Rapid acclimation of juvenile corals to CO2-mediated acidification by upregulation of heat shock protein and Bcl-2 genes

Corals play a key role in ocean ecosystems and carbonate balance, but their molecular response to ocean acidification remains unclear. The only previous whole-transcriptome study documented extensive disruption of gene expression, particularly of genes encoding skeletal organic matrix proteins, in juvenile corals (Acropora millepora) after short-term (3 d) exposure to elevated pCO2. In this study, whole-transcriptome analysis was used to compare the effects of such 'acute' (3 d) exposure to elevated pCO2 with a longer ('prolonged'; 9 d) period of exposure beginning immediately post-fertilization. Far fewer genes were differentially expressed under the 9-d treatment, and although the transcriptome data implied wholesale disruption of metabolism and calcification genes in the acute treatment experiment, expression of most genes was at control levels after prolonged treatment. There was little overlap between the genes responding to the acute and prolonged treatments, but heat shock proteins (HSPs) and heat shock factors (HSFs) were over-represented amongst the genes responding to both treatments. Amongst these was an HSP70 gene previously shown to be involved in acclimation to thermal stress in a field population of another acroporid coral. The most obvious feature of the molecular response in the 9-d treatment experiment was the upregulation of five distinct Bcl-2 family members, the majority predicted to be anti-apoptotic. This suggests that an important component of the longer term response to elevated CO2 is suppression of apoptosis. It therefore appears that juvenile A. millepora have the capacity to rapidly acclimate to elevated pCO2, a process mediated by upregulation of specific HSPs and a suite of Bcl-2 family members.

Posible interrupción de la autovía GC-2 (Gran Canaria) por deslizamientos y desprendimientos rocosos: análisis de estabilidad del acantilado de el Rincón

En este trabajo se han identificado los deslizamientos y desprendimientos que se pueden desarrollar en el acantilado de El Rincón (Gran Canaria) y sus repercusiones sobre la Autovía GC-2, en la base del acantilado. El análisis de estabilidad realizado para las condiciones actuales indica que la ladera es estable. El análisis a largo plazo considera el sustrato saturado en agua y se limita a los dos bloques rocosos más susceptibles de experimentar deslizamiento: uno a media ladera y otro en coronación. El deslizamiento del bloque de coronación parece más probable ya que sólo requiere la saturación de los piroclastos y está favorecido por la progresiva apertura de la grieta subvertical. Por último, la evaluación de los desprendimientos rocosos que afectan al acantilado indica que los bloques no alcanzarían la calzada de la autovía GC-2; sino que se acumularían en los conos de deyección presentes al pie de la ladera.

Contribución a la caracterización espacial de canales con sistemas MIMO-OFDM en la banda de 2,45 Ghz

La tecnología de múltiples antenas ha evolucionado para dar soporte a los actuales y futuros sistemas de comunicaciones inalámbricas en su afán por proporcionar la calidad de señal y las altas tasas de transmisión que demandan los nuevos servicios de voz, datos y multimedia. Sin embargo, es fundamental comprender las características espaciales del canal radio, ya que son las características del propio canal lo que limita en gran medida las prestaciones de los sistemas de comunicación actuales. Por ello surge la necesidad de estudiar la estructura espacial del canal de propagación para poder diseñar, evaluar e implementar de forma más eficiente tecnologías multiantena en los actuales y futuros sistemas de comunicación inalámbrica. Las tecnologías multiantena denominadas antenas inteligentes y MIMO han generado un gran interés en el área de comunicaciones inalámbricas, por ejemplo los sistemas de telefonía celular o más recientemente en las redes WLAN (Wireless Local Area Network), principalmente por la mejora que proporcionan en la calidad de las señales y en la tasa de transmisión de datos, respectivamente. Las ventajas de estas tecnologías se fundamentan en el uso de la dimensión espacial para obtener ganancia por diversidad espacial, como ya sucediera con las tecnologías FDMA (Frequency Division Multiplexing Access), TDMA (Time Division Multiplexing Access) y CDMA (Code Division Multiplexing Access) para obtener diversidad en las dimensiones de frecuencia, tiempo y código, respectivamente. Esta Tesis se centra en estudiar las características espaciales del canal con sistemas de múltiples antenas mediante la estimación de los perfiles de ángulos de llegada (DoA, Direction-of- Arrival) considerando esquemas de diversidad en espacio, polarización y frecuencia. Como primer paso se realiza una revisión de los sistemas con antenas inteligentes y los sistemas MIMO, describiendo con detalle la base matemática que sustenta las prestaciones ofrecidas por estos sistemas. Posteriormente se aportan distintos estudios sobre la estimación de los perfiles de DoA de canales radio con sistemas multiantena evaluando distintos aspectos de antenas, algoritmos de estimación, esquemas de polarización, campo lejano y campo cercano de las fuentes. Así mismo, se presenta un prototipo de medida MIMO-OFDM-SPAA3D en la banda ISM (Industrial, Scientific and Medical) de 2,45 Ghz, el cual está preparado para caracterizar experimentalmente el rendimiento de los sistemas MIMO, y para caracterizar espacialmente canales de propagación, considerando los esquemas de diversidad espacial, por polarización y frecuencia. Los estudios aportados se describen a continuación. Los sistemas de antenas inteligentes dependen en gran medida de la posición de los usuarios. Estos sistemas están equipados con arrays de antenas, los cuales aportan la diversidad espacial necesaria para obtener una representación espacial fidedigna del canal radio a través de los perfiles de DoA (DoA, Direction-of-Arrival) y por tanto, la posición de las fuentes de señal. Sin embargo, los errores de fabricación de arrays así como ciertos parámetros de señal conlleva un efecto negativo en las prestaciones de estos sistemas. Por ello se plantea un modelo de señal parametrizado que permite estudiar la influencia que tienen estos factores sobre los errores de estimación de DoA, tanto en acimut como en elevación, utilizando los algoritmos de estimación de DOA más conocidos en la literatura. A partir de las curvas de error, se pueden obtener parámetros de diseño para sistemas de localización basados en arrays. En un segundo estudio se evalúan esquemas de diversidad por polarización con los sistemas multiantena para mejorar la estimación de los perfiles de DoA en canales que presentan pérdidas por despolarización. Para ello se desarrolla un modelo de señal en array con sensibilidad de polarización que toma en cuenta el campo electromagnético de ondas planas. Se realizan simulaciones MC del modelo para estudiar el efecto de la orientación de la polarización como el número de polarizaciones usadas en el transmisor como en el receptor sobre la precisión en la estimación de los perfiles de DoA observados en el receptor. Además, se presentan los perfiles DoA obtenidos en escenarios quasiestáticos de interior con un prototipo de medida MIMO 4x4 de banda estrecha en la banda de 2,45 GHz, los cuales muestran gran fidelidad con el escenario real. Para la obtención de los perfiles DoA se propone un método basado en arrays virtuales, validado con los datos de simulación y los datos experimentales. Con relación a la localización 3D de fuentes en campo cercano (zona de Fresnel), se presenta un tercer estudio para obtener con gran exactitud la estructura espacial del canal de propagación en entornos de interior controlados (en cámara anecóica) utilizando arrays virtuales. El estudio analiza la influencia del tamaño del array y el diagrama de radiación en la estimación de los parámetros de localización proponiendo, para ello, un modelo de señal basado en un vector de enfoque de onda esférico (SWSV). Al aumentar el número de antenas del array se consigue reducir el error RMS de estimación y mejorar sustancialmente la representación espacial del canal. La estimación de los parámetros de localización se lleva a cabo con un nuevo método de búsqueda multinivel adaptativo, propuesto con el fin de reducir drásticamente el tiempo de procesado que demandan otros algoritmos multivariable basados en subespacios, como el MUSIC, a costa de incrementar los requisitos de memoria. Las simulaciones del modelo arrojan resultados que son validados con resultados experimentales y comparados con el límite de Cramer Rao en términos del error cuadrático medio. La compensación del diagrama de radiación acerca sustancialmente la exactitud de estimación de la distancia al límite de Cramer Rao. Finalmente, es igual de importante la evaluación teórica como experimental de las prestaciones de los sistemas MIMO-OFDM. Por ello, se presenta el diseño e implementación de un prototipo de medida MIMO-OFDM-SPAA3D autocalibrado con sistema de posicionamiento de antena automático en la banda de 2,45 Ghz con capacidad para evaluar la capacidad de los sistemas MIMO. Además, tiene la capacidad de caracterizar espacialmente canales MIMO, incorporando para ello una etapa de autocalibración para medir la respuesta en frecuencia de los transmisores y receptores de RF, y así poder caracterizar la respuesta de fase del canal con mayor precisión. Este sistema incorpora un posicionador de antena automático 3D (SPAA3D) basado en un scanner con 3 brazos mecánicos sobre los que se desplaza un posicionador de antena de forma independiente, controlado desde un PC. Este posicionador permite obtener una gran cantidad de mediciones del canal en regiones locales, lo cual favorece la caracterización estadística de los parámetros del sistema MIMO. Con este prototipo se realizan varias campañas de medida para evaluar el canal MIMO en términos de capacidad comparando 2 esquemas de polarización y tomando en cuenta la diversidad en frecuencia aportada por la modulación OFDM en distintos escenarios. ABSTRACT Multiple-antennas technologies have been evolved to be the support of the actual and future wireless communication systems in its way to provide the high quality and high data rates required by new data, voice and data services. However, it is important to understand the behavior of the spatial characteristics of the radio channel, since the channel by itself limits the performance of the actual wireless communications systems. This drawback raises the need to understand the spatial structure of the propagation channel in order to design, assess, and develop more efficient multiantenna technologies for the actual and future wireless communications systems. Multiantenna technologies such as ‘Smart Antennas’ and MIMO systems have generated great interest in the field of wireless communications, i.e. cellular communications systems and more recently WLAN (Wireless Local Area Networks), mainly because the higher quality and the high data rate they are able to provide. Their technological benefits are based on the exploitation of the spatial diversity provided by the use of multiple antennas as happened in the past with some multiaccess technologies such as FDMA (Frequency Division Multiplexing Access), TDMA (Time Division Multiplexing Access), and CDMA (Code Division Multiplexing Access), which give diversity in the domains of frequency, time and code, respectively. This Thesis is mainly focus to study the spatial channel characteristics using schemes of multiple antennas considering several diversity schemes such as space, polarization, and frequency. The spatial characteristics will be study in terms of the direction-of-arrival profiles viewed at the receiver side of the radio link. The first step is to do a review of the smart antennas and MIMO systems technologies highlighting their advantages and drawbacks from a mathematical point of view. In the second step, a set of studies concerning the spatial characterization of the radio channel through the DoA profiles are addressed. The performance of several DoA estimation methods is assessed considering several aspects regarding antenna array structure, polarization diversity, and far-field and near-field conditions. Most of the results of these studies come from simulations of data models and measurements with real multiantena prototypes. In the same way, having understand the importance of validate the theoretical data models with experimental results, a 2,4 GHz MIMO-OFDM-SPAA2D prototype is presented. This prototype is intended for evaluating MIMO-OFDM capacity in indoor and outdoor scenarios, characterize the spatial structure of radio channels, assess several diversity schemes such as polarization, space, and frequency diversity, among others aspects. The studies reported are briefly described below. As is stated in Chapter two, the determination of user position is a fundamental task to be resolved for the smart antenna systems. As these systems are equipped with antenna arrays, they can provide the enough spatial diversity to accurately draw the spatial characterization of the radio channel through the DoA profiles, and therefore the source location. However, certain real implementation factors related to antenna errors, signals, and receivers will certainly reduce the performance of such direction finding systems. In that sense, a parameterized narrowband signal model is proposed to evaluate the influence of these factors in the location parameter estimation through extensive MC simulations. The results obtained from several DoA algorithms may be useful to extract some parameter design for directing finding systems based on arrays. The second study goes through the importance that polarization schemes can have for estimating far-field DoA profiles in radio channels, particularly for scenarios that may introduce polarization losses. For this purpose, a narrowband signal model with polarization sensibility is developed to conduct an analysis of several polarization schemes at transmitter (TX) and receiver (RX) through extensive MC simulations. In addition, spatial characterization of quasistatic indoor scenarios is also carried out using a 2.45 GHz MIMO prototype equipped with single and dual-polarized antennas. A good agreement between the measured DoA profiles with the propagation scenario is achieved. The theoretical and experimental evaluation of polarization schemes is performed using virtual arrays. In that case, a DoA estimation method is proposed based on adding an phase reference to properly track the DoA, which shows good results. In the third study, the special case of near-field source localization with virtual arrays is addressed. Most of DoA estimation algorithms are focused in far-field source localization where the radiated wavefronts are assume to be planar waves at the receive array. However, when source are located close to the array, the assumption of plane waves is no longer valid as the wavefronts exhibit a spherical behavior along the array. Thus, a faster and effective method of azimuth, elevation angles-of-arrival, and range estimation for near-field sources is proposed. The efficacy of the proposed method is evaluated with simulation and validated with measurements collected from a measurement campaign carried out in a controlled propagation environment, i.e. anechoic chamber. Moreover, the performance of the method is assessed in terms of the RMSE for several array sizes, several source positions, and taking into account the effect of radiation pattern. In general, better results are obtained with larger array and larger source distances. The effect of the antennas is included in the data model leading to more accurate results, particularly for range rather than for angle estimation. Moreover, a new multivariable searching method based on the MUSIC algorithm, called MUSA (multilevel MUSIC-based algorithm), is presented. This method is proposed to estimate the 3D location parameters in a faster way than other multivariable algorithms, such as MUSIC algorithm, at the cost of increasing the memory size. Finally, in the last chapter, a MIMO-OFDM-SPAA3D prototype is presented to experimentally evaluate different MIMO schemes regarding antennas, polarization, and frequency in different indoor and outdoor scenarios. The prototype has been developed on a Software-Defined Radio (SDR) platform. It allows taking measurements where future wireless systems will be developed. The novelty of this prototype is concerning the following 2 subsystems. The first one is the tridimensional (3D) antenna positioning system (SPAA3D) based on three linear scanners which is developed for making automatic testing possible reducing errors of the antenna array positioning. A set of software has been developed for research works such as MIMO channel characterization, MIMO capacity, OFDM synchronization, and so on. The second subsystem is the RF autocalibration module at the TX and RX. This subsystem allows to properly tracking the spatial structure of indoor and outdoor channels in terms of DoA profiles. Some results are draw regarding performance of MIMO-OFDM systems with different polarization schemes and different propagation environments.

Pasarela peatonal sobre el río Jarama, al norte de la autovía A-2, a la altura del pk 15+400, en el límite entre Madrid y San Fernando de Henares

El presente proyecto versa sobre la construcción de una pasarela peatonal sobre el río Jarama, al norte de la autovía A-2, a la altura del pk 15+400, en el límite entre Madrid y San Fernando de Henares

La Donsayna: Núm. 2 - 8 de diciembre de 1844

At high concentrations, the tubule poison paclitaxel is able to kill cancer cells that express Bcl-2; it inhibits the antiapoptotic activity of Bcl-2 by inducing its phosphorylation. To localize the site on Bcl-2 regulated by phosphorylation, mutant forms of Bcl-2 were constructed. Mutant forms of Bcl-2 with an alteration in serine at amino acid 70 (S70A) or with deletion of a 60-aa loop region between the α1 and α2 helices (Δloop Bcl-2, which also deletes amino acid 70) were unable to be phosphorylated by paclitaxel treatment of MDA-MB-231 cells into which the genes for the mutant proteins were transfected. The Δloop mutant completely inhibited paclitaxel-induced apoptosis. In cells expressing the S70A mutant, paclitaxel induced about one-third the level of apoptosis seen with wild-type Bcl-2. To evaluate the role of mitogen-activated protein kinases (MAPKs) in Bcl-2 phosphorylation, the activation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 was examined. Paclitaxel-induced apoptosis was associated with phosphorylation of Bcl-2 and activation of ERK and JNK MAPKs. If JNK activation was blocked by transfections with either a stress-activated protein kinase kinase dominant-negative (K→R) gene (which prevents the activation of a kinase upstream of JNK) or MAPK phosphatase-1 gene (which dephosphorylates and inactivates JNK), Bcl-2 phosphorylation did not occur, and the cells were not killed by paclitaxel. By contrast, neither an ERK inhibitor (PD098059) nor p38 inhibitors (SB203580 and SB202190) had an effect on Bcl-2 phosphorylation. Thus, our data show that the antiapoptotic effects of Bcl-2 can be overcome by phosphorylation of Ser-70; forms of Bcl-2 lacking the loop region are much more effective at preventing apoptosis than wild-type Bcl-2 because they cannot be phosphorylated. JNK, but not ERK or p38 MAPK, appear to be involved in the phosphorylation of Bcl-2 induced by paclitaxel.

6-Hydroxydopamine (6-OHDA) is widely used to selectively lesion dopaminergic neurons of the substantia nigra (SN) in the creation of animal models of Parkinson’s disease. In vitro, the death of PC-12 cells caused by exposure to 6-OHDA occurs with characteristics consistent with an apoptotic mechanism of cell death. To test the hypothesis that apoptotic pathways are involved in the death of dopaminergic neurons of the SN caused by 6-OHDA, we created a replication-defective genomic herpes simplex virus-based vector containing the coding sequence for the antiapoptotic peptide Bcl-2 under the transcriptional control of the simian cytomegalovirus immediate early promoter. Transfection of primary cortical neurons in culture with the Bcl-2-producing vector protected those cells from naturally occurring cell death over 3 weeks. Injection of the Bcl-2-expressing vector into SN of rats 1 week before injection of 6-OHDA into the ipsilateral striatum increased the survival of neurons in the SN, detected either by retrograde labeling of those cells with fluorogold or by tyrosine hydroxylase immunocytochemistry, by 50%. These results, demonstrating that death of nigral neurons induced by 6-OHDA lesioning may be blocked by the expression of Bcl-2, are consistent with the notion that cell death in this model system is at least in part apoptotic in nature and suggest that a Bcl-2-expressing vector may have therapeutic potential in the treatment of Parkinson’s disease.

Human cytomegalovirus (CMV), a herpesvirus that causes congenital disease and opportunistic infections in immunocompromised individuals, encodes functions that facilitate efficient viral propagation by altering host cell behavior. Here we show that CMV blocks apoptosis mediated by death receptors and encodes a mitochondria-localized inhibitor of apoptosis, denoted vMIA, capable of suppressing apoptosis induced by diverse stimuli. vMIA, a product of the viral UL37 gene, inhibits Fas-mediated apoptosis at a point downstream of caspase-8 activation and Bid cleavage but upstream of cytochrome c release, while residing in mitochondria and associating with adenine nucleotide translocator. These functional properties resemble those ascribed to Bcl-2; however, the absence of sequence similarity to Bcl-2 or any other known cell death suppressors suggests that vMIA defines a previously undescribed class of anti-apoptotic proteins.

The BCL-2 family of proteins is composed of both pro- and antiapoptotic regulators, although its most critical biochemical functions remain uncertain. The structural similarity between the BCL-XL monomer and several ion-pore-forming bacterial toxins has prompted electrophysiologic studies. Both BAX and BCL-2 insert into KCl-loaded vesicles in a pH-dependent fashion and demonstrate macroscopic ion efflux. Release is maximum at ≈pH 4.0 for both proteins; however, BAX demonstrates a broader pH range of activity. Both purified proteins also insert into planar lipid bilayers at pH 4.0. Single-channel recordings revealed a minimal channel conductance for BAX of 22 pS that evolved to channel currents with at least three subconductance levels. The final, apparently stable BAX channel had a conductance of 0.731 nS at pH 4.0 that changed to 0.329 nS when shifted to pH 7.0 but remained mildly Cl− selective and predominantly open. When BAX-incorporated lipid vesicles were fused to planar lipid bilayers at pH 7.0, a Cl−-selective (PK/PCl = 0.3) 1.5-nS channel displaying mild inward rectification was noted. In contrast, BCL-2 formed mildly K+-selective (PK/PCl = 3.9) channels with a most prominent initial conductance of 80 pS that increased to 1.90 nS. Fusion of BCL-2-incorporated lipid vesicles into planar bilayers at pH 7.0 also revealed mild K+ selectivity (PK/PCl = 2.4) with a maximum conductance of 1.08 nS. BAX and BCL-2 each form channels in artificial membranes that have distinct characteristics including ion selectivity, conductance, voltage dependence, and rectification. Thus, one role of these molecules may include pore activity at selected membrane sites.

Bcl-2 is the prototypical member of a large family of apoptosis-regulating proteins, consisting of blockers and promoters of cell death. The three-dimensional structure of a Bcl-2 homologue, Bcl-XL, suggests striking similarity to the pore-forming domains of diphtheria toxin and the bacterial colicins, prompting exploration of whether Bcl-2 is capable of forming pores in lipid membranes. Using chloride efflux from KCl-loaded unilamellar lipid vesicles as an assay, purified recombinant Bcl-2 protein exhibited pore-forming activity with properties similar to those of the bacterial toxins, diphtheria toxin, and colicins, i.e., dependence on low pH and acidic lipid membranes. In contrast, a mutant of Bcl-2 lacking the two core hydrophobic α-helices (helices 5 and 6), predicted to be required for membrane insertion and channel formation, produced only nonspecific effects. In planar lipid bilayers, where detection of single channels is possible, Bcl-2 formed discrete ion-conducting, cation-selective channels, whereas the Bcl-2 (Δh5, 6) mutant did not. The most frequent conductance observed (18 ± 2 pS in 0.5 M KCl at pH 7.4) is consistent with a four-helix bundle structure arising from Bcl-2 dimers. However, larger channel conductances (41 ± 2 pS and 90 ± 10 pS) also were detected with progressively lower occurrence, implying the step-wise formation of larger oligomers of Bcl-2 in membranes. These findings thus provide biophysical evidence that Bcl-2 forms channels in lipid membranes, suggesting a novel function for this antiapoptotic protein.

Fas activation triggers apoptosis in many cell types. Studies with anti-Fas antibodies have produced conflicting results on Fas signaling, particularly the role of the Bcl-2 family in this process. Comparison between physiological ligand and anti-Fas antibodies revealed that only extensive Fas aggregation, by membrane bound FasL or aggregated soluble FasL consistently triggered apoptosis, whereas antibodies could act as death agonists or antagonists. Studies on Fas signaling in cell lines and primary cells from transgenic mice revealed that FADD/MORT1 and caspase-8 were required for apoptosis. In contrast, Bcl-2 or Bcl-xL did not block FasL-induced apoptosis in lymphocytes or hepatocytes, demonstrating that signaling for cell death induced by Fas and the pathways to apoptosis regulated by the Bcl-2 family are distinct.

Bcl-2, which can both reduce apoptosis and retard cell cycle entry, is thought to have important roles in hematopoiesis. To evaluate the impact of its ubiquitous overexpression within this system, we targeted expression of the human bcl-2 gene in mice by using the promoter of the vav gene, which is active throughout this compartment but rarely outside it. The vav-bcl-2 transgene was expressed in essentially all nucleated cells of hematopoietic tissues but not notably in nonhematopoietic tissues. Presumably because of enhanced cell survival, the mice displayed increases in myeloid cells as well as a marked elevation in B and T lymphocytes. The spleen was enlarged and the lymphoid follicles expanded. Although total thymic cellularity was normal, T cell development was altered: cells at the very immature and most mature stages were increased, whereas those at the intermediate stage were decreased. Unexpectedly, blood platelets were reduced by half, suggesting that their production from megakaryocytes is regulated by the Bcl-2 family. Colony formation by myeloid progenitor cells in vitro remained cytokine dependent, and the frequency of most progenitor and preprogenitor cells was normal. Macrophage progenitors were less frequent and yielded smaller colonies, however, perhaps reflecting inhibitory effects of Bcl-2 on cell cycling in specific lineages. After irradiation or factor deprivation, Bcl-2 markedly enhanced clonogenic survival of all tested progenitor and preprogenitor cells. Thus, Bcl-2 has multiple effects on the hematopoietic system. These mice should help to further clarify the role of apoptosis in the development and homeostasis of this compartment.

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.

Attempts to rescue retinal ganglion cells from retrograde degeneration have had limited success, and the residual function of surviving neurons is not known. Recently, it has been found that axotomized retinal ganglion cells die by apoptotic mechanisms. We have used adult transgenic mice overexpressing the Bcl-2 protein, a powerful inhibitor of apoptosis, as a model for preventing injury-induced cell death in vivo. Several months after axotomy, the majority of retinal ganglion cells survived and exhibited normal visual responses. In control wild-type mice, the vast majority of axotomized retinal ganglion cells degenerated, and the physiological responses were abolished. These results suggest that strategies aimed at increasing Bcl-2 expression, or mimicking its function, might effectively counteract trauma-induced cell death in the central nervous system. Neuronal survival is a necessary condition in the challenge for promoting regeneration and eventually restoring neuronal function.

Deletion of the loop region of Bcl-2 completely blocks paclitaxel-induced apoptosis

At high concentrations, the tubule poison paclitaxel is able to kill cancer cells that express Bcl-2; it inhibits the antiapoptotic activity of Bcl-2 by inducing its phosphorylation. To localize the site on Bcl-2 regulated by phosphorylation, mutant forms of Bcl-2 were constructed. Mutant forms of Bcl-2 with an alteration in serine at amino acid 70 (S70A) or with deletion of a 60-aa loop region between the α1 and α2 helices (Δloop Bcl-2, which also deletes amino acid 70) were unable to be phosphorylated by paclitaxel treatment of MDA-MB-231 cells into which the genes for the mutant proteins were transfected. The Δloop mutant completely inhibited paclitaxel-induced apoptosis. In cells expressing the S70A mutant, paclitaxel induced about one-third the level of apoptosis seen with wild-type Bcl-2. To evaluate the role of mitogen-activated protein kinases (MAPKs) in Bcl-2 phosphorylation, the activation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 was examined. Paclitaxel-induced apoptosis was associated with phosphorylation of Bcl-2 and activation of ERK and JNK MAPKs. If JNK activation was blocked by transfections with either a stress-activated protein kinase kinase dominant-negative (K→R) gene (which prevents the activation of a kinase upstream of JNK) or MAPK phosphatase-1 gene (which dephosphorylates and inactivates JNK), Bcl-2 phosphorylation did not occur, and the cells were not killed by paclitaxel. By contrast, neither an ERK inhibitor (PD098059) nor p38 inhibitors (SB203580 and SB202190) had an effect on Bcl-2 phosphorylation. Thus, our data show that the antiapoptotic effects of Bcl-2 can be overcome by phosphorylation of Ser-70; forms of Bcl-2 lacking the loop region are much more effective at preventing apoptosis than wild-type Bcl-2 because they cannot be phosphorylated. JNK, but not ERK or p38 MAPK, appear to be involved in the phosphorylation of Bcl-2 induced by paclitaxel.

Herpes simplex virus vector-mediated expression of Bcl-2 prevents 6-hydroxydopamine-induced degeneration of neurons in the substantia nigra in vivo

6-Hydroxydopamine (6-OHDA) is widely used to selectively lesion dopaminergic neurons of the substantia nigra (SN) in the creation of animal models of Parkinson’s disease. In vitro, the death of PC-12 cells caused by exposure to 6-OHDA occurs with characteristics consistent with an apoptotic mechanism of cell death. To test the hypothesis that apoptotic pathways are involved in the death of dopaminergic neurons of the SN caused by 6-OHDA, we created a replication-defective genomic herpes simplex virus-based vector containing the coding sequence for the antiapoptotic peptide Bcl-2 under the transcriptional control of the simian cytomegalovirus immediate early promoter. Transfection of primary cortical neurons in culture with the Bcl-2-producing vector protected those cells from naturally occurring cell death over 3 weeks. Injection of the Bcl-2-expressing vector into SN of rats 1 week before injection of 6-OHDA into the ipsilateral striatum increased the survival of neurons in the SN, detected either by retrograde labeling of those cells with fluorogold or by tyrosine hydroxylase immunocytochemistry, by 50%. These results, demonstrating that death of nigral neurons induced by 6-OHDA lesioning may be blocked by the expression of Bcl-2, are consistent with the notion that cell death in this model system is at least in part apoptotic in nature and suggest that a Bcl-2-expressing vector may have therapeutic potential in the treatment of Parkinson’s disease.

A cytomegalovirus-encoded mitochondria-localized inhibitor of apoptosis structurally unrelated to Bcl-2

Human cytomegalovirus (CMV), a herpesvirus that causes congenital disease and opportunistic infections in immunocompromised individuals, encodes functions that facilitate efficient viral propagation by altering host cell behavior. Here we show that CMV blocks apoptosis mediated by death receptors and encodes a mitochondria-localized inhibitor of apoptosis, denoted vMIA, capable of suppressing apoptosis induced by diverse stimuli. vMIA, a product of the viral UL37 gene, inhibits Fas-mediated apoptosis at a point downstream of caspase-8 activation and Bid cleavage but upstream of cytochrome c release, while residing in mitochondria and associating with adenine nucleotide translocator. These functional properties resemble those ascribed to Bcl-2; however, the absence of sequence similarity to Bcl-2 or any other known cell death suppressors suggests that vMIA defines a previously undescribed class of anti-apoptotic proteins.

Comparison of the ion channel characteristics of proapoptotic BAX and antiapoptotic BCL-2

The BCL-2 family of proteins is composed of both pro- and antiapoptotic regulators, although its most critical biochemical functions remain uncertain. The structural similarity between the BCL-XL monomer and several ion-pore-forming bacterial toxins has prompted electrophysiologic studies. Both BAX and BCL-2 insert into KCl-loaded vesicles in a pH-dependent fashion and demonstrate macroscopic ion efflux. Release is maximum at ≈pH 4.0 for both proteins; however, BAX demonstrates a broader pH range of activity. Both purified proteins also insert into planar lipid bilayers at pH 4.0. Single-channel recordings revealed a minimal channel conductance for BAX of 22 pS that evolved to channel currents with at least three subconductance levels. The final, apparently stable BAX channel had a conductance of 0.731 nS at pH 4.0 that changed to 0.329 nS when shifted to pH 7.0 but remained mildly Cl− selective and predominantly open. When BAX-incorporated lipid vesicles were fused to planar lipid bilayers at pH 7.0, a Cl−-selective (PK/PCl = 0.3) 1.5-nS channel displaying mild inward rectification was noted. In contrast, BCL-2 formed mildly K+-selective (PK/PCl = 3.9) channels with a most prominent initial conductance of 80 pS that increased to 1.90 nS. Fusion of BCL-2-incorporated lipid vesicles into planar bilayers at pH 7.0 also revealed mild K+ selectivity (PK/PCl = 2.4) with a maximum conductance of 1.08 nS. BAX and BCL-2 each form channels in artificial membranes that have distinct characteristics including ion selectivity, conductance, voltage dependence, and rectification. Thus, one role of these molecules may include pore activity at selected membrane sites.

Channel formation by antiapoptotic protein Bcl-2

Bcl-2 is the prototypical member of a large family of apoptosis-regulating proteins, consisting of blockers and promoters of cell death. The three-dimensional structure of a Bcl-2 homologue, Bcl-XL, suggests striking similarity to the pore-forming domains of diphtheria toxin and the bacterial colicins, prompting exploration of whether Bcl-2 is capable of forming pores in lipid membranes. Using chloride efflux from KCl-loaded unilamellar lipid vesicles as an assay, purified recombinant Bcl-2 protein exhibited pore-forming activity with properties similar to those of the bacterial toxins, diphtheria toxin, and colicins, i.e., dependence on low pH and acidic lipid membranes. In contrast, a mutant of Bcl-2 lacking the two core hydrophobic α-helices (helices 5 and 6), predicted to be required for membrane insertion and channel formation, produced only nonspecific effects. In planar lipid bilayers, where detection of single channels is possible, Bcl-2 formed discrete ion-conducting, cation-selective channels, whereas the Bcl-2 (Δh5, 6) mutant did not. The most frequent conductance observed (18 ± 2 pS in 0.5 M KCl at pH 7.4) is consistent with a four-helix bundle structure arising from Bcl-2 dimers. However, larger channel conductances (41 ± 2 pS and 90 ± 10 pS) also were detected with progressively lower occurrence, implying the step-wise formation of larger oligomers of Bcl-2 in membranes. These findings thus provide biophysical evidence that Bcl-2 forms channels in lipid membranes, suggesting a novel function for this antiapoptotic protein.

Activation of Fas by FasL induces apoptosis by a mechanism that cannot be blocked by Bcl-2 or Bcl-xL

Fas activation triggers apoptosis in many cell types. Studies with anti-Fas antibodies have produced conflicting results on Fas signaling, particularly the role of the Bcl-2 family in this process. Comparison between physiological ligand and anti-Fas antibodies revealed that only extensive Fas aggregation, by membrane bound FasL or aggregated soluble FasL consistently triggered apoptosis, whereas antibodies could act as death agonists or antagonists. Studies on Fas signaling in cell lines and primary cells from transgenic mice revealed that FADD/MORT1 and caspase-8 were required for apoptosis. In contrast, Bcl-2 or Bcl-xL did not block FasL-induced apoptosis in lymphocytes or hepatocytes, demonstrating that signaling for cell death induced by Fas and the pathways to apoptosis regulated by the Bcl-2 family are distinct.

Constitutive Bcl-2 expression throughout the hematopoietic compartment affects multiple lineages and enhances progenitor cell survival

Bcl-2, which can both reduce apoptosis and retard cell cycle entry, is thought to have important roles in hematopoiesis. To evaluate the impact of its ubiquitous overexpression within this system, we targeted expression of the human bcl-2 gene in mice by using the promoter of the vav gene, which is active throughout this compartment but rarely outside it. The vav-bcl-2 transgene was expressed in essentially all nucleated cells of hematopoietic tissues but not notably in nonhematopoietic tissues. Presumably because of enhanced cell survival, the mice displayed increases in myeloid cells as well as a marked elevation in B and T lymphocytes. The spleen was enlarged and the lymphoid follicles expanded. Although total thymic cellularity was normal, T cell development was altered: cells at the very immature and most mature stages were increased, whereas those at the intermediate stage were decreased. Unexpectedly, blood platelets were reduced by half, suggesting that their production from megakaryocytes is regulated by the Bcl-2 family. Colony formation by myeloid progenitor cells in vitro remained cytokine dependent, and the frequency of most progenitor and preprogenitor cells was normal. Macrophage progenitors were less frequent and yielded smaller colonies, however, perhaps reflecting inhibitory effects of Bcl-2 on cell cycling in specific lineages. After irradiation or factor deprivation, Bcl-2 markedly enhanced clonogenic survival of all tested progenitor and preprogenitor cells. Thus, Bcl-2 has multiple effects on the hematopoietic system. These mice should help to further clarify the role of apoptosis in the development and homeostasis of this compartment.

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.

The visual response of retinal ganglion cells is not altered by optic nerve transection in transgenic mice overexpressing Bcl-2

Attempts to rescue retinal ganglion cells from retrograde degeneration have had limited success, and the residual function of surviving neurons is not known. Recently, it has been found that axotomized retinal ganglion cells die by apoptotic mechanisms. We have used adult transgenic mice overexpressing the Bcl-2 protein, a powerful inhibitor of apoptosis, as a model for preventing injury-induced cell death in vivo. Several months after axotomy, the majority of retinal ganglion cells survived and exhibited normal visual responses. In control wild-type mice, the vast majority of axotomized retinal ganglion cells degenerated, and the physiological responses were abolished. These results suggest that strategies aimed at increasing Bcl-2 expression, or mimicking its function, might effectively counteract trauma-induced cell death in the central nervous system. Neuronal survival is a necessary condition in the challenge for promoting regeneration and eventually restoring neuronal function.