Lindane induced cellular dysfunction in MDCK cells: The role of the peripheral benzodiazepine receptor

The central nervous system GABAA/Benzodiazepine (GABAA/BZD) receptors are targets for many pharmaceutical agents and several classes of pesticides. Lindane is an organochlorine pesticide, although banned from production in the U.S. since 1977, still imported for use as an insecticide and pharmaceutically to control ectoparasites (ATSDR, 1994). Lindane functions as a GABA/BZD receptor antagonist within the central nervous system (CNS). Outside of the CNS, peripheral BZD receptors have been localized to the distal tubule of the kidney. Previous research in our laboratory has shown that incubation of renal cortical slices with lindane can produce an increase in kallikrein leakage, suggesting a distal tubular effect. In this study, Madin Darby Canine Kidney (MDCK) cells were used as an in vitro system to assess the toxicity of lindane. This purpose of this study was to determine if interactions between a renal distal tubular BZD-like receptor and lindane could lead to perturbations in renal distal cellular chloride (Cl−) transport and mitochondrial dysfunction and ultimately, cellular death. ^ Pertubations in renal chloride transport were measured indirectly by determining if lindane altered cell function responsiveness following osmotic stress. MDCK cells pre-treated with lindane and then subjected to osmotic stress remained swollen for up to 12 hours post-stress. Lindane-induced dysfunction was assessed through stress protein induction measured by Western Blot analysis. Lindane pretreatment delayed Heat Shock Protein 72 (HSP72) induction by 36 hours in osmotically stressed cells. Pretreatment with 1 × 10 −5 M LIN followed by osmotic stress elevated p38 and Stress Activated Protein Kinase (SAPK/JNK) at 15 minutes which declined at 30 minutes. Lindane appeared to have no effect on Endoplasmic Reticulum Related Kinase (ERK) induction. Lindane did not effect osmotically stressed LLC-PKI cells, a control cell line. ^ Lindane-treated MDCK cells did not exhibit necrosis. Instead, apoptosis was observed in lindane-treated MDCK cells in both time- and dose-dependent manners. LLC-PKI cells were not affected by LIN treatment. ^ To better understand the mechanism of lindane-induced apoptosis, mitochondrial function was measured. No changes in cytochrome c release or mitochondrial membrane potential were observed suggesting the mitochondrial pathway was not involved in lindane-induced apoptosis. ^ Further research will need to be conducted to determine the mechanism of lindane-induced adverse cellular effects. ^

The cGMP -PKG pathway is critical for inducing long-term sensitization of identified nociceptive sensory neurons

In various species, peripheral injury produces long-lasting sensitization of central and peripheral neurons representing the affected area. In Aplysia, memory-like traces (lasting days or weeks) of noxious peripheral stimulation include enhancement of central synaptic transmission and enhanced excitability of the central soma and peripheral branches of nociceptive sensory neurons. An important role for the cAMP-PKA-CREB pathway in consolidating long-term memory and inducing transcription-dependent synaptic potentiation has also been indicated by studies in rodents and Drosophila. ^ Much less attention has been paid to the cGMP-PKG pathway for transcription-dependent plasticity. Nevertheless, the cGMP-PKG pathway has been implicated in activity-dependent neural alterations lasting hours, and may trigger some forms of persistent pain. Recent evidence indicates PKG can regulate gene expression in the brain and several properties make it an attractive candidate for inducing long-term memories. ^ This dissertation reports that brief, noxious stimulation of a behaving, semi-intact preparation from mollusc, Aplysia californica, produces transcription-dependent, long-term hyperexcitability (LTH) of nociceptive sensory neurons that requires a nitric oxide (NO)-cGMP-protein kinase G (PKG) pathway and which lasts for at least 24 hours. Intracellular injection of cGMP is sufficient to induce LTH. Similarly, body wall injury induces LTH which can be blocked with specific inhibitors of the NO-cGMP-PKG pathway such as L-NMMA, ODQ, Rp-8-cGMPS, PKI-G and KT5823 by isolated perfusion of pleural ganglion sensory cells in or directly by intracellular injection. In contrast, specific inhibitors of the cAMP-PKA pathway (Rp-8-cAMPS, PKI-A and H-89) failed to block injury-induced LTH. Interestingly, co-injection of the cAMP-responsive element (CRE) blocked the induction of both cAMP and injury-induced LTH, but not cGMP-induced LTH. Furthermore, co-injection of cAMP and cGMP with the Ca2+ buffer BAPTA in reduced Ca2+ seawater blocked cAMP-, but not cGMP-induced LTH. These findings demonstrate that the NO-cGMP-PKG pathway and at least one other pathway (perhaps mediated by Ca2+), but not the cAMP-PKA pathway, are critical for inducing LTH during transient, noxious stimulation.^

Solución para la delegación de identidad en Sistemas de Gestión de Identidad paneuropeos basada en infraestructuras de certificación y lenguajes formales de asertos de seguridad

La estrategia i2010 de la UE tiene como objetivo garantizar el liderazgo europeo en materia de TIC y poner los beneficios de la Sociedad de la Información al servicio de la economía, la sociedad y la calidad de vida personal, teniendo presente que los éxitos de Europa hasta la fecha se han basado en favorecer la competencia leal en los mercados de las telecomunicaciones y crear un mercado sin fronteras para contenidos y medios de comunicación digitales. En esta línea, la Comisión Europea ha establecido que los distintos estados miembros deben contribuir activamente al desarrollo y uso seguro de los servicios telemáticos entre sus ciudadanos. Más concretamente, atribuye a las Administraciones Públicas, tanto a nivel nacional, regional como local, un papel dinamizador de la Sociedad de la Información que les obliga a ofrecer paulatinamente todos los actos administrativos a los ciudadanos a través de Internet. Como primer paso para el uso seguro de los servicios telemáticos que ofrecen las instituciones públicas se hace preciso dotar a los ciudadanos de una identidad digital que les permita identificarse ante un Proveedor de Servicio o ante otros ciudadanos de manera inequívoca. Por esta razón, la mayoría de países europeos – y otros en el resto del mundo – están promoviendo, sistemas fiables de gestión de identidad electrónica (eIDM), de tal manera que los ciudadanos, las empresas y departamentos gubernamentales (incluso en Estados miembros diferentes) pueden identificar y certificar sus operaciones con precisión, rapidez y sencillez. Sin embargo, la gestión de esta identidad por las Administraciones Públicas supone un importante desafío, acentuado cuando se hace necesaria la interoperabilidad entre Administraciones de diferentes países, puesto que personas y entidades tienen credenciales de identificación diferentes en función de su propio marco jurídico nacional. Consciente del problema, en la Unión Europea se han puesto en marcha una serie de proyectos con el objetivo de conseguir la interoperabilidad de los eIDMs entre las instituciones públicas de diferentes Estados miembros. A pesar de ello, las soluciones adoptadas hasta la fecha son insuficientes porque no prevén todos los posibles casos de interacción del usuario con las instituciones. En concreto, no tienen en cuenta un aspecto muy importante que se ofrece en los distintos sistemas jurídicos nacionales, a saber, la delegación de la identidad, mediante la cual un ciudadano puede autorizar a otro para que actúe en su nombre para acceder a determinados servicios prestados por las instituciones públicas. En esta tesis se realizan un conjunto de aportaciones que dan solución a distintos aspectos de los problemas planteados y que, de forma conjunta, permiten la interoperabilidad y la delegación de identidad en determinados Sistemas de Gestión de Identidad aplicados al entorno de las Administraciones Públicas. En el caso de la delegación, se ha definido un sistema de delegación dinámica de identidad entre dos entidades genéricas que permite solucionar el problema del acceso delegado a los servicios telemáticos ofrecidos por las Administraciones Públicas. La solución propuesta se basa en la generación de un token de delegación, constituido a partir de un Certificado Proxy, que permite a la entidad que delega establecer la delegación de identidad en otra entidad en base a un subconjunto de sus atributos como delegador, estableciendo además, en el propio token de delegación, restricciones en el conjunto de servicios accesibles a la entidad delegada y el tiempo de validez de la delegación. Adicionalmente, se presentan los mecanismos necesarios tanto para poder revocar un token de delegación como para comprobar sin un token de delegación ha sido o no revocado. Para ello se propone una solución para la identificación unívoca de tokens de delegación y la creación de una nueva entidad denominada Autoridad de Revocación de Tokens de Delegación. Entre las características del sistema de delegación propuesto destaca el que es lo suficientemente seguro como para ser utilizado en el entorno de la Administración Pública, que no requiere el uso de mecanismos off‐line para la generación de la delegación y que se puede realizar la delegación de forma instantánea y sin la necesidad de trámites complejos o la participación de un elevado número de entidades. Adicionalmente, el token de delegación propuesto es perfectamente integrable en las infraestructura de clave pública actual lo que hace que, dado que gran parte de las Administraciones Públicas europeas basan sus sistemas de identidad digital en el uso de la PKI y certificados de identidad X.509, la solución pueda ser puesta en marcha en un entorno real sin necesidad de grandes cambios o modificaciones de comportamiento. En lo referente a la interoperabilidad, se realiza un análisis exhaustivo y la correspondiente evaluación de las principales propuestas de Sistemas de Gestión de Identidad orientados a conseguir la interoperabilidad realizadas hasta la fecha en el marco de la Unión Europea y se propone, a alto nivel, una arquitectura de interoperabilidad para la gestión de identidad en las Administraciones Públicas. Dicha arquitectura es lo suficientemente genérica como para poder ser aplicada tanto en el entorno pan‐Europeo como en los entornos nacionales, autonómicos y locales, de tal forma que la interoperabilidad en la gestión de la identidad esté garantizada en todos los niveles de la Administración Pública. Por último, mediante la integración de la solución de delegación dinámica de identidad y la arquitectura de interoperabilidad propuestas se presenta una solución al problema de la delegación en un escenario pan‐Europeo de gestión de identidad, dando lugar a una arquitectura global de interoperabilidad pan‐Europea con soporte a la delegación de identidad. SUMMARY The i2010 European Union Plan aims to ensure European leadership in ICT and to promote the positive contribution that information and communication technologies can make to the economic, social and personal quality of life, bearing in mind that, to date, success in Europe has been based on promoting fair competition in telecommunications markets and on creating a borderless market for contents and digital media. In this line, the European Commission has established that the different member states should contribute actively to the development and secure use of telematic services among their citizens. More specifically, it is attributed to national, regional and local Public Administrations to have a supportive role of the Information Society, requiring them to gradually provide the citizens with Internet‐based access to all administrative procedures acts. As a first step for the secure use of telematic services offered by public institutions, it is necessary to provide the citizens with a digital identity to enable them to identify themselves unequivocally to a Service Provider or to other citizens. For this reason, most European countries ‐ and others in the rest of the world ‐ are promoting reliable systems for managing electronic identity (eIDM), so that citizens, businesses and government departments (even in different Member States) can identify and certify their operations with precision, speed and simplicity. However, the identity management by Public Administrations is a major challenge that becomes more difficult when interoperability between administrations of different countries is needed, due to the fact that individuals and entities have different identification credentials according to their own national legal framework. Aware of the problem, the European Union has launched a series of projects with the aim of achieving interoperability of eIDMs between public institutions of different Member States. However, the solutions adopted to date are insufficient because they do not foresee all possible cases of user interaction with the institutions. In particular, solutions do not take into account a very important aspect that is offered in different national legal systems, namely, the delegation of identity, by which a citizen can authorize another to act on his/her behalf to access certain services provided by public institutions. In this thesis a collection of contributions that provide solution to different aspects of the aforementioned problems are carried out. The solutions, in global, enable interoperability and identity delegation in some of the Identity Management Systems applied to Public Administration environment. In the case of delegation, a dynamic identity delegation system between generic entities is defined. This system makes it possible to solve the problem of delegated access to telematic services offered by Public Administrations. The proposed solution is based on the generation of a piece of information called delegation token. This delegation token, derived from a Proxy Certificate, allows the establishment of identity delegation by an entity that delegates (delegator) in other entity (delegatee) making use of a subset of delegator attributes. It also establishes restrictions on services that can be used by the delegated entity and the expiry date of delegation. In addition to this, the mechanisms necessary to revoke and check the revocation status of a delegation token are presented. To do this, a solution to univocally identify delegation tokens and the creation of a completely new entity, called Token Delegation Revocation Authority, are proposed. The most remarkable characteristics of the proposed delegation system are its security, enough for it to be used in the Public Administration environment, the fact that it does not require off‐line processes in order to generate the delegation, and the possibility of performing the delegation instantaneously and without neither complex processes nor the intervention of a large number of entities. The proposed delegation token can be completely incorporated into current Public Key Infrastructure (PKI). Thus, since most of the European Public Administrations base their digital identity systems on PKI and X.509 identity certificates, the solution can be adopted in a real environment without great changes or performance modifications. Regarding interoperability, an exhaustive analysis and evaluation of most significant proposals on Identity Management Systems that aim to achieve interoperability carried out in the European Union framework until now are performed. A high level identity management interoperability architecture for Public Administrations is also proposed. This architecture is sufficiently generic to be applied to both pan‐European environment and national, regional or local environments, thus interoperability in identity management at all Public Administration levels is guaranteed. Finally, through the integration of the proposed dynamic identity delegation solution and the high level interoperability architecture, a solution to the problem of identity delegation in a pan‐European identity management environment is suggested, leading to a pan‐European global interoperability architecture with identity delegation support.

Identification of protein–protein interfaces by decreased amide proton solvent accessibility

Matrix-assisted laser desorption ionization–time-of-flight mass spectrometry was used to identify peptic fragments from protein complexes that retained deuterium under hydrogen exchange conditions due to decreased solvent accessibility at the interface of the complex. Short deuteration times allowed preferential labeling of rapidly exchanging surface amides so that primarily solvent accessibility changes and not conformational changes were detected. A single mass spectrum of the peptic digest mixture was analyzed to determine the deuterium content of all proteolytic fragments of the protein. The protein–protein interface was reliably indicated by those peptides that retained more deuterons in the complex compared with control experiments in which only one protein was present. The method was used to identify the kinase inhibitor [PKI(5–24)] and ATP-binding sites in the cyclic-AMP-dependent protein kinase. Three overlapping peptides identified the ATP-binding site, three overlapping peptides identified the glycine-rich loop, and two peptides identified the PKI(5–24)-binding site. A complex of unknown structure also was analyzed, human α-thrombin bound to an 83-aa fragment of human thrombomodulin [TMEGF(4–5)]. Five peptides from thrombin showed significantly decreased solvent accessibility in the complex. Three peptides identified the anion-binding exosite I, confirming ligand competition experiments. Two peptides identified a new region of thrombin near the active site providing a potential mechanism of how thrombomodulin alters thrombin substrate specificity.

A cytosolic activity distinct from Crm1 mediates nuclear export of protein kinase inhibitor in permeabilized cells

The leucine-rich nuclear export signal (NES) is used by a variety of proteins to facilitate their delivery from the nucleus to the cytoplasm. One of the best-studied examples, protein kinase inhibitor (PKI), binds to the catalytic subunit of protein kinase A in the nucleus and mediates its rapid export to the cytoplasm. We developed a permeabilized cell assay that reconstitutes nuclear export mediated by PKI, and we used it to characterize the cytosolic factors required for this process. The two-step assay involves an import phase and an export phase, and quantitation is achieved by digital fluorescence microscopy. During the import phase, a fluorescent derivative of streptavidin is imported into the nuclei of digitonin-permeabilized HeLa cells. During the export phase, biotinylated PKI diffuses into the nucleus, binds to fluorescent streptavidin, and mediates export of the complex to the cytoplasm. Nuclear export of the PKI complex is cytosol dependent and can be stimulated by addition of the purified NES receptor, Crm1. HeLa cell cytosol treated with N-ethylmaleimide (NEM) or phenyl-Sepharose to inactivate or deplete Crm1, respectively, is still fully active in the PKI export assay. Significantly, the export activity can be depleted from cytosol by preadsorption with a protein conjugate that contains a functional NES. These data indicate that cytosol contains an export activity that is distinct from Crm1 and is likely to correspond to an NES receptor.

Inhibition of mRNA export in vertebrate cells by nuclear export signal conjugates

Leucine-rich nuclear export signals (NESs) are recognized by the NES receptor exportin 1 and are central to the export of multiple shuttling proteins and RNAs. The export of messenger RNA in vertebrates was, however, thought to occur by a different pathway, because inhibition by injection of a synthetic Rev NES conjugate could not be demonstrated. Here we find that peptide conjugates composed of the NES of either protein kinase A inhibitor protein (PKI) or the HIV-1 Rev protein, when coupled to human serum albumin, are potent inhibitors of mRNA and small nuclear RNA export. These results provide direct evidence that mRNA export in vertebrates depends on interactions between an NES and its cognate NES receptors. PKI NES conjugates are significantly more efficient at inhibiting RNA export than are REV NES conjugates, indicating that different NESs may have different abilities to promote protein and RNA export. Surprisingly, an expected control conjugate containing the mutant Rev NES sequence M10 strongly inhibited the export of intronless dihydrofolate reductase mRNA. Nuclear injection of NES peptide conjugates led to mislocalization to the nucleus of 10–20% of the cytoplasmic Ran GTPase-binding protein (RanBP1) indicating that RanBP1 shuttles between the nucleus and the cytoplasm via an NES pathway. These results demonstrate that in vertebrates the export of mRNA, like that of small nuclear RNA, 5S rRNA, and transport factors such as RanBP1, employs NES-mediated molecular machinery.

The Polo-like Kinase Plx1 Is Required for Activation of the Phosphatase Cdc25C and Cyclin B-Cdc2 in Xenopus Oocytes

In the Xenopus oocyte system mitogen treatment triggers the G2/M transition by transiently inhibiting the cAMP-dependent protein kinase (PKA); subsequently, other signal transduction pathways are activated, including the mitogen-activated protein kinase (MAPK) and polo-like kinase pathways. To study the interactions between these pathways, we have utilized a cell-free oocyte extract that carries out the signaling events of oocyte maturation after addition of the heat-stable inhibitor of PKA, PKI. PKI stimulated the synthesis of Mos and activation of both the MAPK pathway and the Plx1/Cdc25C/cyclin B-Cdc2 pathway. Activation of the MAPK pathway alone by glutathione S-transferase (GST)-Mos did not lead to activation of Plx1 or cyclin B-Cdc2. Inhibition of the MAPK pathway in the extract by the MEK1 inhibitor U0126 delayed, but did not prevent, activation of the Plx1 pathway, and inhibition of Mos synthesis by cycloheximide had a similar effect, suggesting that MAPK activation is the only relevant function of Mos. Immunodepletion of Plx1 completely inhibited activation of Cdc25C and cyclin B-Cdc2 by PKI, indicating that Plx1 is necessary for Cdc25C activation. In extracts containing fully activated Plx1 and Cdc25C, inhibition of cyclin B-Cdc2 by p21Cip1 had no significant effect on either the phosphorylation of Cdc25C or the activity of Plx1. These results demonstrate that maintenance of Plx1 and Cdc25C activity during mitosis does not require cyclin B-Cdc2 activity.

Nuclear export of late HIV-1 mRNAs occurs via a cellular protein export pathway.

The Rev protein of HIV-1 is essential for the nuclear export of incompletely spliced viral mRNAs. This action depends on the mutationally defined Rev activation domain, which both binds the nucleoporin-like human cellular cofactor Rab/hRIP and also functions as a nuclear export signal. Protein kinase inhibitor alpha (PKI) also contains a potent nuclear export signal. However, PKI plays no role in nuclear RNA export and instead induces the nuclear export of a specific protein target, the catalytic subunit of cAMP-dependent protein kinase. Here, it is demonstrated that the nuclear export signal of PKI not only binds the Rab/hRIP cofactor specifically but also can effectively substitute for the Rev activation domain in mediating the nuclear export of HIV-1 mRNAs. We conclude that HIV-1 Rev and PKI act through an identical nuclear export pathway and that Rev, rather than using a dedicated RNA export pathway, is instead acting as an adaptor that allows viral mRNAs to access a cellular protein export pathway.

Protocolo multiplataforma no centralizado para comunicaciones multimedia seguras

En este trabajo se propone y desarrolla una topología en k-hipercubos que resuelve los principales inconvenientes asociados a la topología en hipercubo convencional. Los resultados obtenidos son muy prometedores, con aplicaciones tanto en el campo de la voz sobre IP, como en muchos otros campos que precisen de un intercambio de información muchos a muchos. Sobre la topología propuesta se define el protocolo Darkcube, que es una propuesta de protocolo totalmente distribuido basado en el concepto de darknet, posibilitando la realización de conversaciones muchos a muchos incluyendo audio, vídeo, texto y datos de geoposicionamiento, entre otros. También se propone un método de codificación de coordenadas de geoposicionamiento que resulta especialmente eficiente en el aprovechamiento del ancho de banda sobrante en las comunicaciones muchos a muchos que proporciona Darkcube. Durante el desarrollo de este trabajo, se ha implementado el simulador DarkcubeEmu; herramienta que posibilita la obtención de resultados relevantes en términos de la calidad de la comunicación. Finalmente, utilizando como base el protocolo Darkcube, se propone un protocolo de seguridad que traslada un esquema de infraestructura de clave pública a un protocolo totalmente distribuido, como es Darkcube; garantizando, de esta forma, la confidencialidad en las comunicaciones y la legitimidad de la identidad asociada a cada uno de sus miembros.

Inference of binding affinity from neuronal receptors in humans

Tese de mestrado, Bioinformática e Biologia Computacional (Bioinformática), Universidade de Lisboa, Faculdade de Ciências, 2016

The role of the 8-18 helix of CGRP 8-37 in mediating high affinity binding to CGRP receptors; coulombic and steric interactions

1. The role of individual residues in the 8-18 helix of CGRP 8-37 in promoting high-affinity binding to CGRP 1 receptors expressed on rat L6 and human SK-N-MC cells has been examined. The relative potencies of various derivatives were estimated from their ability to inhibit the human αCGRP-mediated increase in cyclic AMP production and the binding of [ 125I]-human αCGRP. 3. Arg 11 and Arg 18 were replaced by serines to give [Ser 11.18]CGRP 8-37. These bound with pKi values <6 to SK-N-MC cells and had apparent pA 2 values of 5.81 ± 0.04 and 5.31 ± 0.11 on SK-N-MC and L6 cells. CGRP 8-37 had a pKi of 8.22 on SK-N-MC cells and pK b values on the above cell lines of 8.95±0.04 and 8.76±0.04. 3. The arginines were replaced with glutamic acid residues. [Glu 11]CGRP 8-37 had a pK b of 7.14±0.14 on SK-N-MC cells (pKi=7.05±0.05) and 6.99±0.08 on L6 cells. [Glu 18]CGRP 8-37 had a pK b of 7.10±0.0.08 on SK-N-MC cells (pKi=6.91±0.23) and 7.12±0.09 on L6 cells. 4. Leu 12, Leu 15 and Leu 16 were replaced by benzoyl-phenylalanine (bpa) residues. On SK-N-MC cells, the apparent pA 2 values of [bpa 12]-, [bpa 15]- and [bpa 16]CGRP 8-37 were respectively 7.43±0.23, 8.34±0.11 and 5.66±0.16 (pKi values of 7.14±0.17, 7.66±0.21 and <6): on L6 cells they were 7.96±0.36, 8.28±0.21 and 6.09±0.04 (all n=3). 5. It is concluded that the Arg 11 and Arg 18 are involved in specific electrostatic interactions with other residues, either on the CGRP 1 receptors or elsewhere on CGRP 8-37. Leu 16 is in a conformationally restricted site when CGRP 8-37 binds to CGRP 1 receptors, unlike Leu 12 and Leu 15.