The conserved arginine in rho-GTPase-activating protein is essential for efficient catalysis but not for complex formation with Rho.GDP and aluminum fluoride

The Rho family of small GTP-binding proteins are downregulated by an intrinsic GTPase, which is enhanced by GTPase-activating proteins (GAPs). RhoGAPs contain a single conserved arginine residue that has been proposed to be involved in catalysis. Here, the role of this arginine has been elucidated by mutagenesis followed by determination of catalytic and equilibrium binding constants using single-turnover kinetics, isothermal titration calorimetry, and scintillation proximity assays. The turnover numbers for wild-type, R282A, and R282K RhoGAPs were 5.4, 0.023, and 0.010 s-1, respectively. Thus, the function of this arginine could not be replaced by lysine or alanine. Nevertheless, the R282A mutation had a minimal effect on the binding affinity of RhoGAP for either Rho. GTP or Rho.GMPPNP, which confirms the importance of the arginine residue for catalysis as opposed to formation of the protein-protein complex. The R282A mutant RhoGAP still increased the hydrolysis rate of Rho.GTP by 160-fold, whereas the wild-type enzyme increased it by 38000-fold. We conclude that this arginine contributes half of the total reduction of activation energy of catalysis. In the presence of aluminum fluoride, the R282A mutant RhoGAP binds almost as well as the wild type to Rho.GDP, demonstrating that the conserved arginine is not required for this interaction. The affinity of wild-type RhoGAP for the triphosphate form of Rho is similar to that for Rho.GDP with aluminum fluoride. These last two observations show that this complex is not associated with the free energy changes expected for the transition state, although the Rho.GDP.AlF4-.RhoGAP complex might well be a close structural approximation.     

Liquid Chromatography–High-Resolution Mass Spectrometry for Quantitative Analysis of Gangliosides

Gangliosides are a large family of glycosphingolipids that are abundant in the brain, and have been shown to affect neuronal plasticity during development, adulthood, and aging. We developed a fast, efficient, and sensitive liquid chromatography–mass spectrometry method to quantify eight different classes of gangliosides (GM₁, GM₂, GM₃, GD₃, GD_1a, GD_1b, GT_1b, GQ_1b) in the brains of 2-day-old and 80-day-old Wistar rats. The gangliosides were extracted from rat brain using a modified Svennerholm and Fredman method. After ganglioside class separation using a hydrophilic high performance liquid chromatography (HPLC) column, the resolving power of the LTQ-Orbitrap™ mass spectrometer was used to extract and sum the major species of each ganglioside class, generating fully resolved extracted ion current peaks for both standards and samples. The flexibility and the specificity of this method are such that it can be applied to the analysis of other ganglioside species/classes not discussed in this paper, provided appropriate standards are available. The method had good repeatability (coefficient of variation 4.8–12.3%) and mean recoveries in the range 92–107%.     

A longitudinal study of the relationships between haemostatic, lipid, and oestradiol changes during normal human pregnancy

BACKGROUND: Chronic leukemia is a disease characterized by the malignant proliferation of immunologically incompetent lymphocytes. The knowledge of open heart surgery in patients with this disorder is limited. METHODS: Twelve patients with chronic lymphocytic leukemia underwent open heart surgery (nine coronary artery bypass grafting (CABG), two aortic valve replacement (AVR), one CABG and AVR) from September 1991 to September 1996. There were nine males and three females with a mean age of 68 years (41-81 years). Staging was assigned according to the Rai Classification. There were seven Stage 0, two Stage I, zero Stage II, one Stage III and two Stage IV patients. Cardiopulmonary bypass (CPB) was performed using standard techniques of cannulation, moderate hypothermia and antegrade/retrograde cardioplegia. RESULTS: Hospital mortality occurred in two (17%) patients. Both patients died of sepsis. Hospital morbidity occurred in seven (58%) patients. The most common complications were infections. Five patients were found to have other malignancies (basal cell, laryngeal, prostate, bladder and breast cancers). Transfusion of blood products was required in eight (67%) patients. The average length of stay was 15 days (7-50 days). Follow-up was complete. Late mortality occurred in four patients at a mean of 7 months (1-18 months). All deaths were non-cardiac related (ruptured AAA, kidney failure, respiratory failure and sepsis). Six patients remain alive at a mean of 25 months (1-48 months). CONCLUSION: Hospital mortality and morbidity in patients with chronic lymphocytic leukemia undergoing open heart surgery are high. Infection is the leading cause of hospital death, as well as the most common complication. The majority of patients receive blood products during the course of their hospitalization. Late mortality is high and non-cardiac related. Based on these findings, a re-definition of the aims, goals and expectations of open heart surgery in patients with chronic leukemia is necessary. Suggestions in management are presented.     

Arsenite methylation by methylvitamin B12 and glutathione does not require an enzyme

Although inorganic arsenic is methylated enzymatically by arsenic methyltransferases, which have been found in many mammalian livers, the detection of such enzymes has not been successful in surgically removed human livers. Results of the present experiments demonstrated that methylvitamin B12 (methylcobalamin, CH3B12) in the presence of thiols and inorganic arsenite can produce, in vitro, substantial amounts of monomethylarsonic acid (MMA) and small amounts of dimethylarsinic acid (DMA) in the absence of enzymes. Furthermore, this nonenzymatic methylation of inorganic arsenite by CH3B12 was increased substantially by the presence of dimercaptopropanesulfonate (DMPS) and/or sodium selenite. The actions of DMPS and selenite together were additive. The methylation by CH3B12 was neither inhibited nor stimulated by human liver cytosol. Since the amount of MMA produced by the in vitro system described in this study was not small, these results emphasize the need for a properly designed nutritional study in humans exposed to inorganic arsenic as to the relationship between vitamin B12, selenium, and the metabolism of carcinogenic inorganic arsenic.     

Spatio-temporal modelling of climate-sensitive disease risk: Towards an early warning system for dengue in Brazil

This paper considers the potential for using seasonal climate forecasts in developing an early warning system for dengue fever epidemics in Brazil. In the first instance, a generalised linear model (GLM) is used to select climate and other covariates which are both readily available and prove significant in prediction of confirmed monthly dengue cases based on data collected across the whole of Brazil for the period January 2001 to December 2008 at the microregion level (typically consisting of one large city and several smaller municipalities). The covariates explored include temperature and precipitation data on a 2.5^°×2.5^° longitude-latitude grid with time lags relevant to dengue transmission, an El Niño Southern Oscillation index and other relevant socio-economic and environmental variables. A negative binomial model formulation is adopted in this model selection to allow for extra-Poisson variation (overdispersion) in the observed dengue counts caused by unknown/unobserved confounding factors and possible correlations in these effects in both time and space. Subsequently, the selected global model is refined in the context of the South East region of Brazil, where dengue predominates, by reverting to a Poisson framework and explicitly modelling the overdispersion through a combination of unstructured and spatio-temporal structured random effects. The resulting spatio-temporal hierarchical model (or GLMM—generalised linear mixed model) is implemented via a Bayesian framework using Markov Chain Monte Carlo (MCMC). Dengue predictions are found to be enhanced both spatially and temporally when using the GLMM and the Bayesian framework allows posterior predictive distributions for dengue cases to be derived, which can be useful for developing a dengue alert system. Using this model, we conclude that seasonal climate forecasts could have potential value in helping to predict dengue incidence months in advance of an epidemic in South East Brazil.     

REVLD: A coarse-grained model for polymers

A new model for polymers is presented. REVLD (Rigid, Excluded Volume, Langevin Dynamics) is similar to the coarse-grained, bead spring model for linear chains except that the inter-bead distance is rigidly constrained instead of using an inter-bead potential to encapsulate the connectivity. Static and dynamic results support that REVLD accurately reproduces the single-chain behavior of real polymers known from experiment, theory, and published data from existing models. Additionally, a time step can be used that is at least comparable to simulations using a FENE potential without introducing any computational overhead for accessing longer time scale modes. REVLD, and more simply the idea of using constraints in Cartesian coordinates for large simulations, was made computationally viable through the recent development of the algorithm MILC SHAKE. We expect it to improve established techniques and aid in the development of new models of import to large scale simulations that were not practicable before.     

Pharmacological inhibition of protein kinases in intact cells: antagonism of beta adrenergic receptor ligand binding by H-89 reveals limitations of usefulness

The use of pharmacological inhibitors of protein kinases represents a potentially powerful tool in dissecting the regulatory features of intracellular signaling pathways. However, although the in vitro potency, selectivity, and efficacy of numerous kinase inhibitors have been characterized, little is known regarding the usefulness of these compounds as inhibitors in intact cells. In attempting to characterize the role of protein kinase A (PKA) in regulating the beta-2 adrenergic receptor (AR) in human airway cells, we observed a seemingly profound capacity of the isoquinoline H-89, a potent and widely used PKA inhibitor, to attenuate agonist-mediated desensitization of the beta-2 AR. Although additional experiments identified H-89 as an effective inhibitor of intracellular PKA, extended analysis of the compound determined the principal effect of H-89 was via its action as a beta-2 AR antagonist. Pretreatment with or the acute addition of H-89 significantly attenuated isoproterenol-stimulated cAMP accumulation. In cells pretreated with H-89 and then washed extensively, the subsequent dose-dependent response to isoproterenol suggested beta-2 AR antagonism by retained H-89. Competition binding of [125I]iodopindolol established Ki values of approximately 180 nM and 350 nM for H-89 antagonism of beta-2 AR and beta-1 AR, respectively. Additional receptor binding studies suggest selectivity of H-89 for the beta-2 AR and beta-1 AR, although a weak antagonism (Ki values of approximately 10 microM or greater) of other G protein-coupled receptors was observed. Results from additional pharmacological and biochemical analyses of various protein kinase inhibitors further established the need for careful characterization of pharmacological inhibitors when used in intact cell models.     

Television images and adolescent girls' body image disturbance

The impact of media images on adolescents' body image and thin ideal endorsement has been consistently asserted in the body image literature, yet has remained inadequately tested. I used social comparison theory and critical viewing with a sample of 214 high school girls to predict adolescents' body-image disturbance and thin ideal endorsement. Media variables accounted fr 15% of the variance for drive for thinness, 17% for body dissatisfaction, 16% for bulimic behaviors, and 33% for thin ideal endorsement. Results suggest that body image processing is the boy to understanding how television images affect adolescent girls' body-image attitudes and behaviors.