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Summary of the BIOMOVS A4 scenario: testing models of the air-pasture-cow milk pathway using Chernobyl fallout data.

A unique opportunity to test dose assessment models arose after the Chernobyl reactor accident. During the passage of the contaminated plume, concentrations of 131I and 137Cs in air, pasture, and cow's milk were collected at various sites in the northern hemisphere. Afterwards, contaminated pasture and milk samples were analyzed over time. Under the auspices of the Biospheric Model Validation Study (BIOMOVS), data from 13 sites for 131I and 10 sites for 137Cs were used to test model predictions for the air-pasture-cow milk pathway. Calculations were submitted for 23 models, 10 of which were quasi-steady state. The others were time-dependent. Daily predictions and predictions of time-integrated concentrations of 131I and 137Cs in pasture grass and milk for six months post-accident were calculated and compared with observed data. Testing against data from several locations over time for several steps in the air-to-milk pathway resulted in a better understanding of important processes and how they should be modeled. This model testing exercise showed both the strengths and weaknesses of the models and revealed the importance of testing all parts of dose assessment models whenever possible.

Air Pollutants, Radioactive↗

Developmental signaling in Hydra: what does it take to build a "simple" animal?

Developmental processes in multicellular animals depend on an array of signal transduction pathways. Studies of model organisms have identified a number of such pathways and dissected them in detail. However, these model organisms are all bilaterians. Investigations of the roles of signal transduction pathways in the early-diverging metazoan Hydra have revealed that a number of the well-known developmental signaling pathways were already in place in the last common ancestor of Hydra and bilaterians. In addition to these shared pathways, it appears that developmental processes in Hydra make use of pathways involving a variety of peptides. Such pathways have not yet been identified as developmental regulators in more recently diverged animals. In this review I will summarize work to date on developmental signaling pathways in Hydra and discuss the future directions in which such work will need to proceed to realize the potential that lies in this simple animal.

Animals↗

In silico modeling and simulation of bone biology: a proposal.

Contemporary, computer-based mathematical modeling techniques make it possible to represent complex biological mechanisms in a manner that permits hypothesis testing in silico. This perspective shows how such approaches might be applied to bone remodeling and therapeutic research. Currently, the dominant conceptual model applied in bone research involves the dynamic balance between the continual build-up and breakdown of bone matrix by two cell types, the osteoblasts and osteoclasts, acting together as a coordinated, remodeling unit. This conceptualization has served extraordinarily well as a focal point for understanding how mutations, chemical mediators, and mechanical force, as well as external influences (e.g., drugs, diet) affect bone structure and function. However, the need remains to better understand and predict the consequences of manipulating any single factor, or combination of factors, within the context of this complex system's multiple interacting pathways. Mathematical models are a natural extension of conceptual models, providing dynamic, quantitative descriptions of the relationships among interacting components. This formalization creates the ability to simulate the natural behavior of a system, as well as its modulation by therapeutic or dietetic interventions. A number of mathematical models have been developed to study complex bone functions, but most include only a limited set of biological components needed to address a few specific questions. However, it is possible to develop larger, multiscale models that capture the dynamic interactions of many biological components and relate them to important physiological or pathological outcomes that allow broader study. Examples of such models include entelos' physiolab platforms. These models simulate the dynamic, quantitative interactions among a biological system's biochemicals, cells, tissues, and organs and how they give rise to key physiologic and pathophysiologic outcomes. We propose that a similar predictive, dynamical, multiscale mathematical model of bone remodeling and metabolism would provide a better understanding of the mechanisms governing these phenomena as well as serve as an in silico platform for testing pharmaceutical and clinical interventions on metabolic bone disease.

Bone Diseases↗

The JUN kinase/stress-activated protein kinase pathway is required for epidermal growth factor stimulation of growth of human A549 lung carcinoma cells.

Epidermal growth factor (EGF) plays a major role in non-small cell lung cancer cell autocrine growth and has been reported to activate the JUN kinase/stress-activated protein kinase (JNK/SAPK) pathway in model cells. Activation of JNK/SAPK leads to the phosphorylation of c-JUN protooncogene on serines 63 and 73. This mechanism is required for and cooperates in the transformation of rat embryo fibroblasts by Ha-RAS. However, the function of JNK/SAPK in human tumor growth is unknown. We have tested several lung carcinoma cell lines. All exhibited UV-C-inducible JNK/SAPK activity; two exhibited constitutive activity in low serum, and two (M103 and A549) exhibited EGF-inducible JNK/SAPK activity. In A549 cells, EGF induced a rapid and prolonged (up to 24 h) activation of the JNK/SAPK pathway that correlated with a 150-190% growth stimulation. Stably transfected clones of A549 cells expressing c-JUN(S63A,S73A), a transdominant inhibitor of c-JUN, completely blocked the EGF-stimulated proliferation effect but did not alter the basal proliferation rate. Consistent with these results JNK antisense oligonucleotides targeted to JNK1 and JNK2 entirely eliminated the EGF-stimulated JNK/SAPK activity and blocked EGF-stimulated growth but not basal growth. In contrast, specific inhibition of the RAF/ERK pathway by PD98059 (MEK1 inhibitor) completely blocked ERK activation by EGF and basal cell growth but not EGF-stimulated growth, thereby dissociating the growth-promoting roles of each pathway. Our observations indicate, for the first time, that JNK/SAPK may be a preferential effector pathway for the growth properties of EGF in A549 cells.

Animals↗

Grammatical formalization of metabolic processes.

In the field of biotechnology and medicine it is of interest to model and simulate metabolic processes. The usual methods to model metabolic pathways are chemical descriptions and differential equations. Moreover, the graph theoretical aspect is discussed and the development of expert systems is in process. In this paper we present the formalization of metabolic processes. Our formalization is based on the theory of formal languages. This formalization is called genetic grammar and represents an expansion of the Semi-Thue-System.

Computer Simulation↗

Behavioral responses to injections of muscimol into the subthalamic nucleus: temporal changes after nigrostriatal lesions.

Changes in cellular activity in the subthalamic nucleus are a cardinal feature of Parkinson's disease and occur in rodents after lesions of the nigrostriatal pathway, a model of Parkinson's disease. GABA-ergic neurons from the globus pallidus provide a major input to the subthalamic nucleus. Previous electrophysiological studies revealed temporal changes in the activity of pallidal neurons after nigrostriatal lesions in rats. However, little is known about the impact of these changes on GABAergic transmission in the subthalamic nucleus. We have examined the behavioral responses to a local administration of the GABA A agonist muscimol into the subthalamic nucleus. Muscimol (0.01 and 0.1 microg) induced orofacial dyskinesia in normal rats; this response was blunted 2 weeks but enhanced 2 months after a unilateral lesion of the nigrostriatal pathway. The early decrease in the behavioral response occurred at a time when increased expression of mRNA for glutamic acid decarboxylase, the enzyme of GABA synthesis, and burst firing have been reported in the globus pallidus, suggesting an adaptive post-synaptic response to increased GABAergic transmission in the subthalamic nucleus. In contrast, we now show that glutamic acid decarboxylase mRNA is unchanged in the globus pallidus at the later time point, when electrophysiological changes also subside in this region. The increased behavioral response at this later time point may reflect a decreased activity in GABAergic inputs to the subthalamic nucleus. The results show time-dependent changes in behavioral responses to GABA A receptor stimulation in the subthalamic nucleus which may reflect adaptive changes in postsynaptic inhibitory responses after dopaminergic lesions.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗

Significance of skeletal muscle oxidative enzyme enhancement with endurance training.

A theoretical model is proposed to explain how the increase in mitochondrial protein concentration, and therefore of the oxidative enzymes, that occurs with endurance training could operate to alter the choice of substrate during submaximal exercise in a manner such that the oxidation of fatty acids increases, glycogen depletion and lactate production are reduced, and work capacity is enhanced. The model is based on the control of enzyme activities both by enzyme and substrate concentrations. The effect of altering enzyme concentration on reaction velocities is presented on the basis of standard Henri-Michaelis-Menten kinetics. It is shown that the reaction velocity at a given substrate concentration is a function of total enzyme concentration. With an increase in total enzyme concentration there is a parallel increase in reaction velocity at the same substrate level. This would have its greatest impact at substrate levels below the Km of the enzyme. It would have an effect of enhancing fatty acid flux through the oxidative pathways while inhibiting the Embden-Meyerhof pathway. The model, as proposed, is consistent with known alterations in metabolism as they occur in man during submaximal exercise following endurance training.

Adenosine Diphosphate↗

On maintenance models in severely and long-term limited membrane bioreactor cultivations.

Membrane bioreactors (MBRs) are combinations of common bioreactors and membrane separation units for biomass retention. Through increased biomass concentration, they allow increased productivity (or smaller reactor volume, respectively). Besides high biomass concentrations, operation at very low growth rates is typical for MBRs. In this regime, maintenance metabolism where substrate uptake only yields energy for cell survival becomes of higher importance than in processes run at higher growth rates. While thermodynamically based correlations for the prediction of maintenance coefficients are available for chemostat or other medium growth rate processes, some authors have mentioned a change in energy demand in MBRs and a dependence of maintenance parameters on operating conditions. Due to the fact that often mixed cultures are used and resulting from the different evaluation methods used by different authors, views on the possible influences on maintenance parameters differ. However, it is accepted that common models describing microbial growth and production of metabolites or degradation of pollutants do not consider the effects caused by severe limitations and therefore cannot sufficiently be applied to MBRs. In this study, maintenance parameters were determined for a model organism (Ustilago maydis) and results from different evaluation methods were compared. A continuous fit of respiration data gave more consistent results than the traditional method of plotting specific uptake versus growth rate. They suggest that below micro = 10% micro(max) the maintenance coefficient drops to a third of the value in short-term limited cultures.

Biomass↗

Inhibition of p38 mitogen-activated protein kinase augments progression of remnant kidney model by activating the ERK pathway.

p38, a mitogen-activated protein kinase, is a major intracellular signaling molecule involved in inflammation. To test the hypothesis that p38 mediates renal disease progression, we administered a novel p38 alpha inhibitor, NPC31169, to rats with remnant kidneys (RKs). RK rats showed increased p38 activation at 9 weeks (by p38 kinase assay), which was blocked by the inhibitor. In contrast to our expectation, treatment with the NPC31169 resulted in worse renal function, more proteinuria, and more severe glomerulosclerosis and tubulointerstitial injury. p38 inhibition resulted in marked cell proliferation in RK rats, with more proliferating tubular cells, myofibroblasts, and macrophages. In contrast, p38 suppression resulted in less tubular cell apoptosis. Interestingly, Western blot demonstrated increased ERK1/2 phosphorylation in p38-treated rats. No histological changes were observed in p38 inhibited sham-operated rats. Our findings indicate that, whereas blocking p38 usually shows benefit in inflammatory disease, in this model p38 inhibition resulted in accelerated renal progression. We conclude that blocking p38-dependent inflammation may have resulted in enhanced proliferation and increased ERK1/2 activation, and thereby explains the worse renal lesions observed.

Animals↗

Role of cyclic nucleotide phosphodiesterases in resumption of meiosis.

In the follicles of the mammalian and amphibian ovary, oocyte maturation is arrested at the prophase of the first meiotic division. Prior to ovulation, oocytes reenter the cell cycle, complete the meiotic division, and extrude the first polar body. Work from several laboratories including ours has provided evidence that the cAMP-mediated signal transduction pathway plays an important role in regulation of meiosis, the cyclic nucleotide acting as a negative regulator of maturation. Since cAMP can be regulated both at the level of synthesis and degradation, our laboratory is investigating the role of phosphodiesterases (PDE) in the control of cAMP levels of oocytes. Using pharmacological and molecular tools, we have determined that a PDE3 is the enzyme involved in the control of cAMP levels in the oocytes. In vitro and in vivo studies have established that inhibition of the oocyte PDE3 blocks resumption of a PDE is per se sufficient to cause resumption of meiosis in an amphibian oocyte model. The pathways regulating this PDE isoform expressed in the oocyte is under investigation, as they may uncover the physiological signals controlling meiosis.

3',5'-Cyclic-AMP Phosphodiesterases↗

Colon goblet cells lose proliferative response to TGF alpha as they differentiate.

Two cell line models for colon goblet cells expressed 6- to 14-fold elevated levels of the EGF receptor, 3- to 5-fold levels of TGF alpha and 11- to 15-fold levels of amphiregulin compared with 2 cell lines which model colon enterocytic differentiation, suggesting a role for the EGF receptor and its ligands in goblet cell growth control. Two HT29 colon carcinoma sublines were used to model normal goblet cells at different stages of maturation. TGF alpha induced a 2-fold increase in growth of the HD8 subline but inhibited the growth of the more differentiated HD6 subline by 40%. EGF receptors were activated in each line by ligand, but signal transduction varied sharply. Both MAP kinase isoforms, p44 and p42, were markedly activated in HD8 cells for at least 20 min, while only a marginal activation was seen in HD6 cells. In contrast, the more differentiated HD6 cells showed an increase in 105 kDa MBP kinase activity with EGF treatment, while HD8 cells displayed constitutively elevated levels of this kinase. Thus, activated EGF receptors initiated different signalling pathways in model cell lines for colon goblet cells at different stages of maturation. TGF alpha protein levels have been shown by other investigators to be restricted to the top of the cylinder-like colonic crypt, where cells terminally differentiate and cease division, an unexpected location for an epithelial cell mitogen. Our data with model cell lines imply that normal colon goblet cells lose proliferative response to TGF alpha as they differentiate and the elevated levels of TGF alpha at the top of the colonic crypt in vivo serve to inhibit goblet cell growth.

Calcium-Calmodulin-Dependent Protein Kinases↗

Immunomodulated Signaling in Macrophages:Regulation of the MAPK Signaling Pathways by PKA and PKC.

Previous experimental investigation indicated that immuno-suppressor activities of suppressor macrophages on T B lymphocytes and NK cells could be prevented by treatment with LPS but the tumoricidal activities of those macrophages could be kept or even enhanced after the same treatment. During this complicated course LPS-mediated immuno-modulation was accompanied by activation of PKC and MAPK signal pathways. In order to explore the effect of another signal on MAPK pathway this model of immuno-modulated macrophage was utilized to study the regulatory effect on the activation of three family members of MAPK (ERK1/2 JNK and p38) by cAMP/PKA and PMA/PKC. The results showed that 1) LPS-mediated immuno-modulation was accompanied by dynamic changes of intracellular cAMP amount and PKA activity. 2) A specific PKC activator PMA induced strongly the activation of ERK1/2 JNK and p38 MAPK. 3) In contrast the activation of cAMP/PKA mediated a significant inhibiton of the phosphorylation of ERK1/2 JNK and p38 MAPK and ATF-2 but it enhanced the phosphorylation of CREB. These results suggest that a complicated "cross-talk" may exist among PKC and PKA and MAPK signaling pathways in the regulation of murine peritoneal suppressor macrophages by LPS.

Journal Article↗

Cardioprotective effect of angiotensin II receptor antagonists.

Angiotensin II plays a significant role in cell growth and proliferation in model systems and in humans. In addition, angiotensin II appears to facilitate sympathetic activation and the release of endothelin-1, and also to promote apoptosis. The use of angiotensin-converting enzyme (ACE) inhibitors has provided beneficial effects on left ventricular hypertrophy (LVH) regression and on cardiac remodelling in the presence of heart failure. Data from experimental models as well as studies in humans suggest that the increase of bradykinin mediated by ACE inhibitors provides most of the beneficial effects of ACE inhibitors. The new class of angiotensin receptor blocker appears to provide cardioprotective effects that are similar to those of the ACE inhibitors. Most of the beneficial effects provided by these agents appear to be related to a more complete blockade of angiotensin II type 1 (AT1) receptor. However, costimulation of the angiotensin II type 2 (AT2) receptor appears to increase nitric oxide and thus to cause some bradykinin-like effects. Evidence for the role of angiotensin II in promoting LVH and cardiac failure as well as for abnormal regulations of the angiotensin signal transduction pathways in model systems and in humans are reviewed. Second, the mechanisms for the beneficial effects of angiotensin II modulation by ACE inhibitors versus angiotensin II antagonists studied in model systems are presented. Finally, results from pivotal phase II studies such as Evaluation of Losartan In The Elderly (ELITE), as well as an overview of the ongoing phase III trials involving the use of ARB in high risk patients are presented.

Angiotensin Receptor Antagonists↗

Effects of insulin concentration and self-association on the partitioning of its A-21 cyclic anhydride intermediate to desamido insulin and covalent dimer.

PURPOSE: In the pH range 2-5, human insulin degrades via deamidation at the A-21 asn and covalent dimerization. Both products form via a common cyclic anhydride intermediate, a product of intramolecular neucleophilic attack by the A-21 carboxyl terminus. This study examines the influence of [insulin] and self-association on the partitioning of the intermediate to products. METHODS: Insulin self-association was characterized (pH 2-4) by concentration difference spectroscopy. Deamination rates (pH 2-4) and concurrent rates of covalent dimer formation (pH 4) were determined versus [insulin] at 35 degrees C by initial rates. A mathematical model was developed to account for the overall rate and product composition profile versus pH and [insulin]. RESULTS: Between pH 2-4, insulin self-associates to form non-covalent dimers with a pH independent association constant of 1.8 x 10(4) M-1. The overall rate of degradation is governed by intermediate formation, while product distribution is determined by competition between water and the phe B-1 amino group of insulin for the anhydride. In dilute solutions, deamidation is first-order in [insulin] while covalent dimerization is second-order. Thus, deamidation predominates in dilute solutions but the fraction of covalent dimer formed increases with [insulin]. At high [insulin], self-association inhibits covalent dimer formation, preventing exclusive degradation via this pathway. The model accurately predicts a maximum in covalent dimer formation near pH 4. CONCLUSIONS: A mechanism is described which accounts for the complex dependence of insulin's degradation rate and product distribution profile on pH (between 2-5) and [insulin]. If these results can be generalized, they suggest that covalent aggregation in proteins may be inhibited by self-association.

Anhydrides↗

Dopamine activates ERKs in alveolar epithelial cells via Ras-PKC-dependent and Grb2/Sos-independent mechanisms.

Recently it has been described that dopamine (DA), via dopaminergic type 2 receptors (D(2)R), activates the mitogen-activated protein kinase extracellular signal-regulated kinase (MAPK/ERK) proteins in alveolar epithelial cells (AEC), which results in the upregulation of Na(+)-K(+)-ATPase. In the present report, we used AEC to investigate the signaling pathway that links DA with ERK activation. Incubation of AEC with DA resulted in rapid and transient stimulation of ERK activity, which was mediated by Ras proteins and the serine/threonine kinase Raf-1. Pretreatment of AEC with Src homology 3 binding peptide, which blocks the interaction between Grb2 and Sos, did not prevent DA activation of ERK. Diacylglycerol (DAG)-dependent protein kinase C (PKC) isoenzymes, involved in the DA-mediated activation of ERK proteins as pretreatment with either bisindolylmaleimide or Ro-31-8220, prevented the phosphorylation of Elk-1, and quinpirole, a D(2)R activator, stimulates the translocation of PKCepsilon. Together, the data suggest that DA activated MAPK/ERK via Ras, Raf-1 kinase, and DAG-dependent PKC isoenzymes, but, importantly and contrary to the classical model, this pathway did not involve the Grb2-Sos complex formation.

Adaptor Proteins, Signal Transducing↗

Cross-feeding percolation phase transitions of intercellular metabolic networks.

Intercellular cross-talk is essential for the adaptation capabilities of populations of cells. While direct diffusion-driven cell-to-cell exchanges are difficult to map, current nanotechnology enables one to probe single-cell exchanges with the medium. We introduce a mathematical method to reconstruct the dynamic unfolding of intercellular exchange networks from these data, applying it to an experimental coculture system. The exchange network, initially dense, progressively fragments into small disconnected clusters. To explain these dynamics, we develop a maximum-entropy multicellular metabolic model with diffusion-driven exchanges. The model predicts a transition from a dense network to a sparse one as nutrient consumption shifts. We characterize this crossover both numerically, revealing a power-law decay in the cluster-size distribution, and analytically, by connecting to percolation theory. Comparison with data suggests that populations evolve toward the sparse phase by remaining near the crossover. These findings offer insights into the collective organization driving the adaptive dynamics of cell populations.

Metabolic Networks and Pathways↗

Leishmania donovani polyamine biosynthetic enzyme overproducers as tools to investigate the mode of action of cytotoxic polyamine analogs.

A number of anticancer and antiparasitic drugs are postulated to target the polyamine biosynthetic pathway and polyamine function, but the exact mode of action of these compounds is still being elucidated. To establish whether polyamine analogs specifically target enzymes of the polyamine pathway, a model was developed using strains of the protozoan parasite Leishmania donovani that overproduce each of the polyamine biosynthetic enzymes. Promastigotes overexpressing episomal constructs encoding ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (ADOMETDC), or spermidine synthase (SPDSYN) revealed robust overproduction of the corresponding polyamine biosynthetic enzyme. Polyamine pools, however, were either unchanged or only marginally affected, implying that regulatory mechanisms must exist. The ODC, ADOMETDC, and SPDSYN overproducer strains exhibited a high level of resistance to difluoromethylornithine, 5'-{[(Z)-4-amino-2-butenyl]methylamino}-5'-deoxyadenosine, and n-butylamine, respectively, confirming previous observations that these agents specifically target polyamine enzymes. Conversely, augmented levels of polyamine biosynthetic enzymes did not affect the sensitivity of L. donovani promastigotes to pentamidine, berenil, and mitoguazone, drugs that were postulated to target the polyamine pathway, implying alternative and/or additional targets for these agents. The sensitivities of wild-type and overproducing parasites to a variety of polyamine analogs were also tested. The polyamine enzyme-overproducing lines offer a rapid cell-based screen for assessing whether synthetic polyamine analogs exert their mechanism of action predominantly on the polyamine biosynthetic pathway in L. donovani. Furthermore, the drug resistance engendered by the amplification of target genes and the overproduction of the encoded protein offers a general strategy for evaluating and developing therapeutic agents that target specific proteins in Leishmania.

Adenosylmethionine Decarboxylase↗

Multimodal assessment of pain in the esophagus: a new experimental model.

A new multimodal pain assessment model was developed integrating electrical, mechanical, cold, and warmth stimuli into the same device. The device, with a bag and electrodes for electrical stimulation, was positioned in the lower part of the esophagus in 11 healthy subjects. Mechanical stimuli were delivered with an impedance planimetric system. Thermal stimuli were performed by circulating water of different temperatures (5-50 degrees C) inside the bag. All subjects reported both nonpainful and painful local and referred sensations to all stimuli. Temporal summation to repeated electrical stimuli could be studied. For all stimuli, there was a relationship between stimulus intensity and pain intensity. The referred pain area increased with increasing intensity of the electrical and mechanical stimuli. There were several differences between the sensations evoked by the four stimulus modalities, indicating activation of different visceral nerve pathways. This model offers the possibility for controlled multimodal stimuli activating the superficial and deeper layers of the human gut and should be used in basic, clinical, and pharmacological pain studies.

Adult↗