Revealing the molecular determinants of neurotoxin specificity for calcium-activated versus voltage-dependent potassium channels

Potassium channel dysfunction underlies diseases such as epilepsy, hypertension, cardiac arrhythmias, and multiple sclerosis. Neurotoxins that selectively inhibit potassium channels, alpha-KTx, have provided invaluable information for dissecting the contribution of different potassium channels to neurotransmission, vasoconstriction, and lymphocyte proliferation. Thus, alpha-KTx specificity comprises an important first step in potassium channel-directed drug discovery for these diseases. Despite extensive functional and structural studies of alpha-KTx-potassium channel complexes, none have predicted the molecular basis of alpha-KTx specificity. Here we show that by minimizing the differences in binding free energy between selective and nonselective alpha-KTx we are able to identify all of the determinants of alpha-KTx specificity for calcium-activated versus voltage-dependent potassium channels. Because these determinants correspond to unique features of the two types of channels, they provide a way to develop more accurate models of alpha-KTx-potassium channel complexes that can be used to design novel selective alpha-KTx inhibitors.     

Growth factor for cardiac hypertrophy

Cardiac size can be regulated by the balance in activity between cardiac growth factors and inhibiting factors, chalones. This study was undertaken to verify the role of the cardiac growth factor and its purification from hypertrophied hearts. For this propose the hypertrophied hearts of renovascular hypertensive rats were used. The purification was made by using an isoelectric focusing chromatography and the HPLC method. We examined the cardiac growth effect of the isolated fractions with cultured chicken embryonic cardiac myocytes. Simultaneously, the influence of these fractions on the cardiac cell cycle was examined by DNA analysis with the flow cytometric method. If the hearts were overloaded due to hypertension, the growth of the cardiac size could be induced by increased the level of five proteins with different molecular weight and with an isoelectric point of 8.3. The significant growth activities were observed at these five proteins compared to the absence of the fractions. For the appearance of these growth effect, it is necessary that the structure of the protein is there fundamentally as a form with a molecular weight of 27 k dalton. After addition of these isolated fractions, BrdU content is S and G2 phases by flow cytometry was increased. This change indicates that the cardiac myocytes are stimulated in form DNA synthesis.     

Cytoplasmic flow and the establishment of polarity in C. elegans 1-cell embryos

Early Caenorhabditis elegans embryos provide an excellent model for the study of developmental processes. Development can be studied by direct observation under the light microscope and can be perturbed using laser manipulations, drug inhibitor treatments, and genetic mutants. The first division of the C. elegans embryo is asymmetric, generating two daughter cells unequal in size and developmental fate. These distinct fates are generated by the partitioning of cytoplasmic determinants during the first mitotic cell cycle. Partitioning of these determinants is thought to be driven by cytoplasmic flow. Recent studies in C. elegans in the past year have identified a number of components necessary for this flow, giving us a clearer picture of the molecular mechanisms underlying developmental asymmetry.     

Les examens cardiaques en tomographie par émission de positons

Cardiac positron emission tomography (PET) is yet considered as a reference imaging technique but remains poorly used in clinical practice. At the present time, the advantages of cardiac PET investigations are far to be evident, when compared with conventional tomoscintigraphy (SPECT), except for perfusion imaging in the obese and for viability assessment in case of very severe cardiac dysfunction. However, this situation might quickly move because of an enhanced availability of PET imaging, dramatic technical progresses and promising new tracers. In particular, the last-generation PET-cameras allow reaching spatial resolutions and detection sensitivities, which are now spectacularly higher than those from conventional SPECT imaging. In addition, the list mode recording allows the subsequent images reconstruction to be synchronized to cardiac cycle but also to respiratory cycle; and the quantifications of myocardial perfusion flow and of coronary flow reserve are now available in clinical routine. Furthermore, new tracers labelled with fluorine-18 are under development, especially for perfusion investigations, and kinetics properties of these new tracers are dramatically enhanced when compared with current perfusion SPECT tracers.     

Conditional mutations in SERCA, the Sarco-endoplasmic reticulum Ca2+-ATPase, alter heart rate and rhythmicity in Drosophila

To analyze the role of cytosolic calcium in regulating heart beat frequency and rhythm, we studied conditional mutations in Drosophila Sarco-endoplasmic reticulum Ca²⁺-ATPase, believed to be predominantly responsible for sequestering free cytosolic calcium. Abnormalities in the amount or structure of the SERCA protein have been linked to cardiac malfunction in mammals. Drosophila SERCA protein (dSERCA) is highly enriched in Drosophila larval heart with a distinct membrane distribution of SERCA at cardiac Z-lines, suggesting evolutionarily conserved zones for calcium uptake into the sarcoplasmic reticulum. Heart beat frequency is strikingly reduced in mutant animals following dSERCA inactivation, (achieved by a brief exposure of these conditional mutants to non-permissive temperature). Cardiac contractions also show abnormal rhythmicity and electrophysiological recordings from the heart muscle reveal dramatic alterations in electrical activity. Overall, these studies underscore the utility of the Drosophila heart to model SERCA dysfunction dependent cardiac disorders and constitute an initial step towards developing Drosophila as a viable genetic model system to study conserved molecular determinants of cardiac physiology.     

Model-based optimization of a sequencing batch reactor for biological nitrogen removal

An optimal operating mode for a sequencing batch reactor was determined via a model-based optimization. Synthetic wastewater containing mainly organic matter (as glucose) and nitrogen (as ammonium chloride) was treated without any addition of an external carbon source to accomplish denitrification step. A simplified model was used to describe process dynamics, comprised of six ordinary differential equations and an empirical correlation for oxygen consumption rate. Batch cycle time was the chosen objective function to be minimized for a fixed volume of waste to be treated. Furthermore, as SBR operation is divided in two major phases - aerobic and anoxic, to achieve total pollutants removal within minimum time, these phases can be repeatedly alternated. To ensure availability of organic matter necessary for denitrification, these two phases were combined with feed steps. Different feed strategies were tested using one, two or three feed steps. A successive quadratic programming algorithm was used, and maximum values for final COD, nitrate and ammonium concentrations, as well as maximum feed pump flow rate were some the process constraints. One step feed strategy was indicated by the optimization leading to a batch cycle time of 5h.     

Protective role of mast cells in homocysteine-induced cardiac remodeling

Recent reports including those from our laboratories indicate that hyperhomocysteinemia (Hhe) is an independent risk factor for cardiac dysfunction and clinical heart failure. Mast cell accumulation is a prominent feature in our model of Hhe-induced cardiac dysfunction. Because mast cell-derived mediators can potentially attenuate cardiac remodeling, we investigated the possible protective role of mast cells in Hhe-induced cardiac remodeling using a mast cell-deficient rat model that in our recent report did not demonstrate any adverse cardiac function at younger age (6 mo) than mast cell-competent control animals. Mast cell-deficient (Ws/Ws) rats and mast cell-competent (+/+) littermate control animals (3 mo of age) were treated with a Hhe-inducing diet for 10 wk. Cardiac remodeling was assessed structurally utilizing histomorphometric methods and functionally using an isolated Langendorff-perfused heart preparation. The Hhe-inducing diet caused similar elevations of homocysteine levels in the two groups. Compared with Hhe +/+ rats, the Hhe Ws/Ws rats demonstrated strikingly exacerbated adverse cardiac remodeling and myocardial fibrosis. Cardiac function measurement showed worsened diastolic function in Hhe Ws/Ws rats compared with Hhe +/+ rats. The absence of mast cells strikingly exacerbates Hhe-induced adverse cardiac remodeling and diastolic dysfunction. These findings indicate a potential dual rather than sole deleterious role for mast cells in cardiac injury.     

Closing the larval loop: linking larval ecology to the population dynamics of marine benthic invertebrates

The majority of marine benthic invertebrates exhibit a complex life cycle that includes separate planktonic larval, and bottom-dwelling juvenile and adult phases. To understand and predict changes in the spatial and temporal distributions, abundances, population growth rate, and population structure of a species with such a complex life cycle, it is necessary to understand the relative importance of the physical, chemical and biological properties and processes that affect individuals within both the planktonic and benthic phases. To accomplish this goal, it is necessary to study both phases within a common, quantitative framework defined in terms of some common currency. This can be done efficiently through construction and evaluation of a population dynamics model that describes the complete life cycle.

Two forms that such a model might assume are reviewed: a stage-based, population matrix model, and a model that specifies discrete stages of the population, on the bottom and in the water column, in terms of simultaneous differential equations that may be solved in both space and time. Terms to be incorporated in each type of model can be formulated to describe the critical properties and processes that can affect populations within each stage of the life cycle. For both types of model it is shown how this might be accomplished using an idealized balanomorph barnacle as an example species. The critical properties and processes that affect the planktonic and benthic phases are reviewed. For larvae, these include benthic adult fecundity and fertilization success, growth and larval stage duration, mortality, larval behavior, dispersal by currents and turbulence, and larval settlement. It is possible to predict or estimate empirically all of the key terms that should be built into the larval and benthic components of the model. Thus, the challenge of formulating and evaluating a full life cycle model is achievable. Development and evaluation of such a model will be challenging because of the diverse processes which must be considered, and because of the disparities in the spatial and temporal scales appropriate to the benthic and planktonic larval phases. In evaluating model predictions it is critical that sampling schemes be matched to the spatial and temporal scales of model resolution.     

Exercise-induced and nitroglycerin-induced myocardial preconditioning improves hemodynamics in patients with angina

In humans, regional myocardial dysfunction during ischemia may be improved by ischemic and pharmacological preconditioning. We assessed the possibility that exercise- and nitroglycerin-induced myocardial preconditioning may improve global cardiac performance during subsequent efforts in patients with angina. Ten patients suffering from chronic stable angina and ten healthy volunteers were studied. Through impedance cardiography we assessed hemodynamics during a maximal exercise test, which was used as a baseline (Bas test) and considered as a preconditioning exercise. The Bas test was followed by a sequence of maximal efforts performed during the first (FWOP; 30 min after the Bas test) and second (SWOP; 48 h after the Bas test) windows of protection conferred by ischemic preconditioning. Hemodynamics was further evaluated during maximal exercise performed 48 h later with pharmacologically induced SWOP (PI-SWOP) obtained by transdermal administration of 10 mg of nitroglycerin. In the angina patients, FWOP, SWOP, and PI-SWOP delayed the time to ischemia and allowed them to achieve higher workloads compared with the Bas test. Furthermore, heart rate and cardiac output at peak exercise were enhanced during all the preconditioning phases with respect to the Bas test. However, only SWOP and PI-SWOP increased myocardial contractility and stroke volume. No changes in hemodynamics were detectable in the control subjects. This study demonstrates that in patients with stable angina, although hemodynamics during exercise can be positively improved during both FWOP and SWOP, differences exist between these two phases. Furthermore, the mimicking of exercise-induced SWOP by PI-SWOP with transdermal nitroglycerin may represent an important clinical aspect.     

Development of a 4D numerical chest phantom with customizable breathing

Respiratory movement information is useful for radiation therapy, and is generally obtained using 4D scanners (4DCT). In the interest of patient safety, reducing the use of 4DCT could be a significant step in reducing radiation exposure, the effects of which are not well documented. The authors propose a customized 4D numerical phantom representing the organ contours. Firstly, breathing movement can be simulated and customized according to the patient’s anthroporadiametric data. Using learning sets constituted by 4D scanners, artificial neural networks can be trained to interpolate the lung contours corresponding to an unknown patient, and then to simulate its respiration. Lung movement during the breathing cycle is modeled by predicting the lung contours at any respiratory phases. The interpolation is validated comparing the obtained lung contours with 4DCT via Dice coefficient. Secondly, a preliminary study of cardiac and œsophageal motion is also presented to demonstrate the flexibility of this approach. The application may simulate the position and volume of the lungs, the œsophagus and the heart at every phase of the respiratory cycle with a good accuracy: the validation of the lung modeling gives a Dice index greater than 0.93 with 4DCT over a breath cycle.     

Metabolic status rather than cell cycle signals control quiescence entry and exit

Quiescence is defined as a temporary arrest of proliferation, yet it likely encompasses various cellular situations. Our knowledge about this widespread cellular state remains limited. In particular, little is known about the molecular determinants that orchestrate quiescence establishment and exit. Here we show that upon carbon source exhaustion, budding yeast can enter quiescence from all cell cycle phases. Moreover, using cellular structures that are candidate markers for quiescence, we found that the first steps of quiescence exit can be triggered independently of cell growth and proliferation by the sole addition of glucose in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. Importantly, glucose needs to be internalized and catabolized all the way down to glycolysis to mobilize quiescent cell specific structures, but, strikingly, ATP replenishment is apparently not the key signal. Altogether, these findings strongly suggest that quiescence entry and exit primarily rely on cellular metabolic status and can be uncoupled from the cell cycle.     

High Fat Feeding in Mice Is Insufficient to Induce Cardiac Dysfunction and Does Not Exacerbate Heart Failure

Preclinical studies of animals with risk factors, and how those risk factors contribute to the development of cardiovascular disease and cardiac dysfunction, are clearly needed. One such approach is to feed mice a diet rich in fat (i.e. 60%). Here, we determined whether a high fat diet was sufficient to induce cardiac dysfunction in mice. We subjected mice to two different high fat diets (lard or milk as fat source) and followed them for over six months and found no significant decrement in cardiac function (via echocardiography), despite robust adiposity and impaired glucose disposal. We next determined whether antecedent and concomitant exposure to high fat diet (lard) altered the murine heart’s response to infarct-induced heart failure; high fat feeding during, or before and during, heart failure did not significantly exacerbate cardiac dysfunction. Given the lack of a robust effect on cardiac dysfunction with high fat feeding, we then examined a commonly used mouse model of overt diabetes, hyperglycemia, and obesity (db/db mice). db/db mice (or STZ treated wild-type mice) subjected to pressure overload exhibited no significant exacerbation of cardiac dysfunction; however, ischemia-reperfusion injury significantly depressed cardiac function in db/db mice compared to their non-diabetic littermates. Thus, we were able to document a negative influence of a risk factor in a relevant cardiovascular disease model; however, this did not involve exposure to a high fat diet. High fat diet, obesity, or hyperglycemia does not necessarily induce cardiac dysfunction in mice. Although many investigators use such diabetes/obesity models to understand cardiac defects related to risk factors, this study, along with those from several other groups, serves as a cautionary note regarding the use of murine models of diabetes and obesity in the context of heart failure.     

Macrophage migration inhibitory factor mediates late cardiac dysfunction after burn injury

We have recently demonstrated that macrophage migration inhibitory factor (MIF) is a myocardial depressant protein and that MIF mediates late, prolonged cardiac dysfunction after endotoxin challenge in mice. Because many factors, including endotoxin, have been implicated in the pathogenesis of cardiac dysfunction after burn injury, we tested the hypothesis that MIF might also be the mediator of prolonged cardiac dysfunction in this model. At 4 h after 40% total body surface area burn in anesthetized mice, serum MIF levels increased significantly compared with baseline (2.2-fold). This increase was accompanied by a significant decrease in cardiac tissue MIF levels (2.1-fold decrease compared with controls). This pattern was consistent with MIF release from preformed cytoplasmic stores in the heart and other organs. To determine whether MIF mediates cardiac dysfunction after burn injury, mice were pretreated with anti-MIF neutralizing monoclonal antibodies or isotype control antibodies. Beginning 4 h after burn injury (and continuing through 48 h), burned mice demonstrated a significantly depressed left ventricular shortening fraction of 38.6 +/- 1.8%, compared with the normal controls (56.0 +/- 2.6%). Mice treated with anti-MIF displayed an initial depression of cardiac function similar to nontreated animals but then showed rapid restoration of cardiac function with complete recovery by 24 h, which persisted for the duration of the protocol. This study is the first to demonstrate that MIF mediates late, prolonged cardiac dysfunction after burn injury and suggests that MIF blockade should be considered a therapeutic target for the treatment of burn injury.     

Survival characteristics of normal differentiated rat thyroid cells maintained in vitro

The radiation response of a cultured line of differentiated rat thyroid epithelial cells (FRTL-5) was determined by clonogenic assay in vitro and compared to the previously reported response curve for primary rat thyroid cells assayed by transplantation in vivo. The beta components (or D0 estimates) for the two sets of data were not significantly different although the response of the FRTL-5 cells was displaced to the right as a consequence of differences in the shoulder of the survival curves. These results indicate that this in vitro model can be used to study the radiation response of thyroid epithelial cells and by extrapolation perhaps other hormonally responsive, slowly proliferative parenchyma. The cell age response for FRTL-5 cells was determined after separation of the cells into various phases of the cell cycle by centrifugal elutriation. Resistant peaks in mid-G1 and G2M phases of the cell cycle were detected. The cell age response data for other portions of the cell cycle should be interpreted cautiously because less than satisfactory enrichments were achieved.     

Uncoupling of the order of the S and M phases: effects of staurosporine on human cell cycle kinases

The protein kinase inhibitor staurosporine (SSP) was employed to study the involvement of kinases in human cell cycle progression. Thirty to 100 ng/ml SSP blocks entry into S phase and M phase. Lack of entry into S phase is due to impaired activity of the retinoblastoma protein kinase. The requirement for any of the SSP-sensitive kinases for cell cycle progression can be abrogated in tumour cells. Therefore, these kinases act in a checkpoint network negatively controlling the initiation of S phase, M phase and cytokinesis, rather than being inherent parts of a substrate-product chain required for the initiation of the cell cycle phases. As a consequence of the lack of certain checkpoint effectors, tumour cells may endoreduplicate or binucleate in the presence of SSP. The latter processes, as well as meiosis, are naturally occurring in specialized cell types, leading to the idea that this checkpoint network controls the order of the cell cycle phases in normal cells. A model is presented where the cell cycle is envisioned as two independently running cycles, the S and the M cycle, which are controlled by intra and intercycle-dependent checkpoints in human somatic cells. The model accounts for the dependency of S and M phase initiation on the successful completion of the previous M and S phase, respectively, as well as entry into a resting state.     

Interrelations between Time Intervals of a Kinetocardiogram and a Pulsogram

Kinetocardiography and pulsography (sphygmography) of the radial artery were used to determine the phase structure (time intervals) of the cardiac cycle, and the data obtained with the two methods were compared. This analysis showed that the time intervals of the cardiac cycle (such as protodiastole, isovolumetric relaxation, and atrial systole) can be determined more accurately from the first- and second-order derivative curves of the pulse wave than from the real-time kinetocardiogram. Hence, the pumping function of the heart can be assessed more correctly with radial artery pulsography.     

Heterochrony in plant development

Plants proceed through several distinct phases of growth and development in their life cycle. Under normal conditions, one phase terminates as another begins, but the relative time at which the phases initiate and terminate can be altered experimentally. New phenotypes are obtained when two developmental phases are expressed at the same time, as well as when phases are shifted coordinately. By analyzing these phenotypes, we can learn how plants normally regulate the transitions between developmental phases.     

Exogenous ketone ester administration attenuates systemic inflammation and reduces organ damage in a lipopolysaccharide model of sepsis

AimsSepsis is a life-threatening condition of organ dysfunction caused by dysregulated inflammation which predisposes patients to developing cardiovascular disease. The ketone β-hydroxybutyrate is reported to be cardioprotective in cardiovascular disease and this may be due to their signaling properties that contribute to reducing inflammation. While exogenous ketone esters (KE) increase blood ketone levels, it remains unknown whether KEs can reduce the enhanced inflammatory response and multi-organ dysfunction that is observed in sepsis. Thus, this study assesses whether a recently developed and clinically safe KE can effectively improve the inflammatory response and organ dysfunction in sepsis.Methods and resultsTo assess the anti-inflammatory effects of a KE, we utilized a model of lipopolysaccharide (LPS)-induced sepsis in which an enhanced inflammatory response results in multi-organ dysfunction. Oral administration of KE for three days prior to LPS-injection significantly protected mice against the profound systemic inflammation compared to their vehicle-treated counterparts. In assessing organ dysfunction, KE protected mice from sepsis-induced cardiac dysfunction as well as renal dysfunction and fibrosis. Furthermore, KE administration attenuated the sepsis-induced inflammation in the heart, kidney, and liver. Moreover, these protective effects occurred independent of changes to enzymes involved in ketone metabolism.ConclusionThese data show that the use of an exogenous KE attenuates the dysregulated systemic and organ inflammation as well as organ dysfunction in a model of severe inflammation. We postulate that this exogenous KE is an appealing and promising approach to capitalize on the protective anti-inflammatory effects of ketones in sepsis and/or other inflammatory responses.     

Analysis of vagally induced sinus arrhythmias.

Vagal stimulation at precise times in successive cardiac cycles can elicit sinus arrhythmias. Two mechanisms have been identified that can, but do not necessarily, cause these vagally induced sinus arrhythmias. First, changes in cycle length elicited by a given concentration of acetylcholine (ACh) depend on the phase of the pacemaker cell action potential when the ACh binds to muscarinic receptors. Second, acetylcholinesterase degrades ACh rapidly enough for the mean concentration of ACh per cardiac cycle to vary from cycle to cycle. We used a mathematical model of the underlying cellular physiology, to examine whether these mechanisms are responsible for arrhythmogenesis. Computer simulation showed that both mechanisms contribute to the vagally induced sinus arrhythmias.     

Mathematical Models for Cell Migration with Real-Time Cell Cycle Dynamics

The fluorescent ubiquitination-based cell cycle indicator, also known as FUCCI, allows the visualization of the G1 and S/G2/M cell cycle phases of individual cells. FUCCI consists of two fluorescent probes, so that cells in the G1 phase fluoresce red and cells in the S/G2/M phase fluoresce green. FUCCI reveals real-time information about cell cycle dynamics of individual cells, and can be used to explore how the cell cycle relates to the location of individual cells, local cell density, and different cellular microenvironments. In particular, FUCCI is used in experimental studies examining cell migration, such as malignant invasion and wound healing. Here we present, to our knowledge, new mathematical models that can describe cell migration and cell cycle dynamics as indicated by FUCCI. The fundamental model describes the two cell cycle phases, G1 and S/G2/M, which FUCCI directly labels. The extended model includes a third phase, early S, which FUCCI indirectly labels. We present experimental data from scratch assays using FUCCI-transduced melanoma cells, and show that the predictions of spatial and temporal patterns of cell density in the experiments can be described by the fundamental model. We obtain numerical solutions of both the fundamental and extended models, which can take the form of traveling waves. These solutions are mathematically interesting because they are a combination of moving wavefronts and moving pulses. We derive and confirm a simple analytical expression for the minimum wave speed, as well as exploring how the wave speed depends on the spatial decay rate of the initial condition.