Etoposide is a topoisomerase II poison that is utilized to treat a broad spectrum of human cancers. Despite its wide clinical use, 2-3% of patients treated with etoposide eventually develop treatment-related acute myeloid leukemias (t-AMLs) characterized by rearrangements of the MLL gene. The molecular basis underlying the development of these t-AMLs is not well understood; however, previous studies have implicated etoposide metabolites (i.e., etoposide quinone) and topoisomerase IIβ in the leukemogenic process. Although interactions between etoposide quinone and topoisomerase IIα have been characterized, the effects of the drug metabolite on the activity of human topoisomerase IIβ have not been reported. Thus, we examined the ability of etoposide quinone to poison human topoisomerase IIβ. The quinone induced ~4 times more enzyme-mediated DNA cleavage than did the parent drug. Furthermore, the potency of etoposide quinone was ~2 times greater against topoisomerase IIβ than it was against topoisomerase IIα, and the drug reacted ~2-4 times faster with the β isoform. Etoposide quinone induced a higher ratio of double- to single-stranded breaks than etoposide, and its activity was less dependent on ATP. Whereas etoposide acts as an interfacial topoisomerase II poison, etoposide quinone displayed all of the hallmarks of a covalent poison: the activity of the metabolite was abolished by reducing agents, and the compound inactivated topoisomerase IIβ when it was incubated with the enzyme prior to the addition of DNA. These results are consistent with the hypothesis that etoposide quinone contributes to etoposide-related leukemogenesis through an interaction with topoisomerase IIβ.
Abstract During apoptosis, Bak and Bax are activated by BH3-only proteins binding to the α2–α5 hydrophobic groove; Bax is also activated via a rear pocket. Here we report that antibodies can directly activate Bak and mitochondrial Bax by binding to the α1–α2 loop. A monoclonal antibody (clone 7D10) binds close to α1 in non-activated Bak to induce conformational change, oligomerization, and cytochrome c release. Anti-FLAG antibodies also activate Bak containing a FLAG epitope close to α1. An antibody (clone 3C10) to the Bax α1–α2 loop activates mitochondrial Bax, but blocks translocation of cytosolic Bax. Tethers within Bak show that 7D10 binding directly extricates α1; a structural model of the 7D10 Fab bound to Bak reveals the formation of a cavity under α1. Our identification of the α1–α2 loop as an activation site in Bak paves the way to develop intrabodies or small molecules that directly and selectively regulate these proteins.
Abstract Polyhedral oligomeric silsesquioxane (POSS) nanostructured chemicals, when incorporated at low levels in thermoplastics, provide processability enhancement and viscosity reduction without compromising other bulk physical properties. POSS has been relatively unexplored in high performance polymers, and there is incomplete understanding of the mechanisms by which POSS produces flow improvements. In this study, polyethersulfone (PES) was melt‐blended with trisilanolphenyl (TSP)‐POSS and dodecaphenyl (DP)‐POSS; and rheological, dielectric spectroscopy, and scanning electron microscopy evaluations were conducted to identify structure/property/processing relationships. TSP‐POSS yielded greater processability improvements and viscosity reductions than DP‐POSS, suppressed low temperature relaxations to a larger extent, and displayed a greater degree of nanoscale dispersion in the polymer matrix. The findings are evaluated in terms of competing theories of POSS viscosity reduction.
FLAME TEMPERATURE IMAGING OF A LOW NOx BURNER VIA LASER RAYLEIGH SCATTERING Nicholas A. Smith, B.S.M.E. Marquette University, 2013 Federal and global legislation are requiring increasingly stringent emission regulation on household appliances and in particular water heater burners. Emissions like (NO and ) are a growing concern due to their adverse health effects and contribution to tropospheric ozone, acid rain, and smog formation. As is more closely controlled, appliance manufacturers are developing low emission burners for use in water heaters. Flame temperature is strongly correlated to production. Hence, characterizing flame temperatures in new burners is a key part of improving upon burners used today and the development of future burners. Temperature measurements applied to a new, radiant, ultralowburner are thus the focus of this research. Laser Rayleigh scattering allows us to make near-instantaneous, 2-D measurements using an unobtrusive technique. The application of this technique resulted in flame temperature images in three locations, above and across the burner surface ranging from 800-1600 K in general with an uncertainty of 9.6%. The fluctuation of the flame temperature was also found ranging from 200-800 K, indicating the presence of large scale hot and cold gas mixing. Other temperature measuring techniques were applied to the burner as well. A type-K thermocouple 5 cm above the center of the burner measured a point gas temperature of 1508 K after an estimated radiative correction was applied. This measurement was within 5.3% of the laser Rayleigh scattering measurement of 1428 K at the same location. An IR camera did not provide quantitative temperature measurements, but the videos indicated similar flame structure and mixing behavior when compared to a series of single-shot laser Rayleigh scattering images. It was concluded that the large amount of excess air (equivalence ratio of 0.725) was primarily responsible for reducing the flame temperature by 436 K in comparison with the adiabatic flame temperature under stoichiometric conditions. The radiative emission by the burner was estimated from the thermocouple and laser Rayleigh scattering measurements to decrease the temperature further by an average of 420 K relative to the stoichiometric adiabatic flame temperature.
Apoptosis, the intrinsic programmed cell death process, is mediated by the Bcl-2 family members Bak and Bax. Activation via formation of symmetric core dimers and oligomerization on the mitochondrial outer membrane (MOM) leads to permeabilization and cell death. Although this process is linked to the MOM, the role of the membrane in facilitating such pores is poorly understood. We recently described Bak core domain dimers, revealing lipid binding sites and an initial role of lipids in oligomerization. Here we describe simulations that identified localized clustering and interaction of triacylglycerides (TAGs) with a minimized Bak dimer construct. Coalescence of TAGs occurred beneath this Bak dimer, mitigating dimer-induced local membrane thinning and curvature in representative coarse-grain MOM and model membrane systems. Furthermore, the effects observed as a result of coarse-grain TAG cluster formation was concentration dependent, scaling from low physiological MOM concentrations to those found in other organelles. We find that increasing the TAG concentration in liposomes mimicking the MOM decreased the ability of activated Bak to permeabilize these liposomes. These results suggest that the presence of TAGs within a Bak-lipid membrane preserves membrane integrity and is associated with reduced membrane stress, suggesting a possible role of TAGs in Bak-mediated apoptosis.
Durchaus vergleichbar: Eine Graphen-Monoschicht dient als Substrat für den Aufbau von zweidimensionalen wasserstoffverbrückten supramolekularen Strukturen (siehe STM-Bild). Der Bildung der ausgedehnten Strukturen liegt ein kommensurables Verhalten zwischen den Abmessungen der Superstrukturen und dem vom Graphen gebildeten Moiré-Muster zugrunde.
Abstract BAK and BAX execute intrinsic apoptosis by permeabilising the mitochondrial outer membrane. Their activity is regulated through interactions with pro-survival BCL-2 family proteins and with non-BCL-2 proteins including the mitochondrial porin VDAC2. VDAC2 is important for bringing both BAK and BAX to mitochondria where they execute their apoptotic function. Despite this important function in apoptosis, whilst interactions with pro-survival family members are well characterised and have culminated in the development of drugs that target these interfaces to induce cancer cell apoptosis, the interaction between BAK and VDAC2 remains largely undefined. Deep scanning mutagenesis coupled with cysteine linkage identified key residues in the interaction between BAK and VDAC2. Obstructive labelling of specific residues in the BH3 domain or hydrophobic groove of BAK disrupted this interaction. Conversely, mutating specific residues in a cytosol-exposed region of VDAC2 stabilised the interaction with BAK, and inhibited BAK apoptotic activity. Thus, this VDAC2–BAK interaction site can potentially be targeted to either inhibit BAK-mediated apoptosis in scenarios where excessive apoptosis contributes to disease, or to promote BAK-mediated apoptosis for cancer therapy.
Abstract Background Human cytomegalovirus (HCMV) resides latently in cells of the myeloid compartment, including CD34 + hematopoietic progenitor cells and circulating monocytes. Healthy hosts maintain the virus latently, and this infection is, for the most part, asymptomatic. However, given the proper external cues, HCMV reactivates from latency, at which point the virus disseminates, causing disease. The viral and cellular factors dictating the balance between these phases of infection are incompletely understood, though a large body of literature support a role for viral-mediated manipulation of host cell signaling. Main body To establish and maintain latency, HCMV has evolved various means by which it usurps host cell factors to alter the cellular environment to its own advantage, including altering host cell signaling cascades. As early as virus entry into myeloid cells, HCMV usurps cellular signaling to change the cellular milieu, and this regulation includes upregulation, as well as downregulation, of different signaling cascades. Indeed, given proper reactivation cues, this signaling is again altered to allow for transactivation of viral lytic genes. Conclusions HCMV modulation of host cell signaling is not binary, and many of the cellular pathways altered are finely regulated, wherein the slightest modification imparts profound changes to the cellular milieu. It is also evident that viral-mediated cell signaling differs not only between these phases of infection, but also is myeloid cell type specific. Nonetheless, understanding the exact pathways and the means by which HCMV mediates them will undoubtedly provide novel targets for therapeutic intervention.
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