New approaches to modelling and analysis of biochemical reactions, pathways and networks.
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Results for “Pathway modelling”
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Application of Petri net based analysis techniques to signal transduction pathways.
BACKGROUND: Signal transduction pathways are usually modelled using classical quantitative methods, which are based on ordinary differential equations (ODEs). However, some difficulties are inherent in this approach. On the one hand, the kinetic parameters involved are often unknown and have to be estimated. With increasing size and complexity of signal transduction pathways, the estimation of missing kinetic data is not possible. On the other hand, ODEs based models do not support any explicit insights into possible (signal-) flows within the network. Moreover, a huge amount of qualitative data is available due to high-throughput techniques. In order to get information on the systems behaviour, qualitative analysis techniques have been developed. Applications of the known qualitative analysis methods concern mainly metabolic networks. Petri net theory provides a variety of established analysis techniques, which are also applicable to signal transduction models. In this context special properties have to be considered and new dedicated techniques have to be designed. METHODS: We apply Petri net theory to model and analyse signal transduction pathways first qualitatively before continuing with quantitative analyses. This paper demonstrates how to build systematically a discrete model, which reflects provably the qualitative biological behaviour without any knowledge of kinetic parameters. The mating pheromone response pathway in Saccharomyces cerevisiae serves as case study. RESULTS: We propose an approach for model validation of signal transduction pathways based on the network structure only. For this purpose, we introduce the new notion of feasible t-invariants, which represent minimal self-contained subnets being active under a given input situation. Each of these subnets stands for a signal flow in the system. We define maximal common transition sets (MCT-sets), which can be used for t-invariant examination and net decomposition into smallest biologically meaningful functional units. CONCLUSION: The paper demonstrates how Petri net analysis techniques can promote a deeper understanding of signal transduction pathways. The new concepts of feasible t-invariants and MCT-sets have been proven to be useful for model validation and the interpretation of the biological system behaviour. Whereas MCT-sets provide a decomposition of the net into disjunctive subnets, feasible t-invariants describe subnets, which generally overlap. This work contributes to qualitative modelling and to the analysis of large biological networks by their fully automatic decomposition into biologically meaningful modules.
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Signaling pathways regulating murine cardiac CREB phosphorylation.
Using the mouse Langendorff heart perfusion model, the signaling pathways that regulate cardiac CREB-S133 phosphorylation have been defined. In mouse hearts stimulated with isoproterenol (ISO) (10(-8) M), endothelin-1 (ET-1) (10(-8) M), and phorbol 12-myristate 13-acetate (TPA) (10(-7) M), CREB-S133 phosphorylation was attained only by TPA-treatment. Activation of protein kinase A (PKA) was achieved by ISO. ISO- and ET-1-stimulation activated Ca2+/calmodulin-dependent kinase II (CaMKII). Protein kinase C (PKC) and p90(RSK) were activated with all three stimuli. Inhibition of ERK1/2 with PD98059 (10(-5) M) completely inhibited the activation of p90(RSK), but did not block CREB-S133 phosphorylation in TPA-perfused heart, indicating that PKA, CaMKII, and p90(RSK) do not phosphorylate CREB-S133 in the murine heart. PKC activation is signal specific. Analyses of PKC isoforms suggest that CREB phosphorylation is mediated by PKC epsilon translocating into nucleus only with TPA stimulation. These results, unlike those reported in other tissues, demonstrate that cardiac CREB is not a multi-signal target.
Amino Acid Sequence↗
Antithrombotic properties of a truncated recombinant tissue factor pathway inhibitor in an experimental venous thrombosis model.
The aim of this study was to investigate whether a truncated recombinant tissue factor pathway inhibitor (rTFPI1-161) had an antithrombotic effect comparable to low-molecular-weight (LMW) heparin. A randomized double-dummy study was conducted with 40 rabbits in 6 groups. An experimental thrombosis was induced in the jugular veins by a combination of destroyed endothelium and restricted blood flow. Group 1 was given placebo; group 2, LMW heparin 60 anti-factor Xa units/kg; group 3, rTFPI1-161 0.1 mg/kg; group 4, rTFPI1-161 1.0 mg/kg and group 5, rTFPI1-161 10.0 mg/kg. rTFPI1-161 reduced the thrombus weights in all treated groups, with a significant effect in doses between 1.0 and 10.0 mg/kg compared to placebo. The frequency of thrombosis was significantly reduced in all treated groups. No hemorrhagic side effects were noted. In conclusion, rTFPI1-161 (1.0-10.0 mg/kg) has an antithrombotic effect comparable to that of LMW heparin.
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Mechanisms for gene conversion and homologous recombination: the double-strand break repair model and the successive half crossing-over model.
Two mechanisms for gene conversion and homologous recombination were discussed. (1) The double-strand break repair model. A double-strand break is expanded to a gap, which is then repaired by copying a homologous sequence. The gene conversion is often accompanied by crossing-over of the flanking sequences. We obtained evidence for this model in Red pathway of bacteriophage lambda and RecE pathway of E. coli. (2) The successive half crossing-over model. Half crossing-over leaves one recombinant duplex and one or two end(s) out of two parental duplexes. The resulting ends are, in turn, recombinogenic. Successive rounds of the half crossing-over mechanism explains why apparent plasmid gene conversion in RecF pathway of E. coli is not accompanied by crossing-over. This model can explain chromosomal gene conversion if we assume that the donor is first replicated. Gene conversion during mating-type switching in yeast, antigenic variation in unicellular microorganisms, and chromosomal gene conversion in mammalian somatic cells are explained by this model. Distinguishing between these two mechanisms is important in understanding recombination in yeast and mammalian cells and also in its application to gene targeting.
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Parallel and distributed neural models of the ideomotor principle: an investigation of imitative cortical pathways.
Humans' capacity to imitate has been extensively investigated through a wide-range of behavioral and developmental studies. Yet, despite the huge amount of phenomenological evidence gathered, we are still unable to relate this behavioral data to any specific neural substrate. In this paper, we investigate how principles from psychology can be the result of neural computations and therefore attempt to bridge the gap between monkey neurophysiology and human behavioral data, and hence between these two complementary disciplines. Specifically, we address the principle of ideomotor compatibility, by which 'observing the movements of others influences the quality of one's own performance' and develop two neural models which account for a set of related behavioral studies [Brass, M., Bekkering, H., Wohlschläger, A., & Prinz, W. (2000). Compatibility between observed and executed finger movements: comparing symbolic, spatial and imitative cues. Brain and Cognition 44, 124-143]. We show that the ideomotor effect could be the result of two distinct cognitive pathways, which can be modeled by means of biologically plausible neural architectures. Furthermore, we propose a novel behavioral experiment to confirm or refute either of the two model pathways.
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The Caenorhabditis elegans CED-9 protein does not directly inhibit the caspase CED-3, in vitro nor in yeast.
A genetically defined pathway orchestrates the removal of 131 of the 1090 somatic cells generated during the development of the hermaphrodite nematode Caenorhabditis elegans. Regulation of apoptosis is highly evolutionarily conserved and the nematode cell death pathway is a valuable model for studying mammalian apoptotic pathways, the dysregulation of which can contribute to numerous diseases. The nematode caspase CED-3 is ultimately responsible for the destruction of worm cells in response to apoptotic signals, but it must first be activated by CED-4. CED-9 inhibits programmed cell death and considerable data have demonstrated that CED-9 can directly bind and inhibit CED-4. However, it has been suggested that CED-9 may also directly inhibit CED-3. In this study, we used a yeast-based system and biochemical approaches to explore this second potential mechanism of action. While we confirmed the ability of CED-9 to inhibit CED-4, our data argue that CED-9 can not directly inhibit CED-3.
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Functional interaction between the PKC1 pathway and CDC31 network of SPB duplication genes.
The earliest known step in yeast spindle pole body (SPB) duplication requires Cdc31p and Kar1p, two physically interacting SPB components, and Dsk2p and Rad23p, a pair of ubiquitin-like proteins. Components of the PKC1 pathway were found to interact with these SPB duplication genes in two independent genetic screens. Initially, SLG1 and PKC1 were obtained as high-copy suppressors of dsk2Delta rad23Delta and a mutation in MPK1 was synthetically lethal with kar1-Delta17. Subsequently, we demonstrated extensive genetic interactions between the PKC1 pathway and the SPB duplication mutants that affect Cdc31p function. The genetic interactions are unlikely to be related to the cell-wall integrity function of the PKC1 pathway because the SPB mutants did not exhibit cell-wall defects. Overexpression of multiple PKC1 pathway components suppressed the G2/M arrest of the SPB duplication mutants and mutations in MPK1 exacerbated the cell cycle arrest of kar1-Delta17, suggesting a role for the PKC1 pathway in SPB duplication. We also found that mutations in SPC110, which encodes a major SPB component, showed genetic interactions with both CDC31 and the PKC1 pathway. In support of the model that the PKC1 pathway regulates SPB duplication, one of the phosphorylated forms of Spc110p was absent in pkc1 and mpk1Delta mutants.
Blotting, Western↗
An in vitro pathway from embryonic stem cells to neurons and glia.
Mouse embryonic stem (ES) cells can be induced to differentiate into neurons and glia in vitro. Induction protocols are straightforward and involve culture in the presence of retinoic acid. They result in an efficient conversion of undifferentiated ES cells to neural cells. Mature neurons produced have the key physiological, morphological and molecular properties of primary cultured neurons derived from the central nervous system. Most significantly, they form functional chemical synapses that utilize either glutamate, GABA or glycine as neurotransmitters. ES cell-derived glial cells also correspond well with their normal counterparts. During induction, ES cells undergo a series of developmental steps that resemble key stages in the early mouse embryo. This supports the hypothesis that the in vitro pathway is a valid model of the normal developmental pathway leading to neurons and glia. The in vitro system combines three experimental strengths. It is suitable for genetic manipulation, affords large numbers of cells and allows precise manipulation of the culture environment. It is thus suitable for a wide variety of mechanistic studies in the areas of neural development and cell biology.
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Generating and modifying DiGeorge syndrome-like phenotypes in model organisms: is there a common genetic pathway?
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[Establishment of high-throughput drug screening cell models based on JAK-STAT signal pathway].
AIM: To discover new drugs which may be applied to diseases of the immune system, hemogenesis system diseases and tumors, several high-throughput drug screening cell models based on JAK-STAT signal pathway have been established. METHODS: Four repeats of STAT DNA binding conserved sequences were synthesized, subcloned into pGL-Luc reporter vector and stably transfected into cell lines in vitro. Cell clones with high copy numbers of STAT binding sites and reporter genes were chosen as high-throughput drug screening cell models. The cell models were tested with known anti-allergic drugs and anti-tumor drugs by determining luciferase activity. The reaction was performed in 96 well micro-plates with a final volume of 50 microL. RESULTS: The cell models by performing rapid fluorescence assay were shown to be highly sensitive and stable after testing with cytokine and drugs. The modification of the expression plasmid simplified this method and made it more practical. It also provided good linear correlation, wide range of assay, highly sensitive and good reproducibility. CONCLUSION: The method can be performed by high-throughput drug screening for effective extraction of Chinese traditional herbs.
Anti-Allergic Agents↗
Pathways and bottlenecks in the web of inflammatory adhesion molecules and chemoattractants.
Leukocyte recruitment requires capture, rolling, activation, adhesion, and transmigration. Adhesion molecules and chemoattractants have been identified that mediate each of these steps. Their functions often overlap, but the combined absence of some molecules can lead to severe spontaneous phenotypes, as seen in CD18-/- mice, or early lethality, as in CD18-/- E-selectin-/- mice. These groups of molecules define bottlenecks that restrict the inflammatory response. Adhesion molecules and activation mechanisms can also form groups of preferential usage, or pathways. Based on these findings, a web-like model may represent the inflammatory process better than the linear cascade model. Bottlenecks and pathways depend on the degree and nature of overlapping functions, the disease process, tissue site, and the inflammatory stimulus.
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Virtual models of the HLA class I antigen processing pathway.
Antigen recognition by cytotoxic CD8 T cells is dependent upon a number of critical steps in MHC class I antigen processing including proteosomal cleavage, TAP transport into the endoplasmic reticulum, and MHC class I binding. Based on extensive experimental data relating to each of these steps there is now the capacity to model individual antigen processing steps with a high degree of accuracy. This paper demonstrates the potential to bring together models of individual antigen processing steps, for example proteosome cleavage, TAP transport, and MHC binding, to build highly informative models of functional pathways. In particular, we demonstrate how an artificial neural network model of TAP transport was used to mine a HLA-binding database so as to identify HLA-binding peptides transported by TAP. This integrated model of antigen processing provided the unique insight that HLA class I alleles apparently constitute two separate classes: those that are TAP-efficient for peptide loading (HLA-B27, -A3, and -A24) and those that are TAP-inefficient (HLA-A2, -B7, and -B8). Hence, using this integrated model we were able to generate novel hypotheses regarding antigen processing, and these hypotheses are now capable of being tested experimentally. This model confirms the feasibility of constructing a virtual immune system, whereby each additional step in antigen processing is incorporated into a single modular model. Accurate models of antigen processing have implications for the study of basic immunology as well as for the design of peptide-based vaccines and other immunotherapies.
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Threonine synthesis from aspartate in Escherichia coli cell-free extracts: pathway dynamics.
We have developed an experimental model of the whole threonine pathway that allows us to study the production of threonine from aspartate under different conditions. The model consisted of a desalted crude extract of Escherichia coli to which we added the substrates and necessary cofactors of the pathway: aspartate, ATP and NADPH. In this experimental model we measured not only the production of threonine, but also the time dependence of all the intermediate metabolites and of the initial substrates, aspartate, ATP and NADPH. A stoichiometric conversion of precursors into threonine was observed. We have derived conditions in which a quasi steady state can be transiently observed and used to simulate physiological conditions of functioning of the pathway in the cell. The dependence of threonine synthesis and of the aspartate and NADPH consumption on the initial aspartate and threonine concentrations exhibits greater sensitivity to the aspartate concentration than to the threonine concentration in these non-steady-state conditions. A response to threonine is only observed in a narrow concentration range from 0.23 to 2 mM.
Adenosine Triphosphate↗
Testing computational hypotheses of brain systems function: a case study with the basal ganglia.
We develop a methodology for testing computational hypotheses about neural functionality articulated in models at the systems level of description. In this approach, the first step is to attempt the construction of a model of the underlying brain system which is consistent with the known anatomy and physiology, but which is also able to exhibit functional properties consistent with a putative computational hypothesis. If this is successful, the second step consists of including additional known pathways into the model and testing the new models to see whether they show an improvement in functional performance (using appropriate performance metrics). A positive outcome is taken as evidence in support of the hypothesis. A final step is to construct 'control' models by including pathways that are not consistent with biological data. In this case a performance detriment is taken as support for the hypothesis. The methodology is applied to the basal ganglia, and builds on a previously published model of this system (Gurney et al 2001 Biol. Cybern. 84 401-23) which was based on the hypothesis that the basal ganglia perform action selection. The realistically constrained models show a selection benefit, while control models show a decrement in selection ability. These results, taken together, provide further validation of our selection hypothesis of basal ganglia function.
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Use of kinetic analysis and mathematical modeling in the study of metabolic pathways in vivo. Applications to hepatic organic anion metabolism. (First of two parts).
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Native and non-native interactions along protein folding and unfolding pathways.
Gō-type models, which include only native contact interactions in the energy function, are being used increasingly to describe the protein folding reaction. To investigate the validity of such models, we determine the role of native and non-native interactions along folding and unfolding pathways. For this purpose, we use a molecular mechanics energy function with an implicit solvation model (an effective energy function or potential of mean force) that can be expressed in a pairwise decomposable form. We find that for the native state and a wide range of other configurations, the contact energy is an accurate description, in part due to the cancellation of non-zero contributions from more distant residues. However, significant errors in the energy are introduced for non-native structures if the energy is calculated from the native contacts alone. Non-native contacts tend to make a significant contribution, particularly for molten globules and collapsed states along the unfolding pathways. The implication of these results for the use of Gō-type models in studies of protein folding are discussed.
Amino Acids↗
Biochemical mapping of the noradrenergic projection from the locus coeruleus. A model for studies of brain neuronal pathways.
Mapping of the noradrenergic projection from neurons in the rat locus coeruleus has been examined by combining a sensitive radioisotopic assay for catecholamines with a microdissection technique to remove multiple separate brain nuclei. The effect of a unilateral locus coeruleus lesion on norepinephrine concentration in 19 brain regions ipsilateral and contralateral to the lesion was determined. Evidence for ipsilateral and bilateral innervation to specific regions is presented, and many regions appear to receive combined innervation from other noradrenergic loci, in addition to that from the locus coeruleus. Fluorescence rating was correlated with biochemical measurement of amine content with these techniques and proportionality was observed over a narrow range. With this proportionality taken into consideration, mapping results obtained by biochemical and fluorescence methods are compared.
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