Interplay between Kinase Domain Autophosphorylation and F-Actin Binding Domain in Regulating Imatinib Sensitivity and Nuclear Import of BCR-ABL

BACKGROUND: The constitutively activated BCR-ABL tyrosine kinase of chronic myeloid leukemia (CML) is localized exclusively to the cytoplasm despite the three nuclear localization signals (NLS) in the ABL portion of this fusion protein. The NLS function of BCR-ABL is re-activated by a kinase inhibitor, imatinib, and in a kinase-defective BCR-ABL mutant. The mechanism of this kinase-dependent inhibition of the NLS function is not understood. METHODOLOGY/PRINCIPAL FINDINGS: By examining the subcellular localization of mutant BCR-ABL proteins under conditions of imatinib and/or leptomycin B treatment to inhibit nuclear export, we have found that mutations of three specific tyrosines (Y232, Y253, Y257, according to ABL-1a numbering) in the kinase domain can inhibit the NLS function of kinase-proficient and kinase-defective BCR-ABL. Interestingly, binding of imatinib to the kinase-defective tyrosine-mutant restored the NLS function, suggesting that the kinase domain conformation induced by imatinib-binding is critical to the re-activation of the NLS function. The C-terminal region of ABL contains an F-actin binding domain (FABD). We examined the subcellular localization of several FABD-mutants and found that this domain is also required for the activated kinase to inhibit the NLS function; however, the binding to F-actin per se is not important. Furthermore, we found that some of the C-terminal deletions reduced the kinase sensitivity to imatinib. CONCLUSIONS/SIGNIFICANCE: Results from this study suggest that an autophosphorylation-dependent kinase conformation together with the C-terminal region including the FABD imposes a blockade of the BCR-ABL NLS function. Conversely, conformation of the C-terminal region including the FABD can influence the binding affinity of imatinib for the kinase domain. Elucidating the structural interactions among the kinase domain, the NLS region and the FABD may therefore provide insights on the design of next generation BCR-ABL inhibitors for the treatment of CML

Power efficiency of outer hair cell somatic electromotility

© 2009 Rabbitt et al. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS Computational Biology 5 (2009): e1000444, doi:10.1371/journal.pcbi.1000444.Cochlear outer hair cells (OHCs) are fast biological motors that serve to enhance the vibration of the organ of Corti and increase the sensitivity of the inner ear to sound. Exactly how OHCs produce useful mechanical power at auditory frequencies, given their intrinsic biophysical properties, has been a subject of considerable debate. To address this we formulated a mathematical model of the OHC based on first principles and analyzed the power conversion efficiency in the frequency domain. The model includes a mixture-composite constitutive model of the active lateral wall and spatially distributed electro-mechanical fields. The analysis predicts that: 1) the peak power efficiency is likely to be tuned to a specific frequency, dependent upon OHC length, and this tuning may contribute to the place principle and frequency selectivity in the cochlea; 2) the OHC power output can be detuned and attenuated by increasing the basal conductance of the cell, a parameter likely controlled by the brain via the efferent system; and 3) power output efficiency is limited by mechanical properties of the load, thus suggesting that impedance of the organ of Corti may be matched regionally to the OHC. The high power efficiency, tuning, and efferent control of outer hair cells are the direct result of biophysical properties of the cells, thus providing the physical basis for the remarkable sensitivity and selectivity of hearing.This work was supported by NIDCD R01 DC04928 (Rabbitt), NIDCD R01 DC00384 (Brownell) and NASA Ames GSRA56000135 (Breneman)

an experiment with ten duopoly games and beat-the-average behavior

abstract (concluding remarks): compared with a matrix game, this business game is quite rich, complicated and has a complex environment. by the device of briefing the players to try to maximize the difference in their scores, the game is turned intoa two-person zero-sum game with a saddle point. the strategy space is multidimensional. an extremely strong prisoner's dilemma game is built in on advertising and the evidence obtained was that this was reflected in the actual play. in previous experiments where the players were trying to maximize individual payoffs and the game was truly non-constant sum this effect was also observed. the economic solution predictions were quite good in predicting the range to which price and advertising proceeded. profits were lower than the steady state prediction. this could be explained by the dydnamic aspects of the game which were manifested in the learning and control problems of the players. inventories were in general too high. it took time to work out the implications of advertising. the value of production scheduling and its relationship to management of prices was probably not appreciated by many of the players.

Interplay between kinase domain autophosphorylation and F-actin binding domain in regulating imatinib sensitivity and nuclear import of BCR-ABL.

BackgroundThe constitutively activated BCR-ABL tyrosine kinase of chronic myeloid leukemia (CML) is localized exclusively to the cytoplasm despite the three nuclear localization signals (NLS) in the ABL portion of this fusion protein. The NLS function of BCR-ABL is re-activated by a kinase inhibitor, imatinib, and in a kinase-defective BCR-ABL mutant. The mechanism of this kinase-dependent inhibition of the NLS function is not understood.Methodology/principal findingsBy examining the subcellular localization of mutant BCR-ABL proteins under conditions of imatinib and/or leptomycin B treatment to inhibit nuclear export, we have found that mutations of three specific tyrosines (Y232, Y253, Y257, according to ABL-1a numbering) in the kinase domain can inhibit the NLS function of kinase-proficient and kinase-defective BCR-ABL. Interestingly, binding of imatinib to the kinase-defective tyrosine-mutant restored the NLS function, suggesting that the kinase domain conformation induced by imatinib-binding is critical to the re-activation of the NLS function. The C-terminal region of ABL contains an F-actin binding domain (FABD). We examined the subcellular localization of several FABD-mutants and found that this domain is also required for the activated kinase to inhibit the NLS function; however, the binding to F-actin per se is not important. Furthermore, we found that some of the C-terminal deletions reduced the kinase sensitivity to imatinib.Conclusions/significanceResults from this study suggest that an autophosphorylation-dependent kinase conformation together with the C-terminal region including the FABD imposes a blockade of the BCR-ABL NLS function. Conversely, conformation of the C-terminal region including the FABD can influence the binding affinity of imatinib for the kinase domain. Elucidating the structural interactions among the kinase domain, the NLS region and the FABD may therefore provide insights on the design of next generation BCR-ABL inhibitors for the treatment of CML

Combined antigen-specific interferon-γ and interleukin-2 release assay (FluoroSpot) for the diagnosis of Mycobacterium tuberculosis infection.

To evaluate interleukin (IL)-2 and interferon (IFN)-γ secreting T-cells in parallel for the differentiation of latent infection with Mycobacterium tuberculosis infection (LTBI) from active tuberculosis.Following ex-vivo stimulation of peripheral blood mononuclear cells (PBMC) with M. tuberculosis-specific antigens early secretory antigenic target (ESAT)-6 and culture filtrate protein (CFP)-10, immune responses were assessed by enzyme-linked immunospot IFN-γ release assay (EliSpot-IGRA) and a novel dual cytokine detecting fluorescence-linked immunospot (FluoroSpot) in 18 patients with pulmonary tuberculosis, 10 persons with previously cured tuberculosis, 25 individuals with LTBI and 16 healthy controls.Correlation of IFN-γ+ spot-forming cells in EliSpot-IGRA and FluoroSpot were R2 = 0.67 for ESAT-6 and R2 = 0.73 for CFP-10. The number of IL-2- IFN-γ+ producing cells was higher in patients with tuberculosis compared with past tuberculosis (CFP-10-induced p = 0.0068) or individuals with LTBI (ESAT-6-induced p = 0.0136). A cutoff value of >16 CFP-10-induced IFN-γ+ secreting cells/200.000 PBMC in the EliSpot-IGRA discriminated with highest sensitivity and specificity (89% and 76%, respectively). However, overlap in cytokine responses precludes distinction between the cohorts on an individual basis.Combined analysis of IFN-γ and IL-2 secretion by antigen specific T-cells does not allow a reliable differentiation between different states of M. tuberculosis infection in clinical practice

Demographic characteristics of study subjects by groups.

<p>TB = tuberculosis.</p><p>LTBI = latent infection with <i>M</i>. <i>tuberculosis</i>.</p><p>m = male.</p><p>f = female.</p><p>n = number of cases.</p><p>Demographic characteristics of study subjects by groups.</p

ESAT-6 and CFP-10- induced cytokine response in FluoroSpot.

<p>ESAT-6 induced cytokine immune response in 200.000 PBMCs/well in participants with active tuberculosis (TB, circle, n = 18), past tuberculosis (past TB, inverted triangle, n = 10), latent infection with <i>M</i>. <i>tuberculosis</i> (LTBI, square, ESAT-6-induced n = 24, CFP-10-induced n = 22), EliSpot-negative individuals (control, triangle, ESAT-6-induced n = 17, CFP-10-induced n = 19) was analysed. Groups had been defined according to the combination of their ESAT-6 and CFP-10 induced IFN-γ EliSpot-IGRA test result and clinical data. The number of IL-2⁺ (A), INF-γ⁺ (B), IL-2⁺ INF-γ^- (C), IL-2^- INF-γ⁺ (D) and IL-2⁺ INF-γ⁺ (E) spot-forming cells (SFC) were enumerated by FluoroSpot. (F) Mean proportion of ESAT-6 (top row) and CFP-10 (bottom row) -specific cytokine secreting cells for individuals with tuberculosis, past tuberculosis and LTBI are depicted as pie charts (light grey = IL-2⁺ INF-γ^-, black = IL-2^- INF-γ⁺ and dark grey = IL-2⁺ INF-γ⁺ secreting cells). Mann-Whitney U-test for non-parametric data was used for comparative analysis. A p-value of <0.05 was considered significant.</p

Flow chart of patients included in this study.

<p>TB = tuberculosis; LTBI = latent infection with <i>M</i>. <i>tuberculosis;</i> control = healthy individual with negative EliSpot-IGRA result; E = ESAT-6; C = CFP-10; dark grey = positive test result in the EliSpot-IGRA; light grey = negative test result in the EliSpot-IGRA.</p