin conjugates with macromolecular carriers were studied. Bovine serum albumin and copolymers of N-vinylpyrrolidone with crotonic acid and N-hydroxyphthalimide ether of crotonic acid were used as carriers. It was shown that immunization of mice by the conjugates in combination with Freund's adjuvant resulted in production of neomycin antibodies, the titer being 1/80 to 1/130. When the antibiotic conjugates with the copolymers of N-vinylpyrrolidone were used and not the neomycin conjugates with the carrier of the protein nature, the neomycin antibodies were produced in the absence of Freund's full adjuvant. With the use of the isolated antibodies to neomycin a method for indirect solid phase enzyme immunoassay of neomycin was developed at the minimum detectable level of 25 ng/ml.     

Bistability and oscillations in the Huang-Ferrell model of MAPK signaling

Physicochemical models of signaling pathways are characterized by high levels of structural and parametric uncertainty, reflecting both incomplete knowledge about signal transduction and the intrinsic variability of cellular processes. As a result, these models try to predict the dynamics of systems with tens or even hundreds of free parameters. At this level of uncertainty, model analysis should emphasize statistics of systems-level properties, rather than the detailed structure of solutions or boundaries separating different dynamic regimes. Based on the combination of random parameter search and continuation algorithms, we developed a methodology for the statistical analysis of mechanistic signaling models. In applying it to the well-studied MAPK cascade model, we discovered a large region of oscillations and explained their emergence from single-stage bistability. The surprising abundance of strongly nonlinear (oscillatory and bistable) input/output maps revealed by our analysis may be one of the reasons why the MAPK cascade in vivo is embedded in more complex regulatory structures. We argue that this type of analysis should accompany nonlinear multiparameter studies of stationary as well as transient features in network dynamics.     

Autocrine loops with positive feedback enable context-dependent cell signaling

We describe a mechanism for context-dependent cell signaling mediated by autocrine loops with positive feedback. We demonstrate that the composition of the extracellular medium can critically influence the intracellular signaling dynamics induced by extracellular stimuli. Specifically, in the epidermal growth factor receptor (EGFR) system, amplitude and duration of mitogen-activated protein kinase (MAPK) activation are modulated by the positive-feedback loop formed by the EGFR, the Ras-MAPK signaling pathway, and a ligand-releasing protease. The signaling response to a transient input is short-lived when most of the released ligand is lost to the cellular microenvironment by diffusion and/or interaction with an extracellular ligand-binding component. In contrast, the response is prolonged or persistent in a cell that is efficient in recapturing the endogenous ligand. To study functional capabilities of autocrine loops, we have developed a mathematical model that accounts for ligand release, transport, binding, and intracellular signaling. We find that context-dependent signaling arises as a result of dynamic interaction between the parts of an autocrine loop. Using the model, we can directly interpret experimental observations on context-dependent responses of autocrine cells to ionizing radiation. In human carcinoma cells, MAPK signaling patterns induced by a short pulse of ionizing radiation can be transient or sustained, depending on cell type and composition of the extracellular medium. On the basis of our model, we propose that autocrine loops in this, and potentially other, growth factor and cytokine systems may serve as modules for context-dependent cell signaling.