Effect of ion flux on helium retention in helium-irradiated tungsten

Helium retention in irradiated tungsten leads to swelling, pore formation, sample exfoliation and embrittlement with deleterious consequences in many applications. In particular, the use of tungsten in future nuclear fusion plants is proposed due to its good refractory properties. However, serious concerns about tungsten survivability stems from the fact that it must withstand severe irradiation conditions. In magnetic fusion as well as in inertial fusion (particularly with direct drive targets), tungsten components will be exposed to low and high energy ion (helium) irradiation, respectively. A common feature is that the most detrimental situations will take place in pulsed mode, i.e., high flux irradiation. There is increasing evidence on a correlation between a high helium flux and an enhancement of detrimental effects on tungsten. Nevertheless, the nature of these effects is not well understood due to the subtleties imposed by the exact temperature profile evolution, ion energy, pulse duration, existence of impurities and simultaneous irradiation with other species. Physically based Kinetic Monte Carlo is the technique of choice to simulate the evolution of radiation-induced damage inside solids in large temporal and space scales. We have used the recently developed code MMonCa (Modular Monte Carlo simulator), presented in this conference for the first time, to study He retention (and in general defect evolution) in tungsten samples irradiated with high intensity helium pulses. The code simulates the interactions among a large variety of defects and impurities (He and C) during the irradiation stage and the subsequent annealing steps. In addition, it allows us to vary the sample temperature to follow the severe thermo-mechanical effects of the pulses. In this work we will describe the helium kinetics for different irradiation conditions. A competition is established between fast helium cluster migration and trapping at large defects, being the temperature a determinant factor. In fact, high temperatures (induced by the pulses) are responsible for large vacancy cluster formation and subsequent additional trapping with respect to low flux irradiation.

Silica final lens performance in laser fusion facilities: HiPER and LIFE

Nowadays, the projects LIFE (Laser Inertial Fusion Energy) in USA and HiPER (High Power Laser Energy Research) in Europe are the most advanced ones to demonstrate laser fusion energy viability. One of the main points of concern to properly achieve ignition is the performance of the final optics (lenses) under the severe irradiation conditions that take place in fusion facilities. In this paper, we calculate the radiation fluxes and doses as well as the radiation-induced temperature enhancement and colour centre formation in final lenses assuming realistic geometrical configurations for HiPER and LIFE. On these bases, the mechanical stresses generated by the established temperature gradients are evaluated showing that from a mechanical point of view lenses only fulfil specifications if ions resulting from the imploding target are mitigated. The absorption coefficient of the lenses is calculated during reactor startup and steady-state operation. The obtained results reveal the necessity of new solutions to tackle ignition problems during the startup process for HiPER. Finally, we evaluate the effect of temperature gradients on focal length changes and lens surface deformations. In summary, we discuss the capabilities and weak points of silica lenses and propose alternatives to overcome predictable problems

Effect of ion flux on helium retention in helium-irradiated tungsten

Helium retention in irradiated tungsten leads to swelling, pore formation, sample exfoliation and embrittlement with deleterious consequences in many applications. In particular, the use of tungsten in future nuclear fusion plants is proposed due to its good refractory properties. However, serious concerns about tungsten survivability stems from the fact that it must withstand severe irradiation conditions. In magnetic fusion as well as in inertial fusion (particularly with direct drive targets), tungsten components will be exposed to low and high energy ion irradiation (helium), respectively. A common feature is that the most detrimental situations will take place in pulsed mode, i.e., high flux irradiation. There is increasing evidence of a correlation between a high helium flux and an enhancement of detrimental effects on tungsten. Nevertheless, the nature of these effects is not well understood due to the subtleties imposed by the exact temperature profile evolution, ion energy, pulse duration, existence of impurities and simultaneous irradiation with other species. Object Kinetic Monte Carlo is the technique of choice to simulate the evolution of radiation-induced damage inside solids in large temporal and space scales. We have used the recently developed code MMonCa (Modular Monte Carlo simulator), presented at COSIRES 2012 for the first time, to study He retention (and in general defect evolution) in tungsten samples irradiated with high intensity helium pulses. The code simulates the interactions among a large variety of defects and during the irradiation stage and the subsequent annealing steps. The results show that the pulsed mode leads to significantly higher He retention at temperatures higher than 700 K. In this paper we discuss the process of He retention in terms of trap evolution. In addition, we discuss the implications of these findings for inertial fusion.

Fault management techniques for systems with SRAM-based FPGAs

Las Field-Programmable Gate Arrays (FPGAs) SRAM se construyen sobre una memoria de configuración de tecnología RAM Estática (SRAM). Presentan múltiples características que las hacen muy interesantes para diseñar sistemas empotrados complejos. En primer lugar presentan un coste no-recurrente de ingeniería (NRE) bajo, ya que los elementos lógicos y de enrutado están pre-implementados (el diseño de usuario define su conexionado). También, a diferencia de otras tecnologías de FPGA, pueden ser reconfiguradas (incluso en campo) un número ilimitado de veces. Es más, las FPGAs SRAM de Xilinx soportan Reconfiguración Parcial Dinámica (DPR), la cual permite reconfigurar la FPGA sin interrumpir la aplicación. Finalmente, presentan una alta densidad de lógica, una alta capacidad de procesamiento y un rico juego de macro-bloques. Sin embargo, un inconveniente de esta tecnología es su susceptibilidad a la radiación ionizante, la cual aumenta con el grado de integración (geometrías más pequeñas, menores tensiones y mayores frecuencias). Esta es una precupación de primer nivel para aplicaciones en entornos altamente radiativos y con requisitos de alta confiabilidad. Este fenómeno conlleva una degradación a largo plazo y también puede inducir fallos instantáneos, los cuales pueden ser reversibles o producir daños irreversibles. En las FPGAs SRAM, los fallos inducidos por radiación pueden aparecer en en dos capas de arquitectura diferentes, que están físicamente superpuestas en el dado de silicio. La Capa de Aplicación (o A-Layer) contiene el hardware definido por el usuario, y la Capa de Configuración contiene la memoria de configuración y la circuitería de soporte. Los fallos en cualquiera de estas capas pueden hacer fracasar el sistema, lo cual puede ser ás o menos tolerable dependiendo de los requisitos de confiabilidad del sistema. En el caso general, estos fallos deben gestionados de alguna manera. Esta tesis trata sobre la gestión de fallos en FPGAs SRAM a nivel de sistema, en el contexto de sistemas empotrados autónomos y confiables operando en un entorno radiativo. La tesis se centra principalmente en aplicaciones espaciales, pero los mismos principios pueden aplicarse a aplicaciones terrenas. Las principales diferencias entre ambas son el nivel de radiación y la posibilidad de mantenimiento. Las diferentes técnicas para la gestión de fallos en A-Layer y C-Layer son clasificados, y sus implicaciones en la confiabilidad del sistema son analizados. Se proponen varias arquitecturas tanto para Gestores de Fallos de una capa como de doble-capa. Para estos últimos se propone una arquitectura novedosa, flexible y versátil. Gestiona las dos capas concurrentemente de manera coordinada, y permite equilibrar el nivel de redundancia y la confiabilidad. Con el objeto de validar técnicas de gestión de fallos dinámicas, se desarrollan dos diferentes soluciones. La primera es un entorno de simulación para Gestores de Fallos de C-Layer, basado en SystemC como lenguaje de modelado y como simulador basado en eventos. Este entorno y su metodología asociada permite explorar el espacio de diseño del Gestor de Fallos, desacoplando su diseño del desarrollo de la FPGA objetivo. El entorno incluye modelos tanto para la C-Layer de la FPGA como para el Gestor de Fallos, los cuales pueden interactuar a diferentes niveles de abstracción (a nivel de configuration frames y a nivel físico JTAG o SelectMAP). El entorno es configurable, escalable y versátil, e incluye capacidades de inyección de fallos. Los resultados de simulación para algunos escenarios son presentados y comentados. La segunda es una plataforma de validación para Gestores de Fallos de FPGAs Xilinx Virtex. La plataforma hardware aloja tres Módulos de FPGA Xilinx Virtex-4 FX12 y dos Módulos de Unidad de Microcontrolador (MCUs) de 32-bits de propósito general. Los Módulos MCU permiten prototipar Gestores de Fallos de C-Layer y A-Layer basados en software. Cada Módulo FPGA implementa un enlace de A-Layer Ethernet (a través de un switch Ethernet) con uno de los Módulos MCU, y un enlace de C-Layer JTAG con el otro. Además, ambos Módulos MCU intercambian comandos y datos a través de un enlace interno tipo UART. Al igual que para el entorno de simulación, se incluyen capacidades de inyección de fallos. Los resultados de pruebas para algunos escenarios son también presentados y comentados. En resumen, esta tesis cubre el proceso completo desde la descripción de los fallos FPGAs SRAM inducidos por radiación, pasando por la identificación y clasificación de técnicas de gestión de fallos, y por la propuesta de arquitecturas de Gestores de Fallos, para finalmente validarlas por simulación y pruebas. El trabajo futuro está relacionado sobre todo con la implementación de Gestores de Fallos de Sistema endurecidos para radiación. ABSTRACT SRAM-based Field-Programmable Gate Arrays (FPGAs) are built on Static RAM (SRAM) technology configuration memory. They present a number of features that make them very convenient for building complex embedded systems. First of all, they benefit from low Non-Recurrent Engineering (NRE) costs, as the logic and routing elements are pre-implemented (user design defines their connection). Also, as opposed to other FPGA technologies, they can be reconfigured (even in the field) an unlimited number of times. Moreover, Xilinx SRAM-based FPGAs feature Dynamic Partial Reconfiguration (DPR), which allows to partially reconfigure the FPGA without disrupting de application. Finally, they feature a high logic density, high processing capability and a rich set of hard macros. However, one limitation of this technology is its susceptibility to ionizing radiation, which increases with technology scaling (smaller geometries, lower voltages and higher frequencies). This is a first order concern for applications in harsh radiation environments and requiring high dependability. Ionizing radiation leads to long term degradation as well as instantaneous faults, which can in turn be reversible or produce irreversible damage. In SRAM-based FPGAs, radiation-induced faults can appear at two architectural layers, which are physically overlaid on the silicon die. The Application Layer (or A-Layer) contains the user-defined hardware, and the Configuration Layer (or C-Layer) contains the (volatile) configuration memory and its support circuitry. Faults at either layers can imply a system failure, which may be more ore less tolerated depending on the dependability requirements. In the general case, such faults must be managed in some way. This thesis is about managing SRAM-based FPGA faults at system level, in the context of autonomous and dependable embedded systems operating in a radiative environment. The focus is mainly on space applications, but the same principles can be applied to ground applications. The main differences between them are the radiation level and the possibility for maintenance. The different techniques for A-Layer and C-Layer fault management are classified and their implications in system dependability are assessed. Several architectures are proposed, both for single-layer and dual-layer Fault Managers. For the latter, a novel, flexible and versatile architecture is proposed. It manages both layers concurrently in a coordinated way, and allows balancing redundancy level and dependability. For the purpose of validating dynamic fault management techniques, two different solutions are developed. The first one is a simulation framework for C-Layer Fault Managers, based on SystemC as modeling language and event-driven simulator. This framework and its associated methodology allows exploring the Fault Manager design space, decoupling its design from the target FPGA development. The framework includes models for both the FPGA C-Layer and for the Fault Manager, which can interact at different abstraction levels (at configuration frame level and at JTAG or SelectMAP physical level). The framework is configurable, scalable and versatile, and includes fault injection capabilities. Simulation results for some scenarios are presented and discussed. The second one is a validation platform for Xilinx Virtex FPGA Fault Managers. The platform hosts three Xilinx Virtex-4 FX12 FPGA Modules and two general-purpose 32-bit Microcontroller Unit (MCU) Modules. The MCU Modules allow prototyping software-based CLayer and A-Layer Fault Managers. Each FPGA Module implements one A-Layer Ethernet link (through an Ethernet switch) with one of the MCU Modules, and one C-Layer JTAG link with the other. In addition, both MCU Modules exchange commands and data over an internal UART link. Similarly to the simulation framework, fault injection capabilities are implemented. Test results for some scenarios are also presented and discussed. In summary, this thesis covers the whole process from describing the problem of radiationinduced faults in SRAM-based FPGAs, then identifying and classifying fault management techniques, then proposing Fault Manager architectures and finally validating them by simulation and test. The proposed future work is mainly related to the implementation of radiation-hardened System Fault Managers.

CXCR4 and CD4 mediate a rapid CD95-independent cell death in CD4+ T cells

AIDS is characterized by a progressive decrease of CD4+ helper T lymphocytes. Destruction of these cells may involve programmed cell death, apoptosis. It has previously been reported that apoptosis can be induced even in noninfected cells by HIV-1 gp120 and anti-gp120 antibodies. HIV-1 gp120 binds to T cells via CD4 and the chemokine coreceptor CXCR4 (fusin/LESTR). Therefore, we investigated whether CD4 and CXCR4 mediate gp120-induced apoptosis. We used human peripheral blood lymphocytes, malignant T cells, and CD4/CXCR4 transfectants, and found cell death induced by both cell surface receptors, CD4 and CXCR4. The induced cell death was rapid, independent of known caspases, and lacking oligonucleosomal DNA fragmentation. In addition, the death signals were not propagated via p56lck and Giα. However, the cells showed chromatin condensation, morphological shrinkage, membrane inversion, and reduced mitochondrial transmembrane potential indicative of apoptosis. Significantly, apoptosis was exclusively observed in CD4+ but not in CD8+ T cells, and apoptosis triggered via CXCR4 was inhibited by stromal cell-derived factor-1, the natural CXCR4 ligand. Thus, this mechanism of apoptosis might contribute to T cell depletion in AIDS and might have major implications for therapeutic intervention.

Hepatitis B virus HBx protein sensitizes cells to apoptotic killing by tumor necrosis factor α

Persistent infection with hepatitis B virus (HBV) is a leading cause of human liver disease and is strongly associated with hepatocellular carcinoma, one of the most prevalent forms of human cancer. Apoptosis (programmed cell death) is an important mediator of chronic liver disease caused by HBV infection. It is demonstrated that the HBV HBx protein acutely sensitizes cells to apoptotic killing when expressed during viral replication in cultured cells and in transfected cells independently of other HBV genes. Cells that were resistant to apoptotic killing by high doses of tumor necrosis factor α (TNFα), a cytokine associated with liver damage during HBV infection, were made sensitive to very low doses of TNFα by HBx. HBx induced apoptosis by prolonged stimulation of N-Myc and the stress-mediated mitogen-activated-protein kinase kinase 1 (MEKK1) pathway but not by up-regulating TNF receptors. Cell killing was blocked by inhibiting HBx stimulation of N-Myc or mitogen-activated-protein kinase kinase 1 using dominant-interfering forms or by retargeting HBx from the cytoplasm to the nucleus, which prevents HBx activation of cytoplasmic signal transduction cascades. Treatment of cells with a mitogenic growth factor produced by many virus-induced tumors impaired induction of apoptosis by HBx and TNFα. These results indicate that HBx might be involved in HBV pathogenesis (liver disease) during virus infection and that enhanced apoptotic killing by HBx and TNFα might select for neoplastic hepatocytes that survive by synthesizing mitogenic growth factors.

Precursor B cells for autoantibody production in genomically Fas-intact autoimmune disease are not subject to Fas-mediated immune elimination

The Fas/Fas ligand (FasL) system participates in regulation of the immune system through the apoptotic process. However, the extent to which abnormalities in this system are involved in the loss of self-tolerance and development of autoimmune disease not associated with Fas/FasL mutations remains unknown. The present study addresses this issue in Fas/FasL-intact, systemic lupus erythematosus (SLE)-prone (NZB × NZW) (NZB/W) F1 mice. While splenic B cells from 2-month-old mice before overt SLE expressed Fas poorly, in vitro stimulation with an agonistic anti-CD40 mAb up-regulated their Fas expression, thus revealing the existence of two populations: one was Fashigh and highly susceptible to anti-Fas mAb-induced apoptosis, and the other was Faslow and apoptosis-resistant. The Faslow cells were included in the CD5+ B cell subpopulation and contained most of the cells that produced IgM anti-DNA antibodies. The isotype of anti-DNA antibodies switches from IgM to IgG in NZB/W F1 mice at ages beginning at about 6 months. These IgG anti-DNA antibodies were produced almost exclusively by a subpopulation of splenic B cells that spontaneously expressed low levels of Fas in vivo and were apoptosis-resistant. The findings indicate that precursor B cells for autoantibody production and presumably autoantibody-secreting cells in these mice are relatively resistant to Fas-mediated apoptosis, a finding supporting the concept that abnormalities of Fas-mediated apoptotic process are involved in the development of autoreactive B cells in Fas/FasL-intact autoimmune disease.

Selective induction of p53 and chemosensitivity in RB-deficient cells by E1A mutants unable to bind the RB-related proteins

The adenovirus E1A oncoprotein renders primary cells sensitive to the induction of apoptosis by diverse stimuli, including many anticancer agents. E1A-expressing cells accumulate p53 protein, and p53 potentiates drug-induced apoptosis. To determine how E1A promotes chemosensitivity, a series of E1A mutants were introduced into primary human and mouse fibroblasts using high-titer recombinant retroviruses, allowing analysis of E1A in genetically normal cells outside the context of adenovirus infection. Mutations that disrupted apoptosis and chemosensitivity separated into two complementation groups, which correlated precisely with the ability of E1A to associate with either the p300/CBP or retinoblastoma protein families. Furthermore, E1A mutants incapable of binding RB, p107, and p130 conferred chemosensitivity to fibroblasts derived from RB-deficient mice, but not fibroblasts from mice lacking p107 or p130. Hence, inactivation of RB, but not p107 or p130, is required for chemosensitivity induced by E1A. Finally, the same E1A functions that promote drug-induced apoptosis also induce p53. Together, these data demonstrate that p53 accumulation and chemosensitivity are linked to E1A’s oncogenic potential, and identify a strategy to selectively induce apoptosis in RB-deficient tumor cells.

Nuclear translocation of NF-κB is increased in dopaminergic neurons of patients with Parkinson disease

Evidence from postmortem studies suggest an involvement of oxidative stress in the degeneration of dopaminergic neurons in Parkinson disease (PD) that have recently been shown to die by apoptosis, but the relationship between oxidative stress and apoptosis has not yet been elucidated. Activation of the transcription factor NF-κB is associated with oxidative stress-induced apoptosis in several nonneuronal in vitro models. To investigate whether it may play a role in PD, we looked for the translocation of NF-κB from the cytoplasm to the nucleus, evidence of its activation, in melanized neurons in the mesencephalon of postmortem human brain from five patients with idiopathic PD and seven matched control subjects. In PD patients, the proportion of dopaminergic neurons with immunoreactive NF-κB in their nuclei was more than 70-fold that in control subjects. A possible relationship between the nuclear localization of NF-κB in mesencephalic neurons of PD patients and oxidative stress in such neurons has been shown in vitro with primary cultures of rat mesencephalon, where translocation of NF-κB is preceded by a transient production of free radicals during apoptosis induced by activation of the sphingomyelin-dependent signaling pathway with C2-ceramide. The data suggest that this oxidant-mediated apoptogenic transduction pathway may play a role in the mechanism of neuronal death in PD.

Superoxide-mediated clastogenesis and anticlastogenic effects of exogenous superoxide dismutase

Superoxide-mediated clastogenesis is characteristic for various chronic inflammatory diseases with autoimmune reactions and probably plays a role in radiation-induced clastogenesis and in the congenital breakage syndromes. It is consistently prevented by exogenous superoxide dismutase (SOD), but not by heat-inactivated SOD, indicating that the anticlastogenic effect is related to the catalytic function of the enzyme. Increased superoxide production by activated monocytes/macrophages is followed by release of more long-lived metabolites, so-called clastogenic factors, which contain lipid peroxidation products, unusual nucleotides of inosine, and cytokines such as tumor necrosis factor α. Since these components are not only clastogenic, but can stimulate further superoxide production by monocytes and neutrophils, the genotoxic effects are self-sustaining. It is shown here that anticlastogenic effects of exogenous SOD are preserved despite extensive washing of the cells and removal of all extracellular SOD. Using flow cytometry and confocal laser microscopy, rapid adherence of the fluorescently labeled enzyme to the cell surface could be observed with slow uptake into the cell during the following hours. The degree of labeling was concentration and time dependent. It was most important for monocytes, compared with lymphocytes, neutrophils, and fibroblasts. The cytochrome c assay showed significantly diminished O2− production by monocytes, pretreated with SOD and washed thereafter. The preferential and rapid binding of SOD to monocytes may be of importance not only for the superoxide-mediated genotoxic effects, described above, but also from a therapeutic standpoint. It can explain the observation that beneficial effects of injected SOD lasted for weeks and months despite rapid clearance of the enzyme from the blood stream according to pharmacodynamic studies.

Spontaneous mutations recovered as mosaics in the mouse specific-locus test

The specific-locus test (SLT) detects new mutants among mice heterozygous for seven recessive visible markers. Spontaneous mutations can be manifested not only as singleton whole-body mutants in controls (for which we report new data), but as mosaics—either visible (manifesting mottled coat color) in the scored generation (G2) or masked, among the wild-type parental generation (G1). Masked G1 mosaics reveal themselves by producing clusters of whole-body mutants in G2. We provide evidence that most, if not all, mosaics detected in the SLT (both radiation and control progenies) result from a single-strand spontaneous mutation subsequent to the last premeiotic mitosis and before the first postmeiotic one of a parental genome—the “perigametic interval.” Such events in the genomes of the G1 and G0 result, respectively, in visible and masked 50:50 mosaics. Per cell cycle, the spontaneous mutation rate in the perigametic interval is much higher than that in pregamete mitotic divisions. A clearly different locus spectrum further supports the hypothesis of different origin, and casts further doubt on the validity of the doubling-dose risk-estimation method. Because mosaics cannot have arisen in mitotic germ cells, and are not induced by radiation exposure in the perigametic interval, they should not be included in calculations of radiation-induced germ-line mutation rates. For per-generation calculations, inclusion of mosaics yields a spontaneous frequency 1.7 times that calculated from singletons alone for mutations contributed by males; including both sexes, the multiple is 2.2.

Chemokines regulate hippocampal neuronal signaling and gp120 neurotoxicity

The HIV-1 envelope protein gp120 induces apoptosis in hippocampal neurons. Because chemokine receptors act as cellular receptors for HIV-1, we examined rat hippocampal neurons for the presence of functional chemokine receptors. Fura-2-based Ca imaging showed that numerous chemokines, including SDF-1α, RANTES, and fractalkine, affect neuronal Ca signaling, suggesting that hippocampal neurons possess a wide variety of chemokine receptors. Chemokines also blocked the frequency of spontaneous glutamatergic excitatory postsynaptic currents recorded from these neurons and reduced voltage-dependent Ca currents in the same neurons. Reverse transcription–PCR demonstrated the expression of CCR1, CCR4, CCR5, CCR9/10, CXCR2, CXCR4, and CX3CR1, as well as the chemokine fractalkine in these neurons. Both fractalkine and macrophage-derived chemokine (MDC) produced a time-dependent activation of extracellular response kinases (ERK)-1/2, whereas no activation of c-JUN NH2-terminal protein kinase (JNK)/stress-activated protein kinase, or p38 was evident. Furthermore, these two chemokines, as well as SDF-1α, activated the Ca- and cAMP-dependent transcription factor CREB. Several chemokines were able also to block gp120-induced apoptosis of hippocampal neurons, both in the presence and absence of the glial feeder layer. These data suggest that chemokine receptors may directly mediate gp120 neurotoxicity.

Bok is a pro-apoptotic Bcl-2 protein with restricted expression in reproductive tissues and heterodimerizes with selective anti-apoptotic Bcl-2 family members

In the intracellular death program, hetero- and homodimerization of different anti- and pro-apoptotic Bcl-2-related proteins are critical in the determination of cell fate. From a rat ovarian fusion cDNA library, we isolated a new pro-apoptotic Bcl-2 gene, Bcl-2-related ovarian killer (Bok). Bok had conserved Bcl-2 homology (BH) domains 1, 2, and 3 and a C-terminal transmembrane region present in other Bcl-2 proteins, but lacked the BH4 domain found only in anti-apoptotic Bcl-2 proteins. In the yeast two-hybrid system, Bok interacted strongly with some (Mcl-1, BHRF1, and Bfl-1) but not other (Bcl-2, Bcl-xL, and Bcl-w) anti-apoptotic members. This finding is in direct contrast to the ability of other pro-apoptotic members (Bax, Bak, and Bik) to interact with all of the anti-apoptotic proteins. In addition, negligible interaction was found between Bok and different pro-apoptotic members. In mammalian cells, overexpression of Bok induced apoptosis that was blocked by the baculoviral-derived cysteine protease inhibitor P35. Cell killing induced by Bok was also suppressed following coexpression with Mcl-1 and BHRF1 but not with Bcl-2, further indicating that Bok heterodimerized only with selective anti-apoptotic Bcl-2 proteins. Northern blot analysis indicated that Bok was highly expressed in the ovary, testis and uterus. In situ hybridization analysis localized Bok mRNA in granulosa cells, the cell type that underwent apoptosis during follicle atresia. Identification of Bok as a new pro-apoptotic Bcl-2 protein with restricted tissue distribution and heterodimerization properties could facilitate elucidation of apoptosis mechanisms in reproductive tissues undergoing hormone-regulated cyclic cell turnover.

Fas-dependent CD4+ cytotoxic T-cell-mediated pathogenesis during virus infection

β2-Microglobulin-deficient (β2m−) mice generate a CD4+ major histocompatibility complex class II-restricted cytotoxic T-lymphocyte (CTL) response following infection with lymphocytic choriomeningitis (LCM) virus (LCMV). We have determined the cytotoxic mechanism used by these CD4+ CTLs and have examined the role of this cytotoxic activity in pathogenesis of LCM disease in β2m− mice. Lysis of LCMV-infected target cells by CTLs from β2m− mice is inhibited by addition of soluble Fas-Ig fusion proteins or by pretreatment of the CTLs with the protein synthesis inhibitor emetine. In addition, LCMV-infected cell lines that are resistant to anti-Fas-induced apoptosis are refractory to lysis by these virus-specific CD4+ CTLs. These data indicate that LCMV-specific CD4+ CTLs from β2m− mice use a Fas-dependent lytic mechanism. Intracranial (i.c.) infection of β2m− mice with LCMV results in loss of body weight. Fas-deficient β2m−.lpr mice develop a similar wasting disease following i.c. infection. This suggests that Fas-dependent cytotoxicity is not required for LCMV-induced weight loss. A potential mediator of this chronic wasting disease is tumor necrosis factor (TNF)-α, which is produced by LCMV-specific CD4+ CTLs. In contrast to LCMV-induced weight loss, lethal LCM disease in β2m− mice is dependent on Fas-mediated cytotoxicity. Transfer of immune splenocytes from LCMV-infected β2m− mice into irradiated infected β2m− mice results in death of recipient animals. In contrast, transfer of these splenocytes into irradiated infected β2m−.lpr mice does not cause death. Thus a role for CD4+ T-cell-mediated cytotoxicity in virus-induced immunopathology has now been demonstrated.

The ATM homologue MEC1 is required for phosphorylation of replication protein A in yeast

Replication protein A (RPA) is a highly conserved single-stranded DNA-binding protein, required for cellular DNA replication, repair, and recombination. In human cells, RPA is phosphorylated during the S and G2 phases of the cell cycle and also in response to ionizing or ultraviolet radiation. Saccharomyces cerevisiae exhibits a similar pattern of cell cycle-regulated RPA phosphorylation, and our studies indicate that the radiation-induced reactions occur in yeast as well. We have examined yeast RPA phosphorylation during the normal cell cycle and in response to environmental insult, and have demonstrated that the checkpoint gene MEC1 is required for the reaction under all conditions tested. Through examination of several checkpoint mutants, we have placed RPA phosphorylation in a novel pathway of the DNA damage response. MEC1 is similar in sequence to human ATM, the gene mutated in patients with ataxia-telangiectasia (A-T). A-T cells are deficient in multiple checkpoint pathways and are hypersensitive to killing by ionizing radiation. Because A-T cells exhibit a delay in ionizing radiation-induced RPA phosphorylation, our results indicate a functional similarity between MEC1 and ATM, and suggest that RPA phosphorylation is involved in a conserved eukaryotic DNA damage-response pathway defective in A-T.