Apaf-1 and caspase-9 accelerate apoptosis, but do not determine whether factor-deprived or drug-treated cells die

Apoptosis after growth factor withdrawal or drug treatment is associated with mitochondrial cytochrome c release and activation of Apaf-1 and caspase-9. To determine whether loss of Apaf-1, caspase-2, and caspase-9 prevented death of factor-starved cells, allowing them to proliferate when growth factor was returned, we generated IL-3–dependent myeloid lines from gene-deleted mice. Long after growth factor removal, cells lacking Apaf-1, caspase-9 or both caspase-9 and caspase-2 appeared healthy, retained intact plasma membranes, and did not expose phosphatidylserine. However, release of cytochrome c still occurred, and they failed to form clones when IL-3 was restored. Cells lacking caspase-2 alone had no survival advantage. Therefore, Apaf-1, caspase-2, and caspase-9 are not required for programmed cell death of factor-dependent cells, but merely affect its rate. In contrast, transfection with Bcl-2 provided long-term, clonogenic protection, and could act independently of the apoptosome. Unlike expression of Bcl-2, loss of Apaf-1, caspase-2, or caspase-9 would therefore be unlikely to enhance the survival of cancer cells

Choriocapillaris and Choroidal Microvasculature Imaging with Ultrahigh Speed OCT Angiography

We demonstrate in vivo choriocapillaris and choroidal microvasculature imaging in normal human subjects using optical coherence tomography (OCT). An ultrahigh speed swept source OCT prototype at 1060 nm wavelengths with a 400 kHz A-scan rate is developed for three-dimensional ultrahigh speed imaging of the posterior eye. OCT angiography is used to image three-dimensional vascular structure without the need for exogenous fluorophores by detecting erythrocyte motion contrast between OCT intensity cross-sectional images acquired rapidly and repeatedly from the same location on the retina. En face OCT angiograms of the choriocapillaris and choroidal vasculature are visualized by acquiring cross-sectional OCT angiograms volumetrically via raster scanning and segmenting the three-dimensional angiographic data at multiple depths below the retinal pigment epithelium (RPE). Fine microvasculature of the choriocapillaris, as well as tightly packed networks of feeding arterioles and draining venules, can be visualized at different en face depths. Panoramic ultra-wide field stitched OCT angiograms of the choriocapillaris spanning ~32 mm on the retina show distinct vascular structures at different fundus locations. Isolated smaller fields at the central fovea and ~6 mm nasal to the fovea at the depths of the choriocapillaris and Sattler's layer show vasculature structures consistent with established architectural morphology from histological and electron micrograph corrosion casting studies. Choriocapillaris imaging was performed in eight healthy volunteers with OCT angiograms successfully acquired from all subjects. These results demonstrate the feasibility of ultrahigh speed OCT for in vivo dye-free choriocapillaris and choroidal vasculature imaging, in addition to conventional structural imaging.National Institutes of Health (U.S.) (NIH R01-EY011289-27)National Institutes of Health (U.S.) (NIH R01-EY013178-12)National Institutes of Health (U.S.) (NIH R44-EY022864-01)National Institutes of Health (U.S.) (NIH R01-CA075289-16)United States. Air Force Office of Scientific Research (AFOSR FA9550-10-1-0551)United States. Air Force Office of Scientific Research (AFOSR FA9550-12-1-0499

A Genome-Wide Association Study of Diabetic Kidney Disease in Subjects With Type 2 Diabetes

dentification of sequence variants robustly associated with predisposition to diabetic kidney disease (DKD) has the potential to provide insights into the pathophysiological mechanisms responsible. We conducted a genome-wide association study (GWAS) of DKD in type 2 diabetes (T2D) using eight complementary dichotomous and quantitative DKD phenotypes: the principal dichotomous analysis involved 5,717 T2D subjects, 3,345 with DKD. Promising association signals were evaluated in up to 26,827 subjects with T2D (12,710 with DKD). A combined T1D+T2D GWAS was performed using complementary data available for subjects with T1D, which, with replication samples, involved up to 40,340 subjects with diabetes (18,582 with DKD). Analysis of specific DKD phenotypes identified a novel signal near GABRR1 (rs9942471, P = 4.5 x 10(-8)) associated with microalbuminuria in European T2D case subjects. However, no replication of this signal was observed in Asian subjects with T2D or in the equivalent T1D analysis. There was only limited support, in this substantially enlarged analysis, for association at previously reported DKD signals, except for those at UMOD and PRKAG2, both associated with estimated glomerular filtration rate. We conclude that, despite challenges in addressing phenotypic heterogeneity, access to increased sample sizes will continue to provide more robust inference regarding risk variant discovery for DKD.Peer reviewe

O31 Integrative analysis reveals a molecular stratification of systemic autoimmune diseases

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Heat shock-induced signal transduction in hematopoietic cells

Heat shock is a cellular stress that induces a characteristic set of signalling events, many of which are highly conserved throughout evolution. This includes the increased synthesis of a set of protein chaperones known as heat shock proteins (hsps) which facilitates survival during harsh environmental conditions. In eukaryotes, heat shock also leads to the stimulation of signalling pathways that negatively regulate translation via phosphorylation of the eukaryotic initiation factor 2α and also activate protein kinases of the MAPK superfamily. In mammals, the JNK and p38 MAPK signalling cascades have been found to be activated in response to numerous forms of environmental stress, including heat shock, however the mechanisms governing their activation in response to these stresses are poorly understood. In this work, the activation status of the JNK signalling cascade was investigated in a variety of cell types following heat shock treatment. Surprisingly, we have found that the degree of JNK activation that occurs in response to heat shock varies markedly in different murine cell types. Thus, while heat shock induced strong activation in macrophages and mast cells, a dramatically reduced activation was noted in T and B lymphocytes. Despite the lack of heat-induced JNK activation in murine lymphocytes, they could respond to heat shock in terms of the induction of the heat shock protein response which includes phosphorylation of the heat shock factor 1 (HSF1) and subsequent upregulation of the heat shock protein, Hsp70. In addition, we found that heat shock in murine lymphocytes, as in other cells, led to the phosphorylation of the eukaryotic initiation factor 2α (elF2α). These findings suggest that JNK signalling may be dispensable for the activation of the heat shock protein response and translational inhibition mediated by elF2α. To help understand the mechanistic basis for the altered JNK signalling response in murine lymphocytes, the potential involvement of various proximal signalling events in the activation of JNK during heat shock was investigated. It was found that both of the JNK kinases, MKK4 and MKK7 were activated during heat shock in most cells although not in murine lymphocytes. We also discovered that, unlike all other cell types tested, murine lymphocytes failed to activate JNK in response to the ribosomal toxin anisomycin, suggesting that there may be a common mechanistic link between the effects of this compound and heat shock on JNK activation. To address the possible biological significance of attenuated JNK signalling in murine lymphocytes in response to heat shock, we compared the induction of apoptosis in these cells with non-lymphoid cells. These studies indicated that murine lymphoid cells were, in fact, more susceptible to undergoing apoptosis as compared to non-lymphoid cells during heat shock. Thus, our evidence suggests that JNK activation is not likely to be the main factor influencing the progression of apoptosis in cells exposed to heat shock. We speculate that the attenuation of JNK signalling in murine lymphocytes during heat shock is due to the possible negative influence of this pathway on aspects of lymphocyte function during this form of stress or others that mimic its effects.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat

High spin polarization of optically-oriented trions in p-doped GaAs-AlGaAs quantum wells

We performed cw-photoluminescence (PL) measurements on a 15 nm wide p-doped Al0.3Ga0.7As-GaAs-Al0.3Ga0.7As quantum well structure at low temperatures. By irradiating the sample with circularly polarized light and analyzing the degree of circular polarization of the PL signal, we found a significantly higher degree of spin polarization for charged excitons (trions) than for neutral excitons. Time-resolved pump-probe and Faraday-rotation experiments give an additional information concerning the exciton and trion resonant photoexcitation

A New Simulation Package to Model Detector Systems with Fragmentation Reactions and Ion Separators: Application to the LYCCA-0 System

A Monte-Carlo simulation package has been developed to model the response of a detector system for ion identification used in conjunction with ion separators following nuclear reactions. The simulation is written predominantly using the GEANT4 framework but utilises the ion transport code MOCADI for accurate separator and reaction modelling. A novel MOCADI-GEANT4 interface has been developed to utilise the parameter file output option of MOCADI as an event generator for the GEANT4 detector simulation. Test simulation results have been compared with experimental data and excellent agreement was observed. The simulation has successfully been used to model a new particle detection system prototype (Lund-York-Cologne CAlorimeter (LYCCA)-0) and validate a method of ion identification using energy and time-of-flight with this system. (C) 2009 Elsevier B.V. All rights reserved