Quantification of short term signaling by the epidermal growth factor receptor.

During the past decade, our knowledge of molecular mechanisms involved in growth factor signaling has proliferated almost explosively. However, the kinetics and control of information transfer through signaling networks remain poorly understood. This paper combines experimental kinetic analysis and computational modeling of the short term pattern of cellular responses to epidermal growth factor (EGF) in isolated hepatocytes. The experimental data show transient tyrosine phosphorylation of the EGF receptor (EGFR) and transient or sustained response patterns in multiple signaling proteins targeted by EGFR. Transient responses exhibit pronounced maxima, reached within 15-30 s of EGF stimulation and followed by a decline to relatively low (quasi-steady-state) levels. In contrast to earlier suggestions, we demonstrate that the experimentally observed transients can be accounted for without requiring receptor-mediated activation of specific tyrosine phosphatases, following EGF stimulation. The kinetic model predicts how the cellular response is controlled by the relative levels and activity states of signaling proteins and under what conditions activation patterns are transient or sustained. EGFR signaling patterns appear to be robust with respect to variations in many elemental rate constants within the range of experimentally measured values. On the other hand, we specify which changes in the kinetic scheme, rate constants, and total amounts of molecular factors involved are incompatible with the experimentally observed kinetics of signal transfer. Quantitation of signaling network responses to growth factors allows us to assess how cells process information controlling their growth and differentiation.     

The topology design principles that determine the spatiotemporal dynamics of G-protein cascades

Small monomeric G-proteins control cellular behavior, cycling between inactive GDP-bound and active GTP-bound states. Activating and deactivating transitions are regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), respectively. G-proteins can control different GEF and GAP activities, thereby creating GTPase signaling cascades. Here, we characterize all 128 different wiring topologies of two-layer cascades, which include feedforward/feedback interactions and an auto-regulatory loop. Exclusion of "mirror" designs leaves 64 topologies, which are classified into eight groups. We demonstrate that eight different cascades in each group generate the same number of steady states and similar spatiotemporal dynamics. Two groups (featuring 16 topologies) can generate three distinct dynamics: (i) bistable switches, (ii) excitable behavior, and (iii) sustained oscillations, giving rise to propagating waves of G-protein activation switches and pulses. Four other groups can produce switch-like, bistable behaviors and trigger waves. The remaining two groups have a single steady state. This first, complete classification of all possible interaction circuitries systematically links topological design to the spatiotemporal dynamics of G-protein cascades, predicting and explaining experimentally observed behavior.     

Resistance of Human Liver Mesenchymal Stem Cells to FAS-Induced Cell Death

Mesenchymal stem cells (MSCs) have a pronounced therapeutic potential in various pathological conditions. Though therapeutic effects of MSC transplantation have been studied for a long time, the underlying mechanisms are still not clear. It has been shown that transplanted MSCs are rapidly eliminated, presumably by apoptosis. As the mechanisms of MSC apoptosis are not fully understood, in the present work we analyzed MSC sensitivity to Fas-induced apoptosis using MSCs isolated from the biopsies of liver fibrosis patients (L-MSCs). The level of cell death was analyzed by flow cytometry in the propidium iodide test. The luminescent ATP assay was used to measure cellular ATP levels; and the mitochondrial membrane potential was assessed using the potential-dependent dye JC-1. We found that human L-MSCs were resistant to Fas-induced cell death over a wide range of FasL and anti-Fas mAb concentrations. At the same time, intrinsic death signal inducers CoCl₂ and staurosporine caused apoptosis of L-MSCs in a dose-dependent manner. Despite the absence of Fas-induced cell death treatment of L-MSCs with low concentrations of FasL or anti-Fas mAb resulted in a cellular ATP level decrease, while high concentrations of the inducers caused a decline of the mitochondrial membrane potential. Pre-incubation of L-MSCs with the pro-inflammatory cytokine TNF-α did not promote L-MSC cell death. Our data indicate that human L-MSCs have increased resistance to receptor-mediated cell death even under inflammatory conditions.     

Phenotype characteristic of the ionomycin-resistant CD4+ subset of the peripheral blood T-lymphocytes

The differential sensitivity of peripheral blood (PB) CD4+ T lymphocytes to the calcium ionophore ionomycin was investigated. Effect of ionomycin exerted on T cells was time- and dose-dependent. We have shown that resistant cells belonged to some distinct T cell subsets. The resting naive CD4+CD45RA+ T cells showed a little, if any, resistance to ionomycin treatment. The primed CD4+CD45R0+ memory T cells behaved similarly as did ionomycin-resistant (IR) cells. Although IR CD4+ T cells had a typical "memory" phenotype, some quantitative differences were found in expression of CD11a, CD28, CD29, CD62L and CD243 markers between PB CD4+CD45R0+ T cells and corresponding IR cells.     

Cellular information transfer regarded from a stoichiometry and control analysis perspective

Metabolic control analysis (MCA) allows one to formalize important aspects of information processing in living cells. For example, information processing via multi-level enzyme cascades can be quantified in terms of the response coefficient of a cellular target to a signal. In many situations, control and response coefficients cannot be determined exactly for all enzymes involved, owing to difficulties in 'observing' all enzymes experimentally. Here, we review a number of qualitative approaches that were developed to cope with such situations. The usefulness of the concept of null-space of the stoichiometry matrix for analysing the structure of intracellular signaling networks is discussed. It is shown that signal transduction operates very efficiently when the network structure is such that the null-space matrix can be block-diagonalized (which may or may not imply that the network consists of several disconnected parts) and some enzymes have low elasticities to their substrates.     

Chemical-microbiological diagnosis of stress-corrosive damage to pipelines]

Samples of soil, ground, electrolyte, corrosion products, and protective coating were taken after excavating pipelines. The depth of stress corrosion cracks of the pipe steel was mostly related to the numbers of sulfate-reducing and denitrifying bacteria. In certain types of soil, damage correlated with the number of acid-producing microorganisms and aerobic chemoorganotrophs (saprophytes). A correlation was found between the extent of stress-corrosion damage to pipelines and the contents of reduced iron, sulfides, and organic carbon in surrounding ground.     

Effect of space flight conditions on properties of hydrocarbon-oxidizing bacteria

Results of experiments on the Mir space station (EO-25 and EO-26) demonstrated that the conditions of orbital flight, primarily the cosmic radiation, was a mutagenic factor affecting both the genotype and phenotype of an oil-oxidizing bacterial strain, Mycobacterium flavescens EX-91. The emerging mutants differed from original culture by the rate of colony growth and the ability to ferment certain carbohydrates or synthesize beta-galactosidase. Changes in the rate of utilization of raw oil and individual hydrocarbon types (constituting model mixtures) suggest that cosmic radiation may serve as a means of obtaining mutant clones of microorganisms with new properties.     

Study of the integral toxicity of aqueous media, polluted by petroleum and petroleum products using bacterial tests

A biotest kit was used to assess the integral toxicity level of aquatic medium contamination with petroleum and petroleum-based products. The integral toxicity dynamics was also monitored during biodegradation of petroleum and petroleum-based products by an association of petroleum-degrading strains including Acinetobacter sp., Mycobacterium flavescens, and Rhodococcus sp. The following bacterial tests were used: the bioluminescence (BL) test based on Photobacterium leiognathi; electro-orientation (EO), optoosmotic (OO), and growth test; as well as the reducing activity (RA) test based on the Agrobacterium radiobacter culture. No significant increase in the integral toxicity level of aquatic medium was observed when diesel fuel and kerosene contamination had been subjected to biodegradation. Although express biotests (EO, OO, RA, and BL) detected a pronounced increase in the integral toxicity of aquatic medium, long-term growth biotest revealed no statistically significant increase in the toxicity level.