A fluorescence lifetime-based assay for abelson kinase

We present a novel homogeneous in vitro assay format and apply it to the quantitative determination of the enzymatic activity of a tyrosine kinase. The assay employs a short peptidic substrate containing a single tyrosine and a single probe attached via a cysteine side chain. The structural flexibility of the peptide allows for the dynamic quenching of the probe by the nonphosphorylated tyrosine side chain. The probe responds with changes in its fluorescence lifetime depending on the phosphorylation state of the tyrosine. We use this effect to directly follow the enzymatic phosphorylation of the substrate, without having to resort to additional assay components such as an antibody against the phosphotyrosine. As an example for the application of this assay principle, we present results from the development of an assay for Abelson kinase (c-Abl) used for compound profiling. Adjustments in the peptide sequence would make this assay format suitable to a wide variety of other tyrosine kinases.     

Mendelian breeding units versus standard sampling strategies: Mitochondrial DNA variation in southwest Sardinia

We report a sampling strategy based on Mendelian Breeding Units (MBUs), representing an interbreeding group of individuals sharing a common gene pool. The identification of MBUs is crucial for case-control experimental design in association studies. The aim of this work was to evaluate the possible existence of bias in terms of genetic variability and haplogroup frequencies in the MBU sample, due to severe sample selection. In order to reach this goal, the MBU sampling strategy was compared to a standard selection of individuals according to their surname and place of birth. We analysed mitochondrial DNA variation (first hypervariable segment and coding region) in unrelated healthy subjects from two different areas of Sardinia: the area around the town of Cabras and the western Campidano area. No statistically significant differences were observed when the two sampling methods were compared, indicating that the stringent sample selection needed to establish a MBU does not alter original genetic variability and haplogroup distribution. Therefore, the MBU sampling strategy can be considered a useful tool in association studies of complex traits.     

Computing the partition function for kinetically trapped RNA secondary structures

An RNA secondary structure is locally optimal if there is no lower energy structure that can be obtained by the addition or removal of a single base pair, where energy is defined according to the widely accepted Turner nearest neighbor model. Locally optimal structures form kinetic traps, since any evolution away from a locally optimal structure must involve energetically unfavorable folding steps. Here, we present a novel, efficient algorithm to compute the partition function over all locally optimal secondary structures of a given RNA sequence. Our software, RNAlocopt runs in O(n3) time and O(n2) space. Additionally, RNAlocopt samples a user-specified number of structures from the Boltzmann subensemble of all locally optimal structures. We apply RNAlocopt to show that (1) the number of locally optimal structures is far fewer than the total number of structures--indeed, the number of locally optimal structures approximately equal to the square root of the number of all structures, (2) the structural diversity of this subensemble may be either similar to or quite different from the structural diversity of the entire Boltzmann ensemble, a situation that depends on the type of input RNA, (3) the (modified) maximum expected accuracy structure, computed by taking into account base pairing frequencies of locally optimal structures, is a more accurate prediction of the native structure than other current thermodynamics-based methods. The software RNAlocopt constitutes a technical breakthrough in our study of the folding landscape for RNA secondary structures. For the first time, locally optimal structures (kinetic traps in the Turner energy model) can be rapidly generated for long RNA sequences, previously impossible with methods that involved exhaustive enumeration. Use of locally optimal structure leads to state-of-the-art secondary structure prediction, as benchmarked against methods involving the computation of minimum free energy and of maximum expected accuracy. Web server and source code available at http://bioinformatics.bc.edu/clotelab/RNAlocopt/.     

Moral concepts set decision strategies to abstract values

Persons have different value preferences. Neuroimaging studies where value-based decisions in actual conflict situations were investigated suggest an important role of prefrontal and cingulate brain regions. General preferences, however, reflect a superordinate moral concept independent of actual situations as proposed in psychological and socioeconomic research. Here, the specific brain response would be influenced by abstract value systems and moral concepts. The neurobiological mechanisms underlying such responses are largely unknown. Using functional magnetic resonance imaging (fMRI) with a forced-choice paradigm on word pairs representing abstract values, we show that the brain handles such decisions depending on the person's superordinate moral concept. Persons with a predominant collectivistic (altruistic) value system applied a "balancing and weighing" strategy, recruiting brain regions of rostral inferior and intraparietal, and midcingulate and frontal cortex. Conversely, subjects with mainly individualistic (egocentric) value preferences applied a "fight-and-flight" strategy by recruiting the left amygdala. Finally, if subjects experience a value conflict when rejecting an alternative congruent to their own predominant value preference, comparable brain regions are activated as found in actual moral dilemma situations, i.e., midcingulate and dorsolateral prefrontal cortex. Our results demonstrate that superordinate moral concepts influence the strategy and the neural mechanisms in decision processes, independent of actual situations, showing that decisions are based on general neural principles. These findings provide a novel perspective to future sociological and economic research as well as to the analysis of social relations by focusing on abstract value systems as triggers of specific brain responses.     

Autophagy and ATP-induced anti-apoptosis in antigen presenting cells (APC) follows the cytokine storm in patients after major trauma

Severe trauma and the systemic inflammatory response syndrome (SIRS) occur as a result of a cytokine storm which is in part due to ATP released from damaged tissue. This pathology also leads to increased numbers of immature antigen presenting cells (APC) sharing properties of dendritic cells (DC) or macrophages (MΦ). The occurrence of immature APC appears to coincide with the reactivation of herpes virus infections such as Epstein Barr virus (EBV). The aim of this study was the comparative analysis of the ultrastructural and functional characteristics of such immature APC. In addition, we investigated EBV infection/ reactivation and whether immature APC might be targets for natural killers (NK). Significant macroautophagy, mitochondrial degradation and multivesicular body formation together with the identification of herpes virus particles were morphological findings associated with immature APC. Exogenous stressors such as ATP further increased morphological signs of autophagy, including LC3 expression. Functional tests using fluorescent bacteria proved impaired phagolysosome fusion. However, immature APC were susceptible to NK-92-mediated cytolysis. We found evidence for EBV latency state II infection by detecting EBV-specific LMP1 and EBNA2 in immature APC and in whole blood of these patients. In summary, trauma-induced cytokine storms may induce maturation arrest of APC, promote ATP-induced autophagy, support EBV persistence and impair the degradation of phagocytozed bacteria through inefficient phagolysosome fusion. The susceptibility to NK-mediated cytolysis supports the hypothesis that NK function is likely to contribute to immune reconstitution after major trauma by regulating immature APC, and ATP-induced autophagy and survival.     

Blood pressure maintenance in NHE3-deficient mice with transgenic expression of NHE3 in small intestine

NHE3 Na(+)/H(+) exchanger knockout (Nhe3(-/-)) mice have severe absorptive deficits in the kidney proximal tubule and intestinal tract. The resulting hypovolemia has confounded efforts to carefully evaluate the specific effects of NHE3 deficiency on kidney function. Development of mice with transgenic expression of NHE3 in the small intestine (tgNhe3(-/-)) has allowed us to analyze the role of renal NHE3 in overall maintenance of blood pressure, pressure natriuresis, and autoregulation of both glomerular filtration rate (GFR) and renal blood flow (RBF). Ambulatory blood pressure, measured by telemetry, was lower in tgNhe3(-/-) mice than in wild-type controls (tgNhe3(+/+)) when the mice were maintained on a normal NaCl diet but was normalized when they were provided with a high NaCl intake. Furthermore, administration of the AT1-receptor blocker losartan showed that circulating ANG II plays a major role in maintaining blood pressure in tgNhe3(-/-) mice fed normal NaCl but not in those receiving high NaCl. Clearance studies revealed a blunted pressure-natriuresis response in tgNhe3(-/-) mice at lower blood pressures but a robust response at higher blood pressures. Autoregulation of GFR and RBF was normal in tgNhe3(-/-) mice. These results show that dietary NaCl loading normalizes blood pressure in awake tgNhe3(-/-) mice and that alterations in NHE3 activity are not essential for normal autoregulation of GFR and RBF. Furthermore, the data strongly support the hypothesis that NHE3 plays an important role in the diuretic and natriuretic responses to increases in blood pressure but also show that mechanisms not involving NHE3 mediate pressure natriuresis in the higher range of blood pressures studied.