Modulation of adenylate cyclase activity by sulfated glycosaminoglycans II. Effects of mucopolysaccharides and dextran sulfate on the activity of adenylate cyclase derived from various tissues

Heparin was found to be the most potent inhibitor of rat ovarian luteinizing hormone-sensitive adenylate cyclase (I₅₀ = 2 μg/ml) when compared to other naturally occurring glycosaminoglycans. This inhinibition was also appparent when this enzyme was stimulated by follicle-stimulating hormone or prostaglandin E ₂. Heparin was also found to inhibit glucagon-sensitive rat hepatice adenylate cyclase, and the prostaglandin E₁-sensitive enzyme from rat ileum and human platelets. In contrast, heparin stimulated the dopamine sensitive adenylate cyclase from rat caudate nucleus. The sulfade polysugar dextran sulfate exerts similar effects on adenylate cyclase activity of the rat ovary was shown to inhibit hormone binding to rat ovarian plasma membrane in a manner similar to that exerted by heparin. In contrast to heparin, dextran sulfate inhibited dopamine-sensitive adenylate cyclase from rat caudate nucleus.     

Protein kinase C (PKC) level is increased in PC12 cells overexpressing transfected liver-type phosphofructokinase

Summary— PC12 cells which overexpress transfected liver-type phosphofructokinase (PFKL) have previously been described as a model system for PFKL overexpression in Down's syndrome and have been shown to perform glycolysis at enhanced rates. Here we report that levels of protein kinase C (PKC) in PC 12-PFKL cells were almost doubled, as estimated from in vitro activity and phorbol ester binding experiments and from an increase found in PKC-alpha mRNA levels. Most of the added PKC was found to be associated with the cellular membrane while the cytoplasmic levels of PKC were barely increased. The steady-state levels of 1,2-sn-diacylglycerol in PC12-PFKL cells were found to be unaltered, suggesting that enhanced glycolysis in these cells did not influence PKC by altering the amounts of this compound. PFKL is one of several genes known to be overexpressed in Down's syndrome. Upregulation of PKC due to PFKL overexpression could result in widespread disturbances of gene expression and play a part in causing some of the many symptoms of the disease.     

Analysis of Serum Inflammatory Mediators Identifies Unique Dynamic Networks Associated with Death and Spontaneous Survival in Pediatric Acute Liver Failure

Background

Tools to predict death or spontaneous survival are necessary to inform liver transplantation (LTx) decisions in pediatric acute liver failure (PALF), but such tools are not available. Recent data suggest that immune/inflammatory dysregulation occurs in the setting of acute liver failure. We hypothesized that specific, dynamic, and measurable patterns of immune/inflammatory dysregulation will correlate with outcomes in PALF.

Methods

We assayed 26 inflammatory mediators on stored serum samples obtained from a convenience sample of 49 children in the PALF study group (PALFSG) collected within 7 days after enrollment. Outcomes were assessed within 21 days of enrollment consisting of spontaneous survivors, non-survivors, and LTx recipients. Data were subjected to statistical analysis, patient-specific Principal Component Analysis (PCA), and Dynamic Bayesian Network (DBN) inference.

Findings

Raw inflammatory mediator levels assessed over time did not distinguish among PALF outcomes. However, DBN analysis did reveal distinct interferon-gamma-related networks that distinguished spontaneous survivors from those who died. The network identified in LTx patients pre-transplant was more like that seen in spontaneous survivors than in those who died, a finding supported by PCA.

Interpretation

The application of DBN analysis of inflammatory mediators in this small patient sample appears to differentiate survivors from non-survivors in PALF. Patterns associated with LTx pre-transplant were more like those seen in spontaneous survivors than in those who died. DBN-based analyses might lead to a better prediction of outcome in PALF, and could also have more general utility in other complex diseases with an inflammatory etiology.     

Strong genetic influences on measures of behavioral‐regulation among inbred rat strains

A fundamental challenge for any complex nervous system is to regulate behavior in response to environmental challenges. Three measures of behavioral‐regulation were tested in a panel of eight inbred rat strains. These measures were: (1) sensation seeking as assessed by locomotor response to novelty and the sensory reinforcing effects of light onset, (2) attention and impulsivity, as measured by a choice reaction time task and (3) impulsivity as measured by a delay discounting task. Deficient behavioral‐regulation has been linked to a number of psychopathologies, including ADHD, Schizophrenia, Autism, drug abuse and eating disorders. Eight inbred rat strains (August Copenhagen Irish, Brown Norway, Buffalo, Fischer 344, Wistar Kyoto, Spontaneous Hypertensive Rat, Lewis, Dahl Salt Sensitive) were tested. With n = 9 for each strain, we observed robust strain differences for all tasks; heritability was estimated between 0.43 and 0.66. Performance of the eight inbred rat strains on the choice reaction time task was compared to the performance of outbred Sprague Dawley (n = 28) and Heterogeneous strain rats (n = 48). The results indicate a strong genetic influence on complex tasks related to behavioral‐regulation and indicate that some of the measures tap common genetically driven processes. Furthermore, our results establish the potential for future studies aimed at identifying specific alleles that influence variability for these traits. Identification of such alleles could contribute to our understanding of the molecular genetic basis of behavioral‐regulation, which is of fundamental importance and likely contributes to multiple psychiatric disorders .     

Determinants of Beat-to-Beat Variability of Repolarization Duration in the Canine Ventricular Myocyte: A Computational Analysis

Beat-to-beat variability of repolarization duration (BVR) is an intrinsic characteristic of cardiac function and a better marker of proarrhythmia than repolarization prolongation alone. The ionic mechanisms underlying baseline BVR in physiological conditions, its rate dependence, and the factors contributing to increased BVR in pathologies remain incompletely understood. Here, we employed computer modeling to provide novel insights into the subcellular mechanisms of BVR under physiological conditions and during simulated drug-induced repolarization prolongation, mimicking long-QT syndromes type 1, 2, and 3. We developed stochastic implementations of 13 major ionic currents and fluxes in a model of canine ventricular-myocyte electrophysiology. Combined stochastic gating of these components resulted in short- and long-term variability, consistent with experimental data from isolated canine ventricular myocytes. The model indicated that the magnitude of stochastic fluctuations is rate dependent due to the rate dependence of action-potential (AP) duration (APD). This process (the “active” component) and the intrinsic nonlinear relationship between membrane current and APD (“intrinsic component”) contribute to the rate dependence of BVR. We identified a major role in physiological BVR for stochastic gating of the persistent Na⁺ current (I_Na) and rapidly activating delayed-rectifier K⁺ current (I_Kr). Inhibition of I_Kr or augmentation of I_Na significantly increased BVR, whereas subsequent β-adrenergic receptor stimulation reduced it, similar to experimental findings in isolated myocytes. In contrast, β-adrenergic stimulation increased BVR in simulated long-QT syndrome type 1. In addition to stochastic channel gating, AP morphology, APD, and beat-to-beat variations in Ca²⁺ were found to modulate single-cell BVR. Cell-to-cell coupling decreased BVR and this was more pronounced when a model cell with increased BVR was coupled to a model cell with normal BVR. In conclusion, our results provide new insights into the ionic mechanisms underlying BVR and suggest that BVR reflects multiple potentially proarrhythmic parameters, including increased ion-channel stochasticity, prolonged APD, and abnormal Ca²⁺ handling.     

Variability of Bacterial Community Composition on Leaves Between and Within Plant Species

The phyllosphere is one of the largest habitats for terrestrial microorganisms. To gain a better insight into the factors underlying the composition of bacterial communities inhabiting leaf surfaces we performed culture-dependent and independent (Denaturing Gradient Gel Electrophoresis) analyses on the bacteria associated with the leaves of three plant species: Amygdalus communis, Citrus paradisi, and Nicotiana glauca. We found that the culturable classes Bacilli and Actinobacteria were the predominant classes on the phyllosphere of all three plant species. In contrast to this consistency on the bacterial class level, we found a significant variation on the bacterial species-level based on the culturable methods. Although some variation was detected among individual plants within one plant species, the inter-specific variability exceeded the intra-specific variability. C. paradisi leaf surface had the highest predicted total species richness (Chao 2 and ICE) and the highest species diversity (βw) among the three plant species. Our findings demonstrate that environmental conditions, mainly the plant species within a site, govern the bacterial community composition on leaf surfaces.     

Evaluation of helmet and goggle designs by modeling non-penetrating projectile impacts

Despite the progress in developing personal combat-protective gear, eye and brain injuries are still widely common and carry fatal or long-term repercussions. The complex nature of the cranial tissues suggests that simple methods (e.g. crash-dummies) for testing the effectiveness of personal protective gear against non-penetrating impacts are both expensive and ineffective, and there are ethical issues in using animal or cadavers. The present work presents a versatile testing framework for quantitatively evaluating protective performances of head and eye combat-protective gear, against non-penetrating impacts. The biomimetic finite element (FE) head model that was developed provides realistic representation of cranial structure and tissue properties. Simulated crash impact results were validated against a former cadaveric study and by using a crash-phantom developed in our lab. The model was then fitted with various helmet and goggle designs onto which a non-penetrating ballistic impact was applied. Example data show that reduction of the elastic and shear moduli by 30% and 80% respectively of the helmet outer Kevlar-29 layer, lowered intracranial pressures by 20%. Our modeling suggests that the level of stresses that develop in brain tissues, which ultimately cause the brain damage, cannot be predicted solely by the properties of the helmet/goggle materials. We further found that a reduced contact area between goggles and face is a key factor in reducing the mechanical loads transmitted to the optic nerve and eye balls following an impact. Overall, this work demonstrates the simplicity, flexibility and usefulness for development, evaluation, and testing of combat-protective equipment using computational modeling.

• Highlights

• A finite element head model was developed for testing head gear.

• Reduced helmet’s outer layer elastic and shear moduli lowered intracranial stresses.

• Gear material properties could not fully predict impact-related stress in the brain.

• Reduced goggles-face contact lowered transmitted loads to the optic nerve and eyes.

     

Detection of light images by simple tissues as visualized by photosensitized magnetic resonance imaging

In this study, we show how light can be absorbed by the body of a living rat due to an injected pigment circulating in the blood stream. This process is then physiologically translated in the tissue into a chemical signature that can be perceived as an image by magnetic resonance imaging (MRI). We previously reported that illumination of an injected photosynthetic bacteriochlorophyll-derived pigment leads to a generation of reactive oxygen species, upon oxygen consumption in the blood stream. Consequently, paramagnetic deoxyhemoglobin accumulating in the illuminated area induces changes in image contrast, detectable by a Blood Oxygen Level Dependent (BOLD)-MRI protocol, termed photosensitized (ps)MRI. Here, we show that laser beam pulses synchronously trigger BOLD-contrast transients in the tissue, allowing representation of the luminous spatiotemporal profile, as a contrast map, on the MR monitor. Regions with enhanced BOLD-contrast (7-61 fold) were deduced as illuminated, and were found to overlap with the anatomical location of the incident light. Thus, we conclude that luminous information can be captured and translated by typical oxygen exchange processes in the blood of ordinary tissues, and made visible by psMRI (Fig. 1). This process represents a new channel for communicating environmental light into the body in certain analogy to light absorption by visual pigments in the retina where image perception takes place in the central nervous system. Potential applications of this finding may include: non-invasive intra-operative light guidance and follow-up of photodynamic interventions, determination of light diffusion in opaque tissues for optical imaging and possible assistance to the blind.     

Simultaneous determination of 6 beta-blockers, 3 calcium-channel antagonists, 4 angiotensin-II antagonists and 1 antiarrhythmic drug in post-mortem whole blood by automated solid phase extraction and liquid chromatography mass spectrometry. Method development and robustness testing by experimental design

A method for the simultaneous determination of the beta-blockers atenolol, sotalol, metoprolol, bisoprolol, propranolol and carvedilol, the calcium-channel antagonists diltiazem, amlodipine and verapamil, the angiotensin-II antagonists losartan, irbesartan, valsartan and telmisartan, and the antiarrhythmic drug flecainide, in whole blood samples from forensic autopsies was developed. Sample clean-up was achieved by precipitation and solid phase extraction (SPE) with a mixed-mode column. Quantification was performed by reversed phase high performance liquid chromatography with positive electrospray ionization mass spectrometric detection (HPLC-MS). The method has been developed and robustness tested by systematically searching for satisfactory conditions using experimental designs including factorial and response surface designs. With the exception of amlodipine, the concentration limit of quantification (cLOQ) covered low therapeutic concentration levels for all the compounds. Within assay precisions and accuracies (bias) were 3.4-21% RSD and from -24 to 21% for the concentration range 1.00-5.00 microM, respectively. Between assay precisions were 4.4-28% RSD for the concentration range from 0.1 to 5 microM and recoveries varied from 9 to 103%. The method is used for determination of cardiovascular drugs in post-mortem whole blood samples from forensic autopsy cases.