A quantitative model for linking Na+/Ca2+ exchanger to SERCA during refilling of the sarcoplasmic reticulum to sustain [Ca2+] oscillations in vascular smooth muscle

We have developed a quantitative model for the creation of cytoplasmic Ca²⁺ gradients near the inner surface of the plasma membrane (PM). In particular we simulated the refilling of the sarcoplasmic reticulum (SR) via PM–SR junctions during asynchronous [Ca²⁺]_i oscillations in smooth muscle cells of the rabbit inferior vena cava. We have combined confocal microscopy data on the [Ca²⁺]_i oscillations, force transduction data from cell contraction studies and electron microscopic images to build a basis for computational simulations that model the transport of calcium ions from Na⁺/Ca²⁺ exchangers (NCX) on the PM to sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA) pumps on the SR as a three-dimensional random walk through the PM–SR junctional cytoplasmic spaces. Electron microscopic ultrastructural images of the smooth muscle cells were elaborated with software algorithms to produce a very clear and dimensionally accurate picture of the PM–SR junctions. From this study, we conclude that it is plausible and possible for enough Ca²⁺ to pass through the PM–SR junctions to replete the SR during the regenerative Ca²⁺ release, which underlies agonist induced asynchronous Ca²⁺ oscillations in vascular smooth muscle.     

The antioxidative function of eicosapentaenoic acid in a marine bacterium, Shewanella marinintestina IK-1

When the eicosapentaenoic acid (EPA)-deficient mutant strain IK-1Delta8 of the marine EPA-producing Shewanella marinintestina IK-1 was treated with various concentrations of hydrogen peroxide (H(2)O(2)), its colony-forming ability decreased more than that of the wild type. Protein carbonylation, induced by treating cells with 0.01 mM H(2)O(2) under bacteriostatic conditions, was enhanced only in cells lacking EPA. The amount of cells recovered from the cultures was decreased more significantly by the presence of H(2)O(2) for cells lacking EPA than for those producing EPA. Treatment of the cells with 0.1 mM H(2)O(2) resulted in much lower intracellular concentrations of H(2)O(2) being consistently detected in cells with EPA than in those without EPA. These results suggest that cellular EPA can directly protect cells against oxidative damage by shielding the entry of exogenously added H(2)O(2) in S. marinintestina IK-1.     

Mitochondrial nitric oxide localization in H9c2 cells revealed by confocal microscopy.

This study shows the presence of all three nitric oxide synthases (NOSs) and NOS activity in H9c2 cells cultured under non-stimulated conditions. By using the 4,5 diaminofluoresceindiacetate (DAF-2DA) fluorimetric nitric oxide (NO(*)) detection system we observed NO(*) production in H9c2 cells. As revealed by confocal microscopy, NO(*) fluorescence colocalizes in mitochondria labeled with Mito-Tracker Red CM-H(2)Xros. Upon stimulation with acetylcholine (Ach), which increased NOS activity by 75%, the colocalization coefficient C(green) value, calculated as Pearson's correlation, increased from 0.07 to 0.10, demonstrating an augmented presence of NO(*) in mitochondria. Conversely, the presence of NO(*) in mitochondria decreased following cells pretreatment with l-MonoMethylArginine (L-NMMA), a competitive inhibitor of NOS activity, as indicated by the reduction of the C(green) value to 0.02. This work confirms that the presence of NO(*) in mitochondria can be modulated in response to different fluxes of NO(*).     

One-hit stochastic decline in a mechanochemical model of cytoskeleton-induced neuron death II: transition state metastability

This is the second of two papers in which we study a mathematical model of cytoskeleton-induced neuron death. Recent evidence indicates that aggravated assembly or destruction of the cytoskeleton can trigger programmed death in neurons, by mechanisms as yet poorly understood. In our model, assembly control of the neuronal cytoskeleton interacts with both cellular stress levels and cytosolic free radical concentrations to trigger neurodegeneration. This trigger mechanism is further modulated by a diffusible toxic factor released from dying neurons. In the companion report we established that the model relates the observed general patterns of neuron decline to specific scales of cytoskeleton reorganization and cell-cell interaction strength. In this paper we study the transit of neurons through states intermediate between initial viability and cell death in our model. We find that the stochastic flow of neuron fate, from viability to cell death, self-organizes into two distinct temporal phases. There is a rapid relaxation of the initial neuron population to a more disordered phase that is long-lived, or metastable, with respect to the time scales of change in single cells. Strikingly, cellular egress from this metastable phase follows the one-hit kinetic pattern of exponential decline now established as a principal hallmark of cell death in neurodegenerative disorders. Intermediate state metastability may therefore be an important element in the systems biology of one-hit neurodegeneration.     

Drawing inferences about the coancestry coefficient

The coancestry coefficient, also known as the population structure parameter, is of great interest in population genetics. It can be thought of as the intraclass correlation of pairs of alleles within populations and it can serve as a measure of genetic distance between populations. For a general class of evolutionary models it determines the distribution of allele frequencies among populations. Under more restrictive models it can be regarded as the probability of identity by descent of any pair of alleles at a locus within a random mating population. In this paper we review estimation procedures that use the method of moments or are maximum likelihood under the assumption of normally distributed allele frequencies. We then consider the problem of testing hypotheses about this parameter. In addition to parametric and non-parametric bootstrap tests we present an asymptotically-distributed chi-square test. This test reduces to the contingency-table test for equal sample sizes across populations. Our new test appears to be more powerful than previous tests, especially for loci with multiple alleles. We apply our methods to HapMap SNP data to confirm that the coancestry coefficient for humans is strictly positive.     

A multilevel approach to cancer growth modeling

Cancer growth models may be divided into macroscopic models, which describe the tumor as a single entity, and microscopic ones, which consider the tumor as a complex system whose behavior emerges from the local dynamics of its basic components, the neoplastic cells. Mesoscopic models (e.g. as based on the Local Interaction Simulation Approach [Delsanto, P.P., Mignogna, R., Scalerandi, M., Schechter, R., 1998. In: Delsanto, P.P. Saenz, A.W. (Eds.), New Perspectives on Problems in Classical and Quantum Physics, vol. 2. Gordon & Breach, New Delhi, p. 5174]), which explicitly consider the behavior of cell clusters and their interactions, may be used instead of the microscopic ones, in order to study the properties of cancer biology that strongly depend on the interactions of small groups of cells at intermediate spatial and temporal scales. All these approaches have been developed independently, which limits their usefulness, since they all include relevant features and information that should be cross-correlated for a deeper understanding of the mechanisms involved. In this contribution we consider multicellular tumor spheroids as biological reference systems and propose an intermediate model to bridge the gap between a macroscopic formulation of tumor growth and a mesoscopic one. Thus we are able to establish, as an important result of our formalism, a direct correspondence between parameters characterizing processes occurring at different scales. In particular, we analyze their dependence on an important limiting factor to tumor growth, i.e. the extra-cellular matrix pressure. Since the macro and meso-models stem from totally different roots (energy conservation and clinical observations vs. cell groups dynamics), their consistency may be used to validate both approaches. It may also be interesting to note that the proposed formalism fits well into a recently proposed conjecture of growth laws universality.     

Oxidative stress reduces transintestinal transports and (Na+, K+) -ATPase activity in rat jejunum

Because oxidative stress is a component of gastrointestinal injury, we investigated the effect of H(2)O(2) on transintestinal transport using isolated rat jejunum incubated in vitro. Millimolar concentrations of H(2)O(2) inhibited all the tested parameters without inducing any cytotoxic effect. Electrophysiological experiments indicated that H(2)O(2) decreases significantly both short circuit current and transepithelial electrical potential difference without affecting transepithelial resistance. The possibility that H(2)O(2) could influence (Na+, K+) -ATPase activity was explored using isolated basolateral membranes. Besides H(2)O(2), free radicals (O(2)(*-), HO*) were generated using different iron-dependent and independent systems; (Na+, K+) -ATPase activity was inhibited after membrane exposure to all ROS tested. The inhibition was prevented by allopurinol, superoxide dismutase or desferrioxamine. Western blot analysis showed a decreased expression of the alpha(1)-subunit of (Na+, K+) -ATPase. We conclude that H(2)O(2) may be a modulator of jejunal ion and water transport by multiple mechanisms, among which a significant inhibition of the basolateral (Na+, K+) -ATPase.     

Taurine alleviates ochratoxin A-induced pyroptosis in PK-15 cells by inhibiting oxidative stress

Ochratoxin A (OTA) is one of the most harmful mycotoxins, which can cause multiple toxicological effects, especially nephrotoxicity in animals and humans. Taurine is an essential amino acid with various biological functions such as anti-inflammatory and anti-oxidation. However, the protective effect of taurine on OTA-induced nephrotoxicity and pyroptosis had not been reported. Our results showed that OTA exposure induced cytotoxicity and oxidative stress in PK-15 cells, including reactive oxygen species (ROS) accumulation, increased mRNA levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2), and decreased mRNA levels of catalase (CAT), glutathione peroxidase 1 (GPx1), and glutathione peroxidase 4 (GPx4). In addition, OTA treatment induced pyroptosis by increasing the expressions of pyroptosis-related proteins NLRP3, GSDMD, Caspase-1 P20, ASC, Pro-caspase-1, and IL-1β. Meanwhile, taurine could alleviate OTA-induced pyroptosis and cytotoxicity, as well as reduce ROS level, COX-2, and iNOS mRNA levels, and increase the mRNA levels of the antioxidant enzyme in PK-15 cells. Taken together, taurine alleviated OTA-induced pyroptosis in PK-15 cells by inhibiting ROS generation and altering the activity of antioxidant enzymes, thereby attenuating its nephrotoxicity.     

The role of low avidity T cells in the protection against type 1 diabetes: a modeling investigation

Cytotoxic T lymphocytes (CTLs) play a dominant role in the pathogenesis of autoimmune diabetes, commonly denoted Type 1 Diabetes (T1D). These CTLs (notably CD8⁺ T cells) recognize and kill insulin-secreting pancreatic β cells, reducing their number by ∼90%. The resulting reduction of insulin secretion causes the defective regulation of glucose metabolism, leading to the characteristic symptoms of diabetes. Recognition of β cells as targets by CTLs depends on the interactions between MHC-peptide complexes on the surface of β cells and receptors (TCRs) on T cells. Those CTLs with high affinity TCRs (also called high avidity T cells) cause most of the harm, while those with low affinity TCRs (also called low avidity T cells) play a more mysterious role. Recent experimental evidence suggests that low avidity T cells accumulate as memory T cells during the disease and may be protective in NOD mice (a strain prone to developing T1D), delaying disease progression. It has been hypothesized that such low avidity T cells afford disease protection either by crowding the islets of Langerhans, where β cells reside, or by killing antigen presenting cells (APCs).In this paper, we explore the hypothesized mechanisms for this protective effect in the context of a series of models for (1) the interactions of low and high avidity T cells, (2) the effect of APCs and (3) the feedback from β cell killing to autoantigen-induced T cell proliferation. We analyze properties of these models, noting consistency of predictions with observed behaviour. We then use the models to examine the influence of various treatment strategies on the progression of the disease. The model reveals that progressive accumulation of memory low avidity autoreactive T cells during disease progression makes treatments aimed at expanding these protective T cell types more effective close to, or at the onset of clinical disease. It also provides evidence for the hypothesis that low avidity T cells kill APCs (rather than the alternate hypothesis that they crowd the islets).     

Contributions of HERG current to repolarization of the human ventricular action potential

Action potential repolarization in the mammalian heart is governed by interactions of a number of time- and voltage-dependent channel-mediated currents, as well as contributions from the Na⁺/Ca²⁺ exchanger and the Na⁺/K⁺ pump. Recent work has shown that one of the K⁺ currents (HERG) which contributes to repolarization in mammalian ventricle is a locus at which a number of point mutations can have significant functional consequences. In addition, the remarkable sensitivity of this K⁺ channel isoform to inhibition by a variety of pharmacological agents and clinical drugs has resulted in HERG being a major focus for Safety Pharmacology requirements.For these reasons we and others have attempted to define the functional role for HERG-mediated K⁺ currents in repolarization of the action potential in the human ventricle. Here, we describe and evaluate changes in the formulations for two K⁺ currents, I_K1 and HERG (or I_K,r), within the framework of ten Tusscher model of the human ventricular action potential. In this computational study, new mathematical formulations for the two nonlinear K⁺ conductances, I_K1 and HERG, have been developed based upon experimental data obtained from electrophysiological studies of excised human ventricular tissue and/or myocytes. The resulting mathematical model provides much improved simulations of the relative sizes and time courses of the K⁺ currents which modulate repolarization. Our new formulation represents an important first step in defining the mechanism(s) of repolarization of the membrane action potential in the human ventricle. Our overall goal is to understand the genesis of the T-wave of the human electrocardiogram.     

Modulation of ochratoxin A induced DNA-damage in urothelial cell cultures

Despite good evidence for a genotoxic potential of ochratoxin A (OTA), the mechanism of OTA-induced genotoxicity (direct or indirect?) is still unclear. This calls for a further characterization of OTA-related DNA damage, and investigations of factors that may modulate dose-effect relationships in cells. Since bladder epithelium is a target tissue for the toxicity of OTA, its effects were studied in cultures of human bladder carcinoma (H5637) cells. Cytotoxicity of OTA, assessed by Neutral red (NR) uptake or Alamar-Blue assay, is concentration- and time-dependent: Upon 24 h treatment of 5637 cells, NR uptake is reduced by 50% with OTA concentrations of ≥0.2 microM, but not with 3 h treatment of the cells. Since cytotoxicity of OTA was not affected by addition of xenobiotic metabolizing enzymes (S-9 mix), it appears to be unrelated to biotransformation of the mycotoxin. Also, addition of S-9 mix did not significantly affect the genotoxicity of OTA as studied by alkaline single cell gel electrophoresis (Comet assay). DNA damage was detectable after 3 h treatment of cells at OTA concentrations between 0.1 and 1 microM, and increased further at higher concentrations. The magnitude of OTA-induced DNA damage did not increase with longer treatment times (18, 24 h), probably due to repair processes in the cells. Repair of OTA-induced lesions is quite efficient in kidney (Arch Toxicol 2002, 75, 734–741) and in porcine bladder cells (Föllmann and Lebrun, 2005, Mycotoxin Research, this volume). Interestingly, the genotoxicity of OTA is modulated by the pH of the culture medium, with higher damage at pH 5 compared to pH 7.5. In line with this, uptake studies with tritiated OTA show a higher cellular accumulation of the mycotoxin at pH 5 than in buffer of pH 7.5. Thus, bladder cells exposed to OTA in slightly acidic urine (which facilitates reabsorption) may be at higher risk.     

Activity of a KATP+ channel in Arum spadix mitochondria during thermogenesis

This report demonstrates that mitochondria isolated from thermogenic Arum spadices possess an ATP-sensitive potassium channel--responsible for electrical potential (DeltaPsi) collapse and mitochondrial swelling--whose characteristics are similar to those previously described in pea and wheat mitochondria. In order to study the relationship between this K(ATP)(+) channel and the uncoupled respiration, linked to thermogenesis, K(+) transport activities were compared with those of mitochondria that were isolated from pea stems, soybean suspension cell cultures and Arum tubers. The channel from Arum spadices is highly active and its major features are (i) potassium flux is performed primarily in an inward-rectifying manner; (ii) the influx of K(+) is associated with a matrix volume increase in both energized and non-energized mitochondria; and (iii) its activity depends on the redox state of electron transport chain (ETC) and oxygen availability. In particular, this paper shows that the K(ATP)(+) channel is inwardly activated in parallel with the alternative oxidase (AO). The activation is linked to an ETC-oxidized state and to high oxygen consumption. The putative role of this K(ATP)(+) channel is discussed in relation to flowering of thermogenic Arum spadices.     

Free radical oxidation (autoxidation) of alkenones and other lipids in cells of Emiliania huxleyi

Cells of the coccolithophorid Emiliania huxleyi strain CS-57 grown under an atmosphere of air+0.5% CO(2) showed oxidative damage after 10 days growth with concomitant and major changes to the lipid composition. The fatty acid profile was strongly altered and lacked appreciable amounts of the polyunsaturated fatty acids (PUFA: C(18:5), C(18:3) and C(22:6)) typical of healthy cells. Oxidation products of these PUFA could not be detected, but monounsaturated fatty acids proved to be good indicators of oxidative processes. The presence (after NaBH(4)-reduction) of a high proportion of 11-hydroxyoctadec-cis-9-enoic and 8-hydroxyoctadec-cis-9-enoic acids showed that the degradation of oleic acid involved mainly free radical oxidation processes (70-75% autoxidation and 20-25% photooxidation). We also detected large amounts of degradation products of the oxidation product 9,10-epoxyoctadecanoic acid including diols, methoxyhydrins and chlorohydrins. These oxidative effects were found in all the lipid classes examined. Products included significant amounts of chlorophyll side-chain autooxidation products Z- and E-3,7,11,15-tetramethylhexadec-3-ene-1,2-diols and Z-and E-3,7,11,15-tetramethylhexadec-2-ene-1,4-diols, while phytyldiol was present in relatively low proportions. Delta(5)-3beta,7-epimeric unsaturated steroidal diols arising from the autooxidation of the Delta(5) double bond of epi-brassicasterol and minor amounts of Delta(4)-3beta,6-diols were also detected. Long-chain unsaturated ketone (alkenone) content per cell was much higher in the presence of 0.5% CO(2) likely due to carbon storage under these conditions. The proportions of di- and tri-unsaturated alkenones was relatively stable throughout the growth cycle in the absence of additional CO(2), but not when grown with 0.5% CO(2). The detection of characteristic alkenone autoxidation products in cells grown under these latter conditions allowed us to attribute the significant increase in index observed to the involvement of free radical oxidation processes.     

E versus Z geometry in β-d-arabino-hexopyranosidulose oximes

Koenigs–Knorr-type glycosidations of peracylated 2Z-benzoyloxyimino-glycopyranosyl bromides invariably proceed with retention of the Z-geometry. Accordingly, the many β-d-hexosidulose oximes in literature which were prepared in this way and for which the oxime geometry has not been addressed explicitly, are the Z-oximes throughout. By contrast, oximation of β-d-hexopyranosid-2-uloses leads to mixtures of E and Z oximes readily separable and structurally verifiable by ¹H and ¹³C NMR. Configurational assignments rested on comparative evaluation of NMR data of E and Z isomers, and, most notably on an X-ray structural analysis of the pivaloylated isopropyl 2E-benzoyloxyimino-2-deoxy-β-d-arabino-hexopyranoside revealing the unusual ¹S₅^1,4B conformation for the pyranoid ring.     

Patterning, prestress, and peeling dynamics of myocytes

As typical anchorage-dependent cells myocytes must balance contractility against adequate adhesion. Skeletal myotubes grown as isolated strips from myoblasts on micropatterned glass exhibited spontaneous peeling after one end of the myotube was mechanically detached. Such results indicate the development of a prestress in the cells. To assess this prestress and study the dynamic adhesion strength of single myocytes, the shear stress of fluid aspirated into a large-bore micropipette was then used to forcibly peel myotubes. The velocity at which cells peeled from the surface, V(peel), was measured as a continuously increasing function of the imposed tension, T(peel), which ranges from approximately 0 to 50 nN/ micro m. For each cell, peeling proved highly heterogeneous, with V(peel) fluctuating between 0 micro m/s ( approximately 80% of time) and approximately 10 micro m/s. Parallel studies of smooth muscle cells expressing GFP-paxillin also exhibited a discontinuous peeling in which focal adhesions fractured above sites of strong attachment (when pressure peeled using a small-bore pipette). The peeling approaches described here lend insight into the contractile-adhesion balance and can be used to study the real-time dynamics of stressed adhesions through both physical detection and the use of GFP markers; the methods should prove useful in comparing normal versus dystrophic muscle cells.     

p53 activation inhibits ochratoxin A-induced apoptosis in monkey and human kidney epithelial cells via suppression of JNK activation

Ochratoxin A (OTA), one of the major food-borne mycotoxins, induces apoptosis in various types of cells. Induction of apoptosis is suggested to be one of the major cellular mechanisms behind OTA-induced diverse toxic effects. However, the molecular mechanisms involved, especially the role of p53 in OTA-induced apoptosis have not been clearly elucidated. In the present study, we find that p53 activation exerts pro-survival function to inhibit apoptosis induction in MARC-145, Vero monkey kidney cells and HEK293 human kidney cells in response to ochratoxin A treatment. We further demonstrate that the pro-survival activity of p53 is attributed to its ability to suppress JNK activation that mediates apoptotic signaling through down-regulation of Bcl-xL. To our knowledge, this is first report of pro-survival role of p53 in OTA-induced apoptosis in kidney epithelial cells. Our findings provide a novel insight into the mechanisms of OTA-induced apoptosis in kidney epithelial cells.     

Computation of threshold conditions for epidemiological models and global stability of the disease-free equilibrium (DFE)

One goal of this paper is to give an algorithm for computing a threshold condition for epidemiological systems arising from compartmental deterministic modeling. We calculate a threshold condition T(0) of the parameters of the system such that if T(0)<1 the disease-free equilibrium (DFE) is locally asymptotically stable (LAS), and if T(0)>1, the DFE is unstable. The second objective, by adding some reasonable assumptions, is to give, depending on the model, necessary and sufficient conditions for global asymptotic stability (GAS) of the DFE. In many cases, we can prove that a necessary and sufficient condition for the global asymptotic stability of the DFE is R(0)< or =1, where R(0) is the basic reproduction number [O. Diekmann, J.A. Heesterbeek, Mathematical Epidemiology of Infectious Diseases: Model Building, Analysis and Interpretation, Wiley, New York, 2000]. To illustrate our results, we apply our techniques to examples taken from the literature. In these examples we improve the results already obtained for the GAS of the DFE. We show that our algorithm is relevant for high dimensional epidemiological models.