Influence of mycobacterial trehalose 6,6'-dimycolate (TDM) on macrophage responses

Mycobacterium tuberculosis, the causative agent of tuberculosis, survives within macrophages by altering host cell activation and by manipulating phagosomal trafficking and acidification. Part of the success of M. tuberculosis as a major human pathogen has been attributed to its cell wall, a unique structure largely comprised of mycolic acids. Trehalose 6,6′-dimycolate (TDM) is the major glycolipid component on the surface of the mycobacterial cell wall. This study examines the contribution of TDM during mycobacterial infection of murine macrophages. Virulent M. tuberculosis was chemically depleted of surface-exposed TDM using petroleum ether extraction. Compared to their native counterparts, delipidated M. tuberculosis showed similar growth in broth culture. Bone marrow-derived macrophages (BMM) or the murine macrophage-like cell line J774A.1 were infected with delipidated M. tuberculosis, and responses were compared to cells infected with native M. tuberculosis. Delipidated M. tuberculosis demonstrated significantly decreased viability in macrophages by seven days after infection. Reconstitution of delipidated organisms with pure TDM restored viability. Infection with native M. tuberculosis led to high cellular production of cytokines (IL-1β, IL-6, IL-12, and TNF-α) and chemokines (MCP-1 and MIP-1α); infection with delipidated M. tuberculosis significantly abrogated responses. Cytokine and chemokine production were restored when delipidated organisms were reconstituted with TDM. Responses were specifically induced by TDM; all measured cytokines were elicited from macrophages incubated with TDM-coated beads, while control beads coated with bovine serum albumin (BSA) did not induce cytokine production. Visualization of mycobacterial localization in J774A.1 cells using fluorescence microscopy revealed that delipidated M. tuberculosis were significantly more likely to traffic to acidic vesicles (lysosomes) than native organisms. Reconstitution with TDM restored trafficking to non-acidic vesicles. Similarly, TDM-coated beads demonstrated significantly delayed localization to acidic vesicles compared to BSA-coated beads. In summary, the interaction of TDM with macrophages may regulate the outcome of M. tuberculosis infection by influencing cellular cytokine production and intracellular localization of organisms. This research has elucidated a novel and necessary role for TDM in survival of virulent M. tuberculosis in host macrophages during in vitro infection. ^

Regulation of Alix by an autoinhibitory intramolecular interaction

In eukaryotic cells, the ESCRTs (endosomal sorting complexes required for transport) machinery is required for cellular processes such as endosomal sorting, retroviral budding and cytokinesis. The ALG-2 interacting protein Alix is a modular adaptor protein that is critically involved in these ESCRTs-associated cellular processes and consists of an N-terminal Bro1 domain, a middle V domain and C-terminal Pro-rich domain (PRD). In these cellular processes, Alix interacts with the ESCRT-III component CHMP4 at the Bro1 domain, with HIV-1 p6 Gag or EIAV p9Gag at the V domain, and with the ESCRT-I component TSG101 at the Pro-rich domain. Here we demonstrate that the N-terminal Bro1 domain forms an intramolecular interaction with C-terminal PRD within Alix. This Bro1-PRD intramolecular interaction forms a closed conformation of Alix that autoinhibits Alix interaction with all of these partner proteins. Moreover, the binding of Ca2+-activated ALG-2 to the PRD of Alix relieves the autoinhibitory intramolecular interaction, resulting in an open conformation of Alix which is able to interact with all of these partner proteins. The partner proteins bound to Alix in turn maintain Alix in the open conformation after ALG-2 dissociation with Alix. Consistent with the effect of Ca2+-activated ALG-2 on opening/activating Alix in these ESCRTs-associated functions, ALG-2 overexpression accelerates EGF-induced degradation of EGFR in an Alix-dependent manner. These findings discover an intrinsic autoinhibitory mechanism of Alix and a two-step process to activate/open Alix and then keep Alix active/open. This study has solved long-standing issues on the regulations of Alix in ESCRTs-associated functions and the role of ALG-2-Alix interaction, and may serve as the structural basis for further studies about Alix regulations. ^

Crack and powder cocaine federal legislation: A sentencing disparity and potential for policy improvement

Since its introduction into the United States in the 1980s, crack cocaine has been a harsh epidemic that has taken its toll on a countless number of people. This highly addictive, cheap and readily available drug of abuse has permeated many demographic sectors, mostly in low income, lesser educated, and urban communities. This epidemic of crack cocaine use in inner city areas across the Unites States has been described as an expression of economic marginality and “social suffering” coupled with the local and international forces of drug market economies (Agar 2003). As crack cocaine is a derivative of cocaine, it utilizes the psychoactive component of the drug, but delivers it in a much stronger, quicker, and more addictive fashion. This, coupled with its ready availability and cheap price has allowed for users to not only become very addicted very quickly, but to be subject to the stringent and sometimes unequal or inconsistent punishments for possession and distribution of crack-cocaine. ^ There are many public health and social ramifications from the abuse of crack-cocaine, and these epidemics appear to target low income and minority groups. Public health issues relating to the physical, mental, and economic strain will be addressed, as well as the direct and indirect effects of the punishments that come as a result of the disparity in penalties for cocaine and crack-cocaine possession and distribution. ^ Three new policies have recently been introduced into the United Stated Congress that actively address the disparity in sentencing for drug and criminal activities. They are, (1) Powder-Crack Cocaine Penalty Equalization Act of 2009, (HR 18, 111th Cong. 2009), (2) The Drug Sentencing Reform and Cocaine Kingpin Trafficking Act of 2009, (HR 265, 111th Cong. 2009) and (3) The Justice Integrity Act of 2009, (111th Cong. 2009). ^ Although they have only been initiated, if passed, they have potential to not only eliminate the crack-cocaine disparity, but to enact laws that help those affected by this epidemic. The final and overarching goal of this paper is to analyze and ultimately choose the ideal policy that would not only eliminate the cocaine and crack disparity regardless of current or future state statutes, but will provide the best method of rehabilitation, prevention, and justice. ^

Platelets and Anti-Angiogenic Resistance in Ovarian Carcinoma

Background: Resistance to targeted anti-angiogenic therapy is a growing clinical concern given the disappointing clinical impact of anti-angiogenic. Platelets represent a component of the tumor microenvironment that are implicated in metastasis and represent a significant reservoir of angiogenic regulators. Thrombocytosis has been shown to be caused by malignancy and associated with adverse clinical outcomes, however the causal connections between these associations remain to be identified. Materials and Methods: Following IRB approval, patient data were collected on patients from four U.S. centers and platelet levels through and after therapy were considered as indicators of recurrence of disease. In vitro effects of platelets on cancer cell proliferation, apoptosis, and migration were examined. RNA interference was used to query signaling pathways mediating these effects. The necessity of platelet activation for in vitro effect was analyzed. In vivo orthotopic models were used to query the impact of thrombocytosis and thrombocytopenia on the efficacy of cytotoxic chemotherapy, the effect of aspirin on thrombocytosis and cancer, and platelet effect on anti-angiogenic therapy. Results: Platelets were found to increase at the time of diagnosis of ovarian cancer recurrence in a pattern comparable to CA-125. Platelet co-culture increased proliferation, increased migration, and decreased apoptosis in all cell lines tested. RNA interference implicated platelet derived growth factor alpha (PDGFRA) and transforming growth factor beta-receptor 1 (TGFBR1) signaling. Biodistribution studies suggested minimal platelet sequestration of taxanes. Blockade of platelet activation blocked in vitro effects. In vivo, thrombocytosis blocked chemotherapeutic efficacy, thrombocytopenia increased chemotherapeutic efficacy, and aspirin therapy partially blocked the effects of thrombocytosis. In vivo, withdrawal of anti-angiogenic therapy caused loss of therapeutic benefit with evidence of accelerated disease growth. This effect was blocked by use of a small-molecule inhibitor of Focal Adhesion Kinase. Anti-angiogenic therapy was also associated with increased platelet infiltration into tumor that was not seen to the same degree in the control or FAK-inhibitor-treated mice. Conclusions: Platelets are active participants in the growth and metastasis of tumor, both directly and via facilitation of angiogenesis. Blocking platelets, blocking platelet activation, and blocking platelet trafficking into tumor are novel therapeutic avenues supported by this data. Copyright © 2012 Justin Neal Bottsford-Miller, all rights reserved.

Commercial Sexual Exploitation and Missing Children in the Coastal Region of Sao Paulo State, Brazil

The commercial sexual exploitation of children (CSEC) has emerged as one of the world’s most heinous crimes. The problem affects millions of children worldwide and no country or community is fully immune from its effects. This paper reports first generation research of the relationship that exists between CSEC and the phenomenon of missing children living in and around the coastal regions of the state of Sao Paulo, Brazil, the country’s richest State. Data are reported from interviews and case records of 64 children and adolescents, who were receiving care through a major youth serving non-governmental organization (NGO) located in the coastal city of Sao Vicente. Also, data about missing children and adolescents were collected from Police Reports – a total of 858 Police Reports. In Brazil, prostitution is not a crime itself, however, the exploitation of prostitution is a crime. Therefore, the police have no information about children or adolescents in this situation, they only have information about the clients and exploiters. Thus, this investigation sought to accomplish two objectives: 1) to establish the relationship between missing and sexual exploited children; and 2) to sensitize police and child-serving authorities in both the governmental and nongovernmental sectors to the nature, extent, and seriousness of many unrecognized cases of CSEC and missing children that come to their attention. The observed results indicated that the missing children police report are significantly underestimated. They do not represent the number of children that run away and/or are involved in commercial sexual exploitation.

The Role of K63-linked Ubiquitination Cycles in Akt Kinase Activation

Akt (also known as protein kinase B) serves a central regulator in PI3K/Akt signaling pathways to regulate numerous physiological functions including cell proliferation, survival and metabolism. Akt activation requires the binding of Akt to phospholipid PIP3 on the plasma membrane and subsequent phosphorylation of Akt by its kinases. Growth factor-mediated membrane recruitment of Akt is a crucial step for Akt activation. However, the mechanism of Akt membrane translocation is unclear. Protein ubiquitination is a significant posttranslational modification that controls many biological functions such as protein trafficking and signaling activation. Therefore, we hypothesize that ubiquitination may be involved in Akt signaling activation. We have demonstrated that Akt could be conjugated with non-proteolytic K63-linked ubiquitination by TRAF6 ubiquitin E3 ligase. This modification on Akt was required for membrane recruitment, phosphorylation and activation of Akt in response to growth factor stimulation. The human cancer-associated Akt E17K mutant exhibited an increase in K63-linked ubiquitination, which contributes to the enrichment of membrane recruitment and phosphorylation of Akt. Thus, we conclude that K63-linked ubiquitination is a critical step for oncogenic Akt activation and also involved in human cancer development. Notably, the process of protein ubiquitination can be reversed by deubiquitinating enzymes (DUBs), which play a critical role to terminate signaling activation induced by ubiquitination. To further investigate how ubiquitination cycles regulate Akt activation, we have identified that CYLD as a DUB for Akt, and CYLD inhibited growth factor-induced ubiquitination and activation of Akt. Under serum-depletion condition, CYLD interacts with Akt and keep Akt under inactive state by directly removing K63-linked ubiquitination of Akt. CYLD disassociates with Akt upon growth factor stimulation, thereby allowing E3 ligases to induce ubiquitination and activation of Akt. We also demonstrated that CYLD deficiency promoted cancer cell proliferation, survival, glucose metabolism and human prostate cancer development. Therefore, we conclude that CYLD plays a critical role for negatively regulating Akt signaling activation through deubiquitination of Akt. In summary, this study delineated the important mechanism of cycles of ubiquitination and deubiquitination of Akt in regulating membrane translocation and activation of Akt, and TRAF6 and CYLD as central switches for these processes.

The role of the conserved N -terminal domain of STAT3 in STAT3 dephosphorylation and nuclear export

Rapid redistribution of STAT subcellular localization is an essential feature of cytokine signaling. To elucidate the molecular basis of STAT3 function, which plays a critical role in controlling innate immune responses in vivo, we initiated studies to determine the mechanisms controlling STAT3 nuclear trafficking. We found that STAT3 is transported to the nucleus in the absence of cytokine treatment, as judged by indirect immunofluorescence studies in the presence of leptomycin B, an inhibitor of CRM1-dependent nuclear export, suggesting that the non-phosphorylated STAT3 protein contains a functional nuclear import signal. An isoform lacking the STAT3 N-terminal domain (Δ133STAT3) retains the ability to undergo constitutive nuclear localization, indicating that this region is not essential for cytokine-independent nuclear import. Δ133STAT3 is also transported to the nucleus following stimulation with interleukin-6 (IL-6). Interestingly, IL-6-dependent tyrosine phosphorylation of Δ133STAT3 appears to be prolonged and the nuclear export of the protein delayed in cells expressing endogenous STAT3, consistent with defective Δ133STAT3 dephosphorylation. Endogenous STAT3 does not promote the nuclear export of Δ133STAT3, although dimerization between endogenous Stat3 and Δ133STAT3 is detected readily. Thus, the STAT3 N-terminal domain is not required for dimerization with full-length STAT3, yet appears to play a role in proper export of Stat3 from the nucleus following cytokine stimulation. STAT3-deficient cells reconstituted with Δ133STAT3 show enhanced and prolonged Stat1 signaling in response to IL-6, suggesting that induction of the STAT3-dependent negative regulator SOCS3 is impaired. In fact, Δ133STAT3 fails to induce SOCS3 mRNA efficiently. These studies collectively indicate that the STAT3 N-terminal region may be important for IL-6-dependent target gene activation and nuclear dephosphorylation, while dispensable for nuclear import. STAT3 is an oncogene. STAT3 is constitutively activated in primary tumors of many types. Thus far, research in the design of STAT3 protein inhibitors has focused on the SH2 and DNA-binding domains of STAT3. Interference with these domains eliminates all signaling through STAT3. If the N-terminal domain is involved in tetramerization on a subset of target genes, inhibition of this region may lead to a more selective inhibition of some STAT3 functions while leaving others intact. ^

La moda neoliberal : El retorno de los talleres clandestinos de costura

En este artículo se analiza el resurgimiento de talleres de costura clandestinos en grandes ciudades del centro y la periferia mundial, para entender los cambios dados en la industria de la moda durante las últimas cuatro décadas y sus consecuencias sobre los trabajadores. Para ello se realizaron dos estudios de caso: uno en la ciudad de Buenos Aires y otro en la provincia de Prato (Italia). Los resultados de esta investigación demuestran que este sector fue pionero en los procesos de reorganización industrial en la época neoliberal. En ambos estudios de caso, el cierre de fábricas y la utilización masiva de subcontratación a talleres urbanos informales tuvieron como consecuencias una significativa concentración de capital por un lado, y un marcado deterioro de las condiciones de trabajo por el otro. De hecho, la existencia de trata de personas y reducción a la servidumbre de miles de trabajadores inmigrantes es fundamental para el funcionamiento de esta industria

La moda neoliberal : El retorno de los talleres clandestinos de costura

En este artículo se analiza el resurgimiento de talleres de costura clandestinos en grandes ciudades del centro y la periferia mundial, para entender los cambios dados en la industria de la moda durante las últimas cuatro décadas y sus consecuencias sobre los trabajadores. Para ello se realizaron dos estudios de caso: uno en la ciudad de Buenos Aires y otro en la provincia de Prato (Italia). Los resultados de esta investigación demuestran que este sector fue pionero en los procesos de reorganización industrial en la época neoliberal. En ambos estudios de caso, el cierre de fábricas y la utilización masiva de subcontratación a talleres urbanos informales tuvieron como consecuencias una significativa concentración de capital por un lado, y un marcado deterioro de las condiciones de trabajo por el otro. De hecho, la existencia de trata de personas y reducción a la servidumbre de miles de trabajadores inmigrantes es fundamental para el funcionamiento de esta industria

La moda neoliberal : El retorno de los talleres clandestinos de costura

En este artículo se analiza el resurgimiento de talleres de costura clandestinos en grandes ciudades del centro y la periferia mundial, para entender los cambios dados en la industria de la moda durante las últimas cuatro décadas y sus consecuencias sobre los trabajadores. Para ello se realizaron dos estudios de caso: uno en la ciudad de Buenos Aires y otro en la provincia de Prato (Italia). Los resultados de esta investigación demuestran que este sector fue pionero en los procesos de reorganización industrial en la época neoliberal. En ambos estudios de caso, el cierre de fábricas y la utilización masiva de subcontratación a talleres urbanos informales tuvieron como consecuencias una significativa concentración de capital por un lado, y un marcado deterioro de las condiciones de trabajo por el otro. De hecho, la existencia de trata de personas y reducción a la servidumbre de miles de trabajadores inmigrantes es fundamental para el funcionamiento de esta industria

Myanmar migrant laborers in Ranong, Thailand

Thailand is the major destination for migrants in mainland Southeast Asia, and Myanmar (Burmese) migrants account for the dominant share. This paper sheds light on the actual working conditions and the life of Myanmar migrants in Thailand, based on our intensive survey in Ranong in southern Thailand in 2009. We found a wide range of serious problems that Myanmar migrants face in everyday life: very harsh working conditions, low income, heavy indebtedness, risk of being human-trafficking victims, harassment by the police and military (especially of sex workers), high risk of illness including malaria and HIV/AIDS and limited access to affordable medical facilities, and a poor educational environment for their children.

Connexin-43 in the osteogenic BM niche regulates its cellular composition and the bidirectional traffic of hematopoietic stem cells and progenitors

Connexin-43 (Cx43), a gap junction protein involved in control of cell proliferation, differentiation and migration, has been suggested to have a role in hematopoiesis. Cx43 is highly expressed in osteoblasts and osteogenic progenitors (OB/P). To elucidate the biologic function of Cx43 in the hematopoietic microenvironment (HM) and its influence in hematopoietic stem cell (HSC) activity, we studied the hematopoietic function in an in vivo model of constitutive deficiency of Cx43 in OB/P. The deficiency of Cx43 in OB/P cells does not impair the steady state hematopoiesis, but disrupts the directional trafficking of HSC/progenitors (Ps) between the bone marrow (BM) and peripheral blood (PB). OB/P Cx43 is a crucial positive regulator of transstromal migration and homing of both HSCs and progenitors in an irradiated microenvironment. However, OB/P Cx43 deficiency in nonmyeloablated animals does not result in a homing defect but induces increased endosteal lodging and decreased mobilization of HSC/Ps associated with proliferation and expansion of Cxcl12-secreting mesenchymal/osteolineage cells in the BM HM in vivo. Cx43 controls the cellular content of the BM osteogenic microenvironment and is required for homing of HSC/Ps in myeloablated animals

La protección civil municipal: análisis de los distintos tipos de gestión y propuesta metodológica de planificación

Mediante la elaboración de esta tesis doctoral se pretende diseñar un sistema para la realización del plan de autoprotección en industrias con riesgo de incendio dentro de un polígono industrial. Para desarrollar esta línea de investigación se realizará una investigación cartográfica así como un reconocimiento visual de las distintas empresas industriales, de actividades heterogéneas, seleccionadas por su emplazamiento y condiciones. Este tipo de industria es esencial en el sistema económico nacional, ya que dota de recursos y servicios al tejido social, además de tener una relación directa con la generación de riqueza y por tanto incide de forma positiva en el empleo de cualquier región. Estas razones y otras son determinantes para fomentar el progreso, si bien nunca a expensas de la seguridad de los empleados que hacen posible la línea de producción así como de los posibles viandantes que transcurran por la zona de impacto en caso de incidentes como por ejemplo un incendio que afecte a cualquiera de los locales y/o empresas que se emplazen en los denominados polígonos industriales. La tesis incluye trabajos experimentales de los que se extraen recomendaciones y conclusiones encaminadas a la optimización de la instrumentalización utilizada, las técnicas de observación, diseño y cálculo necesarias que determinarán el acercamiento al método propuesto para nuestra valoración del riesgo por incendio. Las posibles emergencias que pueden darse a nivel de industrias localizadas en polígonos industriales son diversas y numerosas. De todas ellas se elige abordar el caso de “incendio” por su casuística más numerosa. No obstante, la planificación orientada desde el prisma de la investigación de esta tesis puede estenderse a cualquier otro riesgo, lo que se aportará como líneas futuras de investigación. Las aproximaciones y etapas de los trabajos que forman parte de esta investigación se han integrado en cada capítulo y son las siguientes: En primer lugar un capítulo de Introducción, en el que se realiza una reflexión justificada de la elección del tema tratado, se formula la hipótesis de partida y se enumeran los distintos objetivos que se pretenden alcanzar. En el segundo capítulo, titulado Aspectos de la Protección Civil en la Constitución Española de 1978, se ha realizado un estudio exhaustivo de la Constitución en todo lo relacionado con el ámbito de la protección, haciendo especial énfasis en las distintas competencias según el tipo de administración que corresponda en cada caso, así como las obligaciones y deberes que corresponden a los ciudadanos en caso de catástrofe. Así mismo se analizan especialmente los casos de Guerra y el estado de Alarma. En el tercer capítulo, titulado Repuesta de las administraciones públicas ante emergencias colectivas, se trata de formular el adecuado sistema de repuesta que sería preciso para tratar de mitigar desastres y catástrofes. Se analizan los distintos sistemas de gestión de emergencias constatando en cada caso los pros y los contras de cada uno. Se pretende con ello servir de ayuda en la toma de decisiones de manera coherente y racional. El capítulo cuarto, denominado Planes Locales de emergencia. Estudio en las distintas administraciones, se ha pormenorizado en la presentación así como puesta en funcionamiento de los planes, comparando los datos obtenidos entre ellos para concluir en un punto informativo que nos lleva a la realidad de la planificación. Para la realización y desarrollo de los capítulos 5 y 6, llamados Análisis de la Norma Básica de Autoprotección y Métodos de evaluación del riesgo de Incendio, se estudia la normativa actual en autoprotección prestando atención a los antecedentes históricos así como a todas las figuras que intervienen en la misma. Se analizan los distintos métodos actuales para la valoración del riesgo por incendio en industrias. Este aspecto es de carácter imprescindible en la protección civil de los ciudadanos pero también es de especial importancia en las compañías aseguradoras. En el capítulo 7, Propuesta metodológica, se propone y justifica la necesidad de establecer una metodología de estudio para estos casos de riesgo por incendio en industrias para así, acortar el tiempo de respuesta de los servicios de emergencia hasta la zona, así como dotar de información imprescindible sobre el riesgo a trabajadores y transeúntes. El último capítulo se refiere a las Conclusiones, donde se establecen y enuncian una serie de conclusiones y resultados como consecuencia de la investigación desarrollada, para finalizar esta tesis doctoral enunciando posibles desarrollos y líneas de investigación futuros. ABSTRACT The development of this thesis is to design a system for the implementation of the plan of self-protection in industries with risk of fire in an industrial park. To develop this line of research will be done cartographic research as well as visual recognition of the distinct and heterogeneous industrial companies selected by its location and conditions. This type of industry is an essential part in the economic national system providing economic resources to society as well as with a direct relationship in unemployment. For this reason it is crucial to promote their progress, but never at the expense of the security of the employees that make the line of production as well as possible walkers that pass by the area of impact in the event of a fire affecting the company. The thesis includes experimental works which are extracted recommendations and conclusions aimed at optimization of used exploit, techniques of observation, design and calculation needed to determine the approach to the method proposed for our assessment of the risk from fire. The approaches and stages of works that are part of this research have been integrated into each chapter and are as follows: In the first chapter, holder introduction, perform a supporting reflection of the choice of the subject matter, is formulated the hypothesis of departure and listed the different objectives that are intended to achieve. In the second chapter, holder aspects of Civil Protection in the Spanish Constitution of 1978, examines an exhaustive study of the Constitution in everything related to the scope of protection. With an emphasis on individual skills according to the type of management with corresponding in each case, as well as the obligations and duties which correspond to citizens in the event of a catastrophe. Also analyzes the particular cases of war and the State of alarm. In the third chapter, holder public administrations collective emergency response, discussed trafficking in defining the proper system response that would be precise to address disasters and catastrophes. We discusses the different systems of emergency management in each case, we pretend the pros and cons of each. We tried that this serve as decision-making aid coherent and rational way. The fourth chapter is holder Local Emergency Plans (LEP). Study on the different administrations, has detailed in the presentation as well as operation of the LEP, comparing the data between them to conclude in an information point that leads us to the reality of planning. For the realization and development of chapters 5 and 6, holder Analysis of the basic rule of self-protection and fire risk assessment methods, paying attention to the historical background as well as all the figures involved in the same studies with the current rules of self-protection. The current methods for the estimation of the risk are analyzed by fire in industries. This aspect is essential in the civil protection of the citizens, but it is also of special importance for insurance companies. The seventh chapter, holder Methodological proposal, we propose and justifies the need to establish a methodology for these cases of risk by fire in industries. That shorts the response time of emergency services to the area, and provides essential information about the risk to workers and walkers. The last chapter refers to the Conclusions, laying down a series of results as a consequence of the previous chapters to complete billing possible developments and research future.

Brefeldin A-inhibited guanine nucleotide-exchange activity of Sec7 domain from yeast Sec7 with yeast and mammalian ADP ribosylation factors

The Saccharomyces cerevisiae Sec7 protein (ySec7p), which is an important component of the yeast secretory pathway, contains a sequence of ≈200 amino acids referred to as a Sec7 domain. Similar Sec7 domain sequences have been recognized in several guanine nucleotide-exchange proteins (GEPs) for ADP ribosylation factors (ARFs). ARFs are ≈20-kDa GTPases that regulate intracellular vesicular membrane trafficking and activate phospholipase D. GEPs activate ARFs by catalyzing the replacement of bound GDP with GTP. We, therefore, undertook to determine whether a Sec7 domain itself could catalyze nucleotide exchange on ARF and found that it exhibited brefeldin A (BFA)-inhibitable ARF GEP activity. BFA is known to inhibit ARF GEP activity in Golgi membranes, thereby causing reversible apparent dissolution of the Golgi complex in many cells. The His6-tagged Sec7 domain from ySec7p (rySec7d) synthesized in Escherichia coli enhanced binding of guanosine 5′-[γ-[35S]thio]triphosphate by recombinant yeast ARF1 (ryARF1) and ryARF2 but not by ryARF3. The effects of rySec7d on ryARF2 were inhibited by BFA in a concentration-dependent manner but not by inactive analogues of BFA (B-17, B-27, and B-36). rySec7d also promoted BFA-sensitive guanosine 5′-[γ-thio]triphosphate binding by nonmyristoylated recombinant human ARF1 (rhARF1), rhARF5, and rhARF6, although the effect on rhARF6 was very small. These results are consistent with the conclusion that the yeast Sec7 domain itself contains the elements necessary for ARF GEP activity and its inhibition by BFA.

Role for G protein-coupled receptor kinase in agonist-specific regulation of μ-opioid receptor responsiveness

The G protein-coupled μ-opioid receptor (μOR) mediates the physiological effects of endogenous opioid peptides as well as the structurally distinct opioid alkaloids morphine and etorphine. An intriguing feature of μOR signaling is the differential receptor trafficking and desensitization properties following activation by distinct agonists, which have been proposed as possible mechanisms related to opioid tolerance. Here we report that the ability of distinct opioid agonists to differentially regulate μOR internalization and desensitization is related to their ability to promote G protein-coupled receptor kinase (GRK)-dependent phosphorylation of the μOR. Although both etorphine and morphine effectively activate the μOR, only etorphine elicits robust μOR phosphorylation followed by plasma membrane translocation of β-arrestin and dynamin-dependent receptor internalization. In contrast, corresponding to its inability to cause μOR internalization, morphine is unable to either elicit μOR phosphorylation or stimulate β-arrestin translocation. However, upon the overexpression of GRK2, morphine gains the capacity to induce μOR phosphorylation, accompanied by the rescue of β-arrestin translocation and receptor sequestration. Moreover, overexpression of GRK2 also leads to an attenuation of morphine-mediated inhibition of adenylyl cyclase. These findings point to the existence of marked differences in the ability of different opioid agonists to promote μOR phosphorylation by GRK. These differences may provide the molecular basis underlying the different analgesic properties of opioid agonists and contribute to the distinct ability of various opioids to induce drug tolerance.