Preparation and characterization of polyester resin-layered silicate containing reactive groups

Polymer nanocomposites, specifically nanoclay-reinforced polymers, have attracted great interest as matrix materials for high temperature composite applications. Nanocomposites require relatively low dispersant loads to achieve significant property enhancements. These enhancements are mainly a consequence of the interfacial effects that result from dispersing the silicate nanolayers in the polymer matrix and the high in-plane strength, stiffness and aspect ratio of the lamellar nanoparticles. The montmorillonite (MMT) clay, modified with organic onium ions with long alkyl chains as Cloisites, has been widely used to obtain nanocomposites. The presence of reactive groups in organic onium ions can form chemical bonds with the polymer matrix which favours a very high exfoliation degree of the clay platelets in the nanocomposite (1,2)

Connexin-43 prevents hematopoietic stem cell senescence through transfer of reactive oxygen species to bone marrow stromal cells

Hematopoietic stem cell (HSC) aging has become a concern in chemotherapy of older patients. Humoral and paracrine signals from the bone marrow (BM) hematopoietic microenvironment (HM) control HSC activity during regenerative hematopoiesis. Connexin-43 (Cx43), a connexin constituent of gap junctions (GJs) is expressed in HSCs, down-regulated during differentiation, and postulated to be a self-renewal gene. Our studies, however, reveal that hematopoietic-specific Cx43 deficiency does not result in significant long-term competitive repopulation deficiency. Instead, hematopoietic Cx43 (H-Cx43) deficiency delays hematopoietic recovery after myeloablation with 5-fluorouracil (5-FU). 5-FU-treated H-Cx43-deficient HSC and progenitors (HSC/P) cells display decreased survival and fail to enter the cell cycle to proliferate. Cell cycle quiescence is associated with down-regulation of cyclin D1, up-regulation of the cyclin-dependent kinase inhibitors, p21cip1. and p16INK4a, and Forkhead transcriptional factor 1 (Foxo1), and activation of p38 mitogen-activated protein kinase (MAPK), indicating that H-Cx43-deficient HSCs are prone to senescence. The mechanism of increased senescence in H-Cx43-deficient HSC/P cells depends on their inability to transfer reactive oxygen species (ROS) to the HM, leading to accumulation of ROS within HSCs. In vivo antioxidant administration prevents the defective hematopoietic regeneration, as well as exogenous expression of Cx43 in HSC/P cells. Furthermore, ROS transfer from HSC/P cells to BM stromal cells is also rescued by reexpression of Cx43 in HSC/P. Finally, the deficiency of Cx43 in the HM phenocopies the hematopoietic defect in vivo. These results indicate that Cx43 exerts a protective role and regulates the HSC/P ROS content through ROS transfer to the HM, resulting in HSC protection during stress hematopoietic regeneration.

Dielectric behaviour of Hf-doped CaCu3Ti4O12 ceramics obtained by conventional synthesis and reactive sintering

CaCu3(Ti4xHfx)O12 ceramics (JC = 0.04, 0.1 and 0.2) were prepared by conventional synthesis (CS) and through reactive sintering (RS), in which synthesis and sintering of the material take place in one single step. The microstructure and the dielectric properties of Hf-doped CCTO (CCTOHf) have been studied by XRD, FE-SEM, AFM, Raman and impedance spectroscopy (IS) in order to correlate the structure, microstructure and the electrical properties. Samples prepared by reactive sintering show slightly higher dielectric constant than those prepared by conventional synthesis in the same way than the pure CCTO. Dielectric constant and dielectric losses decrease slightly increasing Hf content. For CCTOHf ceramics with x> 0.04 for CS and x> 0.1 for RS, a secondary phase HfTi04 appears. As expected, the reactive sintering processing method allows a higher incorporation of Hf in the CCTO lattice than the conventional synthesis one.

Deep reactive ion etching of auxetic structures: present capabilities and challenges

Auxetic materials (or metamaterials) have negative Poisson ratios (NPR) and display the unexpected properties of lateral expansion when stretched, and equal and opposing densification when compressed. Such auxetic materials are being used more frequently in the development of novel products, especially in the fields of intelligent expandable actuators, shape-morphing structures, and minimally invasive implantable devices. Although several micromanufacturing technologies have already been applied to the development of auxetic materials and devices, additional precision is needed to take full advantage of their special mechanical properties. In this study, we present a very promising approach for the development of auxetic materials and devices based on the use of deep reactive ion etching (DRIE). The process stands out for its precision and its potential applications to mass production. To our knowledge, it represents the first time this technology has been applied to the manufacture of auxetic materials with nanometric details. We take into account the present capabilities and challenges linked to the use of DRIE in the development of auxetic materials and auxetic-based devices.

Proactive and reactive thermal aware optimization techniques to minimize the environmental impact of data centers

Data centers are easily found in every sector of the worldwide economy. They are composed of thousands of servers, serving millions of users globally and 24-7. In the last years, e-Science applications such e-Health or Smart Cities have experienced a significant development. The need to deal efficiently with the computational needs of next-generation applications together with the increasing demand for higher resources in traditional applications has facilitated the rapid proliferation and growing of Data Centers. A drawback to this capacity growth has been the rapid increase of the energy consumption of these facilities. In 2010, data center electricity represented 1.3% of all the electricity use in the world. In year 2012 alone, global data center power demand grep 63% to 38GW. A further rise of 17% to 43GW was estimated in 2013. Moreover, Data Centers are responsible for more than 2% of total carbon dioxide emissions.

New insights into the regulation of NADPH oxidase dependent reactive oxygen species signaling during the plant immune response

Las NADPH oxidasas de plantas, denominadas “respiratory burst oxidase homologues” (RBOHs), producen especies reactivas del oxígeno (ROS) que median un amplio rango de funciones. En la célula vegetal, el ajuste preciso de la producción de ROS aporta la especificidad de señal para generar una respuesta apropiada ante las amenazas ambientales. RbohD y RbohF, dos de los diez genes Rboh de Arabidopsis, son pleiotrópicos y median diversos procesos fisiológicos en respuesta a patógenos. El control espacio-temporal de la expresión de los genes RbohD y RbohF podría ser un aspecto crítico para determinar la multiplicidad de funciones de estas oxidasas. Por ello, generamos líneas transgénicas de Arabidopsis con fusiones de los promoters de RbohD y RbohF a los genes delatores de la B-glucuronidasa y la luciferasa. Estas líneas fueron empleadas para revelar el patrón de expresión diferencial de RbohD y RbohF durante la respuesta inmune de Arabidopsis a la bacteria patógena Pseudomonas syringae pv. tomato DC3000, el hongo necrótrofo Plectosphaerella cucumerina y en respuesta a señales relacionadas con la respuesta inmune. Nuestros experimentos revelan un patrón de expresión diferencial de los promotores de RbohD y RbohF durante el desarrollo de la planta y en la respuesta inmune de Arabidopsis. Además hemos puesto de manifiesto que existe una correlación entre el nivel de actividad de los promotores de RbohD y RbohF con la acumulación de ROS y el nivel de muerte celular en respuesta a patógenos. La expression de RbohD y RbohF también es modulada de manera diferencial en respuesta a patrones moleculares asociados a patógenos (PAMPs) y por ácido abscísico (ABA). Cabe destacar que, mediante una estrategia de intercambio de promotores, hemos revelado que la región promotora de RbohD, es necesaria para dirigir la producción de ROS en respuesta a P. cucumerina. Adicionalmente, la activación del promotor de RbohD en respuesta al aislado de P. cucumerina no adaptado a Arabidopsis 2127, nos llevó a realizar ensayos de susceptibilidad con el doble mutante rbohD rbohF que han revelado un papel desconocido de estas oxidasas en resistencia no-huesped. La interacción entre la señalización dependiente de las RBOHs y otros componentes de la respuesta inmune de plantas podría explicar también las distintas funciones que median estas oxidasas en relación con la respuesta inmune. Entre la gran cantidad de señales coordinadas con la actividad de las RBOHs, existen evidencias genéticas y farmacológicas que indican que las proteínas G heterotriméricas están implicadas en algunas de las rutas de señalización mediadas por ROS derivadas de los RBOHs en respuesta a señales ambientales. Por ello hemos estudiado la relación entre estas RBOH-NADPH oxidasas y AGB1, la subunidad β de las proteínas G heterotriméricas en la respuesta inmune de Arabidopsis. Análisis de epistasis indican que las proteínas G heterotriméricas están implicadas en distintas rutas de señalización en defensa mediadas por las RBOHs. Nuestros resultados ilustran la relación compleja entre la señalización mediada por las RBOHs y las proteínas G heterotriméricas, que varía en función de la interacción planta-patógeno analizada. Además, hemos explorado la posible asociación entre AGB1 con RBOHD y RBOHF en eventos tempranos de la respuesta immune. Cabe señalar que experimentos de coímmunoprecipitación apuntan a una posible asociación entre AGB1 y la kinasa citoplasmática reguladora de RBOHD, BIK1. Esto indica un posible mecanismo de control de la función de esta NADPH oxidase por AGB1. En conjunto, estos datos aportan nuevas perspectivas sobre cómo, a través del control transcripcional o mediante la interacción con las proteínas G heterotriméricas, las NADPH oxidases de plantas median la producción de ROS y la señalización por ROS en la respuesta inmune. Nuestro trabajo ejemplifica cómo la regulación diferencial de dos miembros de una familia multigénica, les permite realizar distintas funciones fisiológicas especializadas usando un mismo mecanismo enzimático. ABSTRACT The plant NADPH oxidases, termed respiratory burst oxidase homologues (RBOHs), produce reactive oxygen species (ROS) which mediate a wide range of functions. Fine tuning this ROS production provides the signaling specificity to the plant cell to produce the appropriate response to environmental threats. RbohD and RbohF, two of the ten Rboh genes present in Arabidopsis, are pleiotropic and mediate diverse physiological processes in response to pathogens. One aspect that may prove critical to determine the multiplicity of functions of RbohD and RbohF is the spatio-temporal control of their gene expression. Thus, we generated Arabidopsis transgenic lines with RbohD- and RbohF-promoter fusions to the β-glucuronidase and the luciferase reporter genes. These transgenics were employed to reveal RbohD and RbohF promoter activity during Arabidopsis immune response to the pathogenic bacterium Pseudomonas syringae pv tomato DC3000, the necrotrophic fungus Plectosphaerella cucumerina and in response to immunity-related cues. Our experiments revealed a differential expression pattern of RbohD and RbohF throughout plant development and during Arabidopsis immune response. Moreover, we observed a correlation between the level of RbohD and RbohF promoter activity, the accumulation of ROS and the amount of cell death in response to pathogens. RbohD and RbohF gene expression was also differentially modulated by pathogen associated molecular patterns and abscisic acid. Interestingly, a promoter-swap strategy revealed the requirement for the promoter region of RbohD to drive the production of ROS in response to P. cucumerina. Additionally, since the RbohD promoter was activated during Arabidopsis interaction with a non-adapted P. cucumerina isolate 2127, we performed susceptibility tests to this fungal isolate that uncovered a new role of these oxidases on non-host resistance. The interplay between RBOH-dependent signaling with other components of the plant immune response might also explain the different immunity-related functions mediated by these oxidases. Among the plethora of signals coordinated with RBOH activity, pharmacological and genetic evidence indicates that heterotrimeric G proteins are involved in some of the signaling pathways mediated by RBOH–derived ROS in response to environmental cues. Therefore, we analysed the interplay between these RBOH-NADPH oxidases and AGB1, the Arabidopsis β-subunit of heterotrimeric G proteins during Arabidopsis immune response. We carried out epistasis studies that allowed us to test the implication of AGB1 in different RBOH-mediated defense signaling pathways. Our results illustrate the complex relationship between RBOH and heterotrimeric G proteins signaling, that varies depending on the type of plant-pathogen interaction. Furthermore, we tested the potential association between AGB1 with RBOHD and RBOHF during early immunity. Interestingly, our co-immunoprecipitation experiments point towards an association of AGB1 and the RBOHD regulatory kinase BIK1, thus providing a putative mechanism in the control of the NADPH oxidase function by AGB1. Taken all together, these studies provide further insights into the role that transcriptional control or the interaction with heterotrimeric G-proteins have on RBOH-NADPH oxidase-dependent ROS production and signaling in immunity. Our work exemplifies how, through a differential regulation, two members of a multigenic family achieve specialized physiological functions using a common enzymatic mechanism.

Proactive and reactive thermal aware optimization techniques to minimize the environmental impact of data centers

Los Centros de Datos se encuentran actualmente en cualquier sector de la economía mundial. Están compuestos por miles de servidores, dando servicio a los usuarios de forma global, las 24 horas del día y los 365 días del año. Durante los últimos años, las aplicaciones del ámbito de la e-Ciencia, como la e-Salud o las Ciudades Inteligentes han experimentado un desarrollo muy significativo. La necesidad de manejar de forma eficiente las necesidades de cómputo de aplicaciones de nueva generación, junto con la creciente demanda de recursos en aplicaciones tradicionales, han facilitado el rápido crecimiento y la proliferación de los Centros de Datos. El principal inconveniente de este aumento de capacidad ha sido el rápido y dramático incremento del consumo energético de estas infraestructuras. En 2010, la factura eléctrica de los Centros de Datos representaba el 1.3% del consumo eléctrico mundial. Sólo en el año 2012, el consumo de potencia de los Centros de Datos creció un 63%, alcanzando los 38GW. En 2013 se estimó un crecimiento de otro 17%, hasta llegar a los 43GW. Además, los Centros de Datos son responsables de más del 2% del total de emisiones de dióxido de carbono a la atmósfera. Esta tesis doctoral se enfrenta al problema energético proponiendo técnicas proactivas y reactivas conscientes de la temperatura y de la energía, que contribuyen a tener Centros de Datos más eficientes. Este trabajo desarrolla modelos de energía y utiliza el conocimiento sobre la demanda energética de la carga de trabajo a ejecutar y de los recursos de computación y refrigeración del Centro de Datos para optimizar el consumo. Además, los Centros de Datos son considerados como un elemento crucial dentro del marco de la aplicación ejecutada, optimizando no sólo el consumo del Centro de Datos sino el consumo energético global de la aplicación. Los principales componentes del consumo en los Centros de Datos son la potencia de computación utilizada por los equipos de IT, y la refrigeración necesaria para mantener los servidores dentro de un rango de temperatura de trabajo que asegure su correcto funcionamiento. Debido a la relación cúbica entre la velocidad de los ventiladores y el consumo de los mismos, las soluciones basadas en el sobre-aprovisionamiento de aire frío al servidor generalmente tienen como resultado ineficiencias energéticas. Por otro lado, temperaturas más elevadas en el procesador llevan a un consumo de fugas mayor, debido a la relación exponencial del consumo de fugas con la temperatura. Además, las características de la carga de trabajo y las políticas de asignación de recursos tienen un impacto importante en los balances entre corriente de fugas y consumo de refrigeración. La primera gran contribución de este trabajo es el desarrollo de modelos de potencia y temperatura que permiten describes estos balances entre corriente de fugas y refrigeración; así como la propuesta de estrategias para minimizar el consumo del servidor por medio de la asignación conjunta de refrigeración y carga desde una perspectiva multivariable. Cuando escalamos a nivel del Centro de Datos, observamos un comportamiento similar en términos del balance entre corrientes de fugas y refrigeración. Conforme aumenta la temperatura de la sala, mejora la eficiencia de la refrigeración. Sin embargo, este incremente de la temperatura de sala provoca un aumento en la temperatura de la CPU y, por tanto, también del consumo de fugas. Además, la dinámica de la sala tiene un comportamiento muy desigual, no equilibrado, debido a la asignación de carga y a la heterogeneidad en el equipamiento de IT. La segunda contribución de esta tesis es la propuesta de técnicas de asigación conscientes de la temperatura y heterogeneidad que permiten optimizar conjuntamente la asignación de tareas y refrigeración a los servidores. Estas estrategias necesitan estar respaldadas por modelos flexibles, que puedan trabajar en tiempo real, para describir el sistema desde un nivel de abstracción alto. Dentro del ámbito de las aplicaciones de nueva generación, las decisiones tomadas en el nivel de aplicación pueden tener un impacto dramático en el consumo energético de niveles de abstracción menores, como por ejemplo, en el Centro de Datos. Es importante considerar las relaciones entre todos los agentes computacionales implicados en el problema, de forma que puedan cooperar para conseguir el objetivo común de reducir el coste energético global del sistema. La tercera contribución de esta tesis es el desarrollo de optimizaciones energéticas para la aplicación global por medio de la evaluación de los costes de ejecutar parte del procesado necesario en otros niveles de abstracción, que van desde los nodos hasta el Centro de Datos, por medio de técnicas de balanceo de carga. Como resumen, el trabajo presentado en esta tesis lleva a cabo contribuciones en el modelado y optimización consciente del consumo por fugas y la refrigeración de servidores; el modelado de los Centros de Datos y el desarrollo de políticas de asignación conscientes de la heterogeneidad; y desarrolla mecanismos para la optimización energética de aplicaciones de nueva generación desde varios niveles de abstracción. ABSTRACT Data centers are easily found in every sector of the worldwide economy. They consist of tens of thousands of servers, serving millions of users globally and 24-7. In the last years, e-Science applications such e-Health or Smart Cities have experienced a significant development. The need to deal efficiently with the computational needs of next-generation applications together with the increasing demand for higher resources in traditional applications has facilitated the rapid proliferation and growing of data centers. A drawback to this capacity growth has been the rapid increase of the energy consumption of these facilities. In 2010, data center electricity represented 1.3% of all the electricity use in the world. In year 2012 alone, global data center power demand grew 63% to 38GW. A further rise of 17% to 43GW was estimated in 2013. Moreover, data centers are responsible for more than 2% of total carbon dioxide emissions. This PhD Thesis addresses the energy challenge by proposing proactive and reactive thermal and energy-aware optimization techniques that contribute to place data centers on a more scalable curve. This work develops energy models and uses the knowledge about the energy demand of the workload to be executed and the computational and cooling resources available at data center to optimize energy consumption. Moreover, data centers are considered as a crucial element within their application framework, optimizing not only the energy consumption of the facility, but the global energy consumption of the application. The main contributors to the energy consumption in a data center are the computing power drawn by IT equipment and the cooling power needed to keep the servers within a certain temperature range that ensures safe operation. Because of the cubic relation of fan power with fan speed, solutions based on over-provisioning cold air into the server usually lead to inefficiencies. On the other hand, higher chip temperatures lead to higher leakage power because of the exponential dependence of leakage on temperature. Moreover, workload characteristics as well as allocation policies also have an important impact on the leakage-cooling tradeoffs. The first key contribution of this work is the development of power and temperature models that accurately describe the leakage-cooling tradeoffs at the server level, and the proposal of strategies to minimize server energy via joint cooling and workload management from a multivariate perspective. When scaling to the data center level, a similar behavior in terms of leakage-temperature tradeoffs can be observed. As room temperature raises, the efficiency of data room cooling units improves. However, as we increase room temperature, CPU temperature raises and so does leakage power. Moreover, the thermal dynamics of a data room exhibit unbalanced patterns due to both the workload allocation and the heterogeneity of computing equipment. The second main contribution is the proposal of thermal- and heterogeneity-aware workload management techniques that jointly optimize the allocation of computation and cooling to servers. These strategies need to be backed up by flexible room level models, able to work on runtime, that describe the system from a high level perspective. Within the framework of next-generation applications, decisions taken at this scope can have a dramatical impact on the energy consumption of lower abstraction levels, i.e. the data center facility. It is important to consider the relationships between all the computational agents involved in the problem, so that they can cooperate to achieve the common goal of reducing energy in the overall system. The third main contribution is the energy optimization of the overall application by evaluating the energy costs of performing part of the processing in any of the different abstraction layers, from the node to the data center, via workload management and off-loading techniques. In summary, the work presented in this PhD Thesis, makes contributions on leakage and cooling aware server modeling and optimization, data center thermal modeling and heterogeneityaware data center resource allocation, and develops mechanisms for the energy optimization for next-generation applications from a multi-layer perspective.

Synthetic amino acid copolymers that bind to HLA-DR proteins and inhibit type II collagen-reactive T cell clones

Copolymer 1 [poly(Y,E,A,K)] is a random synthetic amino acid copolymer of l-tyrosine, l-glutamic acid, l-alanine, and l-lysine that is effective both in suppression of experimental allergic encephalomyelitis and in the treatment of relapsing forms of multiple sclerosis. Copolymer 1 binds promiscuously and very efficiently to purified HLA-DR molecules within the peptide-binding groove. In the present study, YEAK and YEAK-related copolymers and type II collagen (CII) peptide 261–273, a candidate autoantigen in rheumatoid arthritis (RA), competed for binding to RA-associated HLA-DR molecules encoded by DRB1*0101 and DRB1*0401. Moreover, these copolymers (particularly YEAK, YAK, and YEK) inhibited the response of DR1- and DR4-restricted T cell clones to the CII epitope 261–273 by >50%. This direct evidence both for competitive interactions of these copolymers and CII peptide with RA-associated HLA-DR molecules and for inhibition of CII-specific T cell responses suggests that these compounds should be evaluated in animal models for rheumatoid arthritis.

Regulation of the human ether-a-gogo related gene (HERG) K+ channels by reactive oxygen species

Human ether-a-gogo related gene (HERG) K+ channels are key elements in the control of cell excitability in both the cardiovascular and the central nervous systems. For this reason, the possible modulation by reactive oxygen species (ROS) of HERG and other cloned K+ channels expressed in Xenopus oocytes has been explored in the present study. Exposure of Xenopus oocytes to an extracellular solution containing FeSO4 (25–100 μM) and ascorbic acid (50–200 μM) (Fe/Asc) increased both malondialdehyde content and 2′,7′-dichlorofluorescin fluorescence, two indexes of ROS production. Oocyte perfusion with Fe/Asc caused a 50% increase of the outward K+ currents carried by HERG channels, whereas inward currents were not modified. This ROS-induced increase in HERG outward K+ currents was due to a depolarizing shift of the voltage-dependence of channel inactivation, with no change in channel activation. No effect of Fe/Asc was observed on the expressed K+ currents carried by other K+ channels such as bEAG, rDRK1, and mIRK1. Fe/Asc-induced stimulation of HERG outward currents was completely prevented by perfusion of the oocytes with a ROS scavenger mixture (containing 1,000 units/ml catalase, 200 ng/ml superoxide dismutase, and 2 mM mannitol). Furthermore, the scavenger mixture also was able to reduce HERG outward currents in resting conditions by 30%, an effect mimicked by catalase alone. In conclusion, the present results seem to suggest that changes in ROS production can specifically influence K+ currents carried by the HERG channels.

Homodimerization of tumor-reactive monoclonal antibodies markedly increases their ability to induce growth arrest or apoptosis of tumor cells

Monoclonal antibodies (mAbs) that exert antitumor activity can do so by virtue of their effector function and/or their ability to signal growth arrest or cell death. In this study, we demonstrate that mAbs which have little or no signaling activity—i.e., anti-CD19, CD20, CD21, CD22 and Her-2—can become potent antitumor agents when they are converted into IgG–IgG homodimers. The homodimers exert antigrowth activity by signaling G0/G1 arrest or apoptosis, depending upon which cell surface molecule they bind. This activity is specific and, in the case of the anti-CD19 mAb, did not require an Fc portion. These results offer the possibility that homodimers of other tumor-reactive mAbs which have little antitumor activity as monomers might be potent, antitumor agents.

Transgenic tobacco plants with reduced capability to detoxify reactive oxygen intermediates are hyperresponsive to pathogen infection

Reactive oxygen intermediates (ROI) play a critical role in the defense of plants against invading pathogens. Produced during the “oxidative burst,” they are thought to activate programmed cell death (PCD) and induce antimicrobial defenses such as pathogenesis-related proteins. It was shown recently that during the interaction of plants with pathogens, the expression of ROI-detoxifying enzymes such as ascorbate peroxidase (APX) and catalase (CAT) is suppressed. It was suggested that this suppression, occurring upon pathogen recognition and coinciding with an enhanced rate of ROI production, plays a key role in elevating cellular ROI levels, thereby potentiating the induction of PCD and other defenses. To examine the relationship between the suppression of antioxidative mechanisms and the induction of PCD and other defenses during pathogen attack, we studied the interaction between transgenic antisense tobacco plants with reduced APX or CAT and a bacterial pathogen that triggers the hypersensitive response. Transgenic plants with reduced capability to detoxify ROI (i.e., antisense APX or CAT) were found to be hyperresponsive to pathogen attack. They activated PCD in response to low amounts of pathogens that did not trigger the activation of PCD in control plants. Our findings support the hypothesis that suppression of ROI-scavenging enzymes during the hypersensitive response plays an important role in enhancing pathogen-induced PCD.

Models of immune memory: On the role of cross-reactive stimulation, competition, and homeostasis in maintaining immune memory

There has been much debate on the contribution of processes such as the persistence of antigens, cross-reactive stimulation, homeostasis, competition between different lineages of lymphocytes, and the rate of cell turnover on the duration of immune memory and the maintenance of the immune repertoire. We use simple mathematical models to investigate the contributions of these various processes to the longevity of immune memory (defined as the rate of decline of the population of antigen-specific memory cells). The models we develop incorporate a large repertoire of immune cells, each lineage having distinct antigenic specificities, and describe the dynamics of the individual lineages and total population of cells. Our results suggest that, if homeostatic control regulates the total population of memory cells, then, for a wide range of parameters, immune memory will be long-lived in the absence of persistent antigen (T1/2 > 1 year). We also show that the longevity of memory in this situation will be insensitive to the relative rates of cross-reactive stimulation, the rate of turnover of immune cells, and the functional form of the term for the maintenance of homeostasis.

The reactive site loop of the serpin SCCA1 is essential for cysteine proteinase inhibition

The high-molecular-weight serine proteinase inhibitors (serpins) are restricted, generally, to inhibiting proteinases of the serine mechanistic class. However, the viral serpin, cytokine response modifier A, and the human serpins, antichymotrypsin and squamous cell carcinoma antigen 1 (SCCA1), inhibit different members of the cysteine proteinase class. Although serpins employ a mobile reactive site loop (RSL) to bait and trap their target serine proteinases, the mechanism by which they inactivate cysteine proteinases is unknown. Our previous studies suggest that SCCA1 inhibits papain-like cysteine proteinases in a manner similar to that observed for serpin–serine proteinase interactions. However, we could not preclude the possibility of an inhibitory mechanism that did not require the serpin RSL. To test this possibility, we employed site-directed mutagenesis to alter the different residues within the RSL. Mutations to either the hinge or the variable region of the RSL abolished inhibitory activity. Moreover, RSL swaps between SCCA1 and the nearly identical serpin, SCCA2 (an inhibitor of chymotrypsin-like serine proteinases), reversed their target specificities. Thus, there were no unique motifs within the framework of SCCA1 that independently accounted for cysteine proteinase inhibitory activity. Collectively, these data suggested that the sequence and mobility of the RSL of SCCA1 are essential for cysteine proteinase inhibition and that serpins are likely to utilize a common RSL-dependent mechanism to inhibit both serine and cysteine proteinases.

The Yeast Saccharomyces cerevisiae Contains Two Glutaredoxin Genes That Are Required for Protection against Reactive Oxygen Species

Glutaredoxins are small heat-stable proteins that act as glutathione-dependent disulfide oxidoreductases. Two genes, designated GRX1 and GRX2, which share 40–52% identity and 61–76% similarity with glutaredoxins from bacterial and mammalian species, were identified in the yeast Saccharomyces cerevisiae. Strains deleted for both GRX1 and GRX2 were viable but lacked heat-stable oxidoreductase activity using β-hydroxyethylene disulfide as a substrate. Surprisingly, despite the high degree of homology between Grx1 and Grx2 (64% identity), the grx1 mutant was unaffected in oxidoreductase activity, whereas the grx2 mutant displayed only 20% of the wild-type activity, indicating that Grx2 accounted for the majority of this activity in vivo. Expression analysis indicated that this difference in activity did not arise as a result of differential expression of GRX1 and GRX2. In addition, a grx1 mutant was sensitive to oxidative stress induced by the superoxide anion, whereas a strain that lacked GRX2 was sensitive to hydrogen peroxide. Sensitivity to oxidative stress was not attributable to altered glutathione metabolism or cellular redox state, which did not vary between these strains. The expression of both genes was similarly elevated under various stress conditions, including oxidative, osmotic, heat, and stationary phase growth. Thus, Grx1 and Grx2 function differently in the cell, and we suggest that glutaredoxins may act as one of the primary defenses against mixed disulfides formed following oxidative damage to proteins.

Inactive conformation of the serpin α1-antichymotrypsin indicates two-stage insertion of the reactive loop: Implications for inhibitory function and conformational disease

The serpins are a family of proteinase inhibitors that play a central role in the control of proteolytic cascades. Their inhibitory mechanism depends on the intramolecular insertion of the reactive loop into β-sheet A after cleavage by the target proteinase. Point mutations within the protein can allow aberrant conformational transitions characterized by β-strand exchange between the reactive loop of one molecule and β-sheet A of another. These loop-sheet polymers result in diseases as varied as cirrhosis, emphysema, angio-oedema, and thrombosis, and we recently have shown that they underlie an early-onset dementia. We report here the biochemical characteristics and crystal structure of a naturally occurring variant (Leu-55–Pro) of the plasma serpin α1-antichymotrypsin trapped as an inactive intermediate. The structure demonstrates a serpin configuration with partial insertion of the reactive loop into β-sheet A. The lower part of the sheet is filled by the last turn of F-helix and the loop that links it to s3A. This conformation matches that of proposed intermediates on the pathway to complex and polymer formation in the serpins. In particular, this intermediate, along with the latent and polymerized conformations, explains the loss of activity of plasma α1-antichymotrypsin associated with chronic obstructive pulmonary disease in patients with the Leu-55–Pro mutation.