The chemokine monocyte chemoattractant protein-1 induces functional responses through dimerization of its receptor CCR2

Cytokines interact with hematopoietin superfamily receptors and stimulate receptor dimerization. We demonstrate that chemoattractant cytokines (chemokines) also trigger biological responses through receptor dimerization. Functional responses are induced after pairwise crosslinking of chemokine receptors by bivalent agonistic antichemokine receptor mAb, but not by their Fab fragments. Monocyte chemoattractant protein (MCP)-1-triggered receptor dimerization was studied in human embryonic kidney (HEK)-293 cells cotransfected with genes coding for the CCR2b receptor tagged with YSK or Myc sequences. After MCP-1 stimulation, immunoprecipitation with Myc-specific antibodies revealed YSK-tagged receptors in immunoblotting. Receptor dimerization also was validated by chemical crosslinking in both HEK-293 cells and the human monocytic cell line Mono Mac 1. Finally, we constructed a loss-of-function CCR2bY139F mutant that acted as a dominant negative, blocking signaling through the CCR2 wild-type receptor. This study provides functional support for a model in which the MCP-1 receptor is activated by ligand-induced homodimerization, allowing discussion of the similarities between bacterial and leukocyte chemotaxis.

Conversion of agonist site to metal-ion chelator site in the β2-adrenergic receptor

Previously metal-ion sites have been used as structural and functional probes in seven transmembrane receptors (7TM), but as yet all the engineered sites have been inactivating. Based on presumed agonist interaction points in transmembrane III (TM-III) and -VII of the β2-adrenergic receptor, in this paper we construct an activating metal-ion site between the amine-binding Asp-113 in TM-III—or a His residue introduced at this position—and a Cys residue substituted for Asn-312 in TM-VII. No increase in constitutive activity was observed in the mutant receptors. Signal transduction was activated in the mutant receptors not by normal catecholamine ligands but instead either by free zinc ions or by zinc or copper ions in complex with small hydrophobic metal-ion chelators. Chelation of the metal ions by small hydrophobic chelators such as phenanthroline or bipyridine protected the cells from the toxic effect of, for example Cu2+, and in several cases increased the affinity of the ions for the agonistic site. Wash-out experiments and structure–activity analysis indicated, that the high-affinity chelators and the metal ions bind and activate the mutant receptor as metal ion guided ligand complexes. Because of the well-understood binding geometry of the small metal ions, an important distance constraint has here been imposed between TM-III and -VII in the active, signaling conformation of 7TM receptors. It is suggested that atoxic metal-ion chelator complexes could possibly in the future be used as generic, pharmacologic tools to switch 7TM receptors with engineered metal-ion sites on or off at will.

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

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

Ligand-dependent activation of transcription in vitro by retinoic acid receptor α/retinoid X receptor α heterodimers that mimics transactivation by retinoids in vivo

All-trans and 9-cis retinoic acids (RA) signals are transduced by retinoic acid receptor/retinoid X receptor (RAR/RXR) heterodimers that act as functional units controlling the transcription of RA-responsive genes. With the aim of elucidating the underlying molecular mechanisms, we have developed an in vitro transcription system using a chromatin template made up of a minimal promoter and a direct repeat with 5-spacing-based RA response element. RARα and RXRα were expressed in and purified from baculovirus-infected Sf9 cells, and transcription was carried out by using naked DNA or chromatin templates. Transcription from naked templates was not affected by the presence of RA and/or RAR/RXR heterodimers. In contrast, very little transcription occurred from chromatin templates in the absence of RA or RAR/RXR heterodimers whereas their addition resulted in a dosage-dependent stimulation of transcription that never exceeded that occurring on naked DNA templates. Most importantly, the addition of synthetic agonistic or antagonistic retinoids to the chromatin transcription system mimicked their stimulatory or inhibitory action in vivo, and activation by a RXR-specific retinoid was subordinated to the binding of an agonist ligand to the RAR partner. Moreover, the addition of the p300 coactivator generated a synergistic enhancement of transcription. Thus, the dissection of this transcription system ultimately should lead to the elucidation of the molecular mechanisms by which RAR/RXR heterodimers control transcription in a ligand-dependent manner.

An NO derivative of ursodeoxycholic acid protects against Fas-mediated liver injury by inhibiting caspase activity

Caspases are key mediators in liver inflammation and apoptosis. In the present study we provide evidence that a nitric oxide (NO) derivative of ursodeoxycholic acid (UDCA), NCX-1000 ([2-(acetyloxy)benzoic acid 3-(nitrooxymethyl)phenyl ester]), protects against liver damage in murine models of autoimmune hepatitis induced by i.v. injection of Con A or a Fas agonistic antibody, Jo2. Con A administration causes CD4+ T lymphocytes to accumulate in the liver and up-regulates FasL expression, resulting in FasL-mediated cytotoxicity. Cotreating mice with NCX-1000, but not with UDCA, protected against liver damage induced by Con A and Jo2, inhibited IL-1β, IL-18, and IFN-γ release and caspase 3, 8, and 9 activation. Studies on HepG2 cells demonstrated that NCX-1000, but not UDCA, directly prevented multiple caspase activation induced by Jo2. Incubating HepG2 cells with NCX-1000 resulted in intracellular NO formation and a DTT-reversible inhibition of proapoptotic caspases, suggesting that cysteine S-nitrosylation was the main mechanism responsible for caspase inhibition. Collectively, these data suggest that NCX-1000 protects against T helper 1-mediated liver injury by inhibiting both the proapoptotic and the proinflammatory branches of the caspase superfamily.

The road not taken*

The annual Janet Doe Lecture was established in l966 to honor Janet Doe, emerita librarian of the New York Academy of Medicine. The lecture focuses on either the history or philosophy of health sciences librarianship. This lecture addresses three fundamental values of the field, highlighting basic beliefs of the profession that are at risk: privacy, intellectual property rights, and access to quality information. It calls upon readers to make the everyday choices required to keep the value system of health sciences librarianship in place. Robert Frost's poignant poem ”The Road Not Taken” provides the metaphor for examining choices in an information economy.

Stimulation via CD40 can substitute for CD4 T cell function in preventing reactivation of a latent herpesvirus

Reactivation of latent herpesviruses is a particular problem in immunocompromised individuals, such as AIDS patients, who lack effective CD4 T helper cell function. An important question is whether residual immune defenses can be mobilized to combat such opportunistic infections, in the absence of CD4 T cells. In the present study, we used a mouse model of opportunistic infection to determine whether stimulation via CD40 could substitute for CD4 T cell function in preventing reactivation of a latent herpesvirus. Treatment with an agonistic antibody to CD40 was highly effective in preventing reactivation of latent murine gammaherpesvirus (MHV-68) in the lungs of CD4 T cell-deficient mice. CD8+ T cells were essential for this effect, whereas virus-specific serum antibody was undetectable and IFN-γ production was unchanged. This demonstration that immunostimulation via CD40 can replace CD4 T cell help in controlling latent virus in vivo has potential implications for the development of novel therapeutic agents to prevent viral reactivation in immunocompromised patients.

Heteromeric association creates a P2Y-like adenosine receptor

Adenosine and its endogenous precursor ATP are main components of the purinergic system that modulates cellular and tissue functions via specific adenosine and ATP receptors (P1 and P2 receptors), respectively. Although adenosine inhibits excitability and ATP functions as an excitatory transmitter in the central nervous system, little is known about the ability of P1 and P2 receptors to form new functional structures such as a heteromer to control the complex purinergic cascade. Here we have shown that Gi/o protein-coupled A1 adenosine receptor (A1R) and Gq protein-coupled P2Y1 receptor (P2Y1R) coimmunoprecipitate in cotransfected HEK293T cells, suggesting the oligomeric association between distinct G protein-coupled P1 and P2 receptors. A1R and P2Y2 receptor, but not A1R and dopamine D2 receptor, also were found to coimmunoprecipitate in cotransfected cells. A1R agonist and antagonist binding to cell membranes were reduced by coexpression of A1R and P2Y1R, whereas a potent P2Y1R agonist adenosine 5′-O-(2-thiotriphosphate) (ADPβS) revealed a significant potency to A1R binding only in the cotransfected cell membranes. Moreover, the A1R/P2Y1R coexpressed cells showed an ADPβS-dependent reduction of forskolin-evoked cAMP accumulation that was sensitive to pertussis toxin and A1R antagonist, indicating that ADPβS binds A1R and inhibits adenylyl cyclase activity via Gi/o proteins. Also, a high degree of A1R and P2Y1R colocalization was demonstrated in cotransfected cells by double immunofluorescence experiments with confocal laser microscopy. These results suggest that oligomeric association of A1R with P2Y1R generates A1R with P2Y1R-like agonistic pharmacology and provides a molecular mechanism for an increased diversity of purine signaling.

Cytotoxic analogs of luteinizing hormone-releasing hormone containing doxorubicin or 2-pyrrolinodoxorubicin, a derivative 500-1000 times more potent.

Doxorubicin (DOX) and its daunosamine-modified derivative, 2-pyrrolino-DOX, which is 500-1000 times more active than DOX, were incorporated into agonistic and antagonistic analogs of luteinizing hormone-releasing hormone (LH-RH). The conjugation of DOX with LH-RH analogs was performed by using N-(9-fluorenylmethoxycarbonyl)-DOX-14-O-hemiglutarate, a dicarboxylic acid ester derivative of DOX. Coupling this derivative covalently to the epsilon-amino group of the D-Lys side chain of agonist [D-Lys6]LH-RH or antagonistic analog AC-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys-Leu-Arg-Pro-D-Ala-NH 2 [where Nal(2) = 3-(2-naphthyl)alanine, Pal(3) = 3-(3-pyridyl)alanine, and Phe(4CI) = 4-chlorophenylalanine] was followed by the removal of the 9-fluorenylmethoxycarbonyl protective group to yield cytotoxic derivatives of LH-RH analogs containing DOX. From these DOX containing LH-RH hybrids, intensely potent analogs with daunosamine-modified derivatives of DOX can be readily formed. Thus, cytotoxic LH-RH agonist containing DOX (AN-152) can be converted in a 66% yield by a reaction with a 30-fold excess of 4-iodobutyraldehyde in N,N-dimethylformamide into a derivative having 2-pyrrolino-DOX (AN-207). Hybrid molecules AN-152 and AN-207 fully preserve the cytotoxic activity of their radicals, DOX or 2-pyrrolino-DOX, respectively, in vitro, and also retain the high binding affinity of the peptide hormone portion of the conjugates to rat pituitary receptors for LH-RH. These highly potent cytotoxic analogs of LH-RH were designed as targeted anti-cancer agents for the treatment of various tumors that possess receptors for the carrier peptide. Initial in vivo studies show that the hybrid molecules are much less toxic than the respective cytotoxic radicals incorporated and significantly more active in inhibiting tumor growth.

Bagaço de cana como enriquecimento ambiental para leitões recém desmamados

O objetivo do estudo foi avaliar o uso de bagaço de cana como enriquecimento ambiental para suínos a partir do comportamento e respostas fisiológicas do estresse causado pelo confinamento e mudança de ambiente, na fase de creche. O projeto foi conduzido no Laboratório de Biometeorologia e Etologia, da Faculdade de Zootecnia e Engenharia de Alimentos da Universidade de São Paulo, Pirassununga-SP, e no setor da Suinocultura da Prefeitura do Campus Administrativo Fernando Costa (PUSP-FC), entre os meses de abril e junho de 2015. Foram utilizados 66 leitões (NK75 X Naïma), machos e fêmeas desmamados aos 28 dias, separados em grupos homogêneos com relação ao peso, transferidos para baias da creche e distribuídos em dois tratamentos: Tratamento Enriquecido (TE) onde as baias foram fornecidas com cama profunda de bagaço de cana, de até 15 cm de profundidade e Tratamento Não Enriquecido (TNE) as baias foram utilizadas da forma convencional, sem cobertura no piso cimentado. Foram avaliadas diferentes respostas fisiológicas, especificamente, níveis de cortisol salivar, temperatura superficial por meio de um termómetro infravermelho e temperatura ocular a través de fotos termográficas. O comportamento dos leitões foi registrado e as análises das observações das atividades foram realizadas pelo efeito dos tratamentos e a interação do tempo. Os dados de desempenho dos animais foram analisados, igualmente como o Ganho de peso diário (GPD) e a conversão alimentar (CA). Animais que receberam enriquecimento ambiental apresentaram concentrações de cortisol mais baixas (P<0,001) durante a primeira semana pós-desmama. A partir da segunda semana pós desmama até o final da fase da creche houve efeito do tratamento (P<0,05), encontrando níveis de cortisol até quatro vezes mais altos para o TNE referentes aos níveis basais, enquanto o TE continua tendo níveis mais baixos (P<0,05). Animais que receberam enriquecimento ambiental tiveram maior frequência em comportamentos exploratórios (P<0,05) e maior atividade brincando (P<0,05) durante toda a fase experimental. Leitões que foram criados em baias pobres manifestaram maior frequência em comportamentos agonísticos (P<0,05) e em repouso (P<0,05). A correlação entre a temperatura superficial do dorso e termografia ocular indicou uma associação moderada positiva (P<0,0001) com a temperatura ocular mínima (r=0,43) e máxima (r=0,41). Apesar de não existir diferença estatística para o desempenho entre tratamentos (P>0,05), o TE apresentou maior ganho de peso diário (0,47±0,015 kg.dia-1) e total (23,47±0,73 kg.dia-1). A conversão alimentar foi maior no TE (2,88±0,25), provavelmente porque os leitões precisavam de mais alimento para compensar a energia gasta pela sua atividade de fuçar e brincar. Ambientes enriquecidos durante a fase da creche melhoram o bem-estar dos animais em confinamento, diminuindo o estresse pela desmama, motivando o animal a expressar comportamentos próprios da espécie suína, tais como fuçar e explorar.

Comportamento e desempenho sexual de suínos reprodutores criados em ambientes enriquecidos

Este trabalho buscou avaliar o comportamento e o desempenho sexual de suínos machos de linhas puras e cruzadas, criados com e sem a utilização de enriquecimento ambiental, na fase de crescimento. A pesquisa foi dividida em duas etapas, que compreenderam a fase de crescimento dos animais e o treinamento para coleta de sêmen. Na fase de crescimento, 128 machos foram alojados em ambientes enriquecidos ou estéreis. Utilizou-se como enriquecimento ambiental correntes suspensas, galão de cinco litros suspenso e um galão de 50 litros solto no piso. Esses objetos foram oferecidos de forma alternada e cada um ficou disponível na baia por um período de 30 dias. Na primeira etapa foram registrados o comportamento dos animais, os escores de lesão e a massa corporal. Após a fase de crescimento, foram escolhidos aleatoriamente 32 animais aprovados na seleção genética para serem avaliados durante o treinamento para coleta de sêmen. O treinamento ocorreu durante seis dias consecutivos e cada animal foi treinado por três vezes em dias alternados. Durante o treinamento para a coleta de sêmen, o comportamento animal, as relações humano-animal, o volume do ejaculado e os níveis de testosterona e cortisol foram registrados. Como respostas na fase de crescimento, verificou-se que, mesmo utilizando uma combinação de objetos, os suínos se habituaram rapidamente a eles e a frequência de manipulação diminuiu após o primeiro período para todos os objetos. Observamos que o ambiente enriquecido foi eficaz na redução dos comportamentos agonísticos e mordedura de cauda e orelha para os animais puros e cruzados, e isso consequentemente reduziu a quantidade e severidade de lesões de pele. Na fase de treinamento para coleta de sêmen, os resultados demonstraram que o comportamento sexual dos animais foi influenciado pelas linhas genéticas, sendo assim, observou-se que os machos de linha cruzada tiveram maior facilidade durante o treinamento para coleta de sêmen e apresentaram maior média do escore de libido, diferindo das linhas puras (P<0,001). Verificou-se que não houve diferença na média do escore de libido entre os tratamentos com e sem enriquecimento ambiental (P=0,276), porém, os tratamentos com enriquecimento tiveram o menor número de animais treinados. Dessa forma, os resultados indicam que o ambiente enriquecido com uma combinação de enriquecimentos pontuais (objetos) é uma estratégia eficaz para aumentar o comportamento exploratório e reduzir os comportamentos agonísticos e anormais na fase de crescimento. Mas, por outro lado, os animais criados em ambientes enriquecidos tiveram um pior desempenho sexual durante o treinamento para coleta de sêmen.

Tecer amizade, habitar o deserto: uma etnografia do quilombo Família Magalhães (GO)

Esta tese é o resultado de uma etnografia sobre a comunidade negra rural Família Magalhães (Nova Roma-GO), originária do território Kalunga. Procurei discutir, tendo em vista o reconhecimento do grupo como quilombola perante o Estado, formas específicas pelas quais ele produz relações entre parentes e não parentes. No último caso, me refiro a agentes do governo federal e estadual, presidentes da república, deputados, procuradores, advogados, prefeitos, vereadores e, também, a conhecidos, vizinhos, compadres e correligionários. Nessa trama, tocar amizade e fazer política aparecem como modos privilegiados de tecer territórios, entendidos em seu caráter relacional, sempre passíveis de serem atravessados por relações de caráter agonístico. Assim, investiguei como são geridos, entre os membros de Família Magalhães, movimentos contínuos de produção de vínculos e segmentações, trazendo à tona agenciamentos específicos do grupo em suas experiências de alteridade.

Time's Social Metaphors: An empirical research

The article addresses the analysis of time images furnished by a qualitative research made in Spain on the relations of working time and family/personal time. The analysis focuses on three widespread time metaphors used in day-to-day speeches by social agents. The first one is the metaphor of time as resource for action. Its value is equally economical, moral and political. Used in different context of action, it may mean something that can be either invested, donated generously to others, appropriated for caring for oneself, or spent without purpose with others. The second metaphor represents time as an external environment to which action must adapt. This metaphor shows many variants that represent time as a dynamic/static, repetitive/innovative, ordered/chaotic environment. In this external environment, the agents must resolve the problems of temporal embeddedness, hierarchy and synchronization of their actions. The third metaphor shows time as a horizon of action intentionality where the agents try to construct the meaning of their action and identity. Within this horizon the construction of a significant narrative connecting past and present experiences with future expectations is possible.

The Sound of the Digital Global City

Madrid has been the center of Spanish musical scene and industry since the 80s, when “la movida” becomes the metaphor for the new colorful, young and cosmopolitan country established with the arrival of democracy. The city, in this way, is basically a place. But this sense of place started to crash with the arrival of digital music. In the new paradigm, intermediaries were supposed to disappear and music was something contained in networks and computers. The question now is how to integrate digital music, a nonphysical, individual experience, with the way in which the city of Madrid is lived through in musical terms. With the advent of digital music, concerts became the primary source of income for musicians. The centrality of the gig can be understood as the confirmation that we are living in an economy of experience. This centrality also reorganized the way in which music is produced and consumed: now, records are produced in order to create the opportunity of a musical event (band promote their tour as presentation of their latest recordings) that can be promoted in social networks and media; concerts are the places where musicians construct their fans’ communities and are the places were records are sold, not a way to know the band but to demonstrate both the support for the band and the status of the listeners. To study the place of music in the process of metropolization in Madrid we need to understand music as a field of tension

Cark

A collection of poems with critical preface. The author expresses concern for responsibilities and obligations resulting from utterance and offers a means of reading poetry in light of such concerns. Lyric theory and the legacy of Language poetry with regards to the lyric are loci in a discussion of contemporary poetics. It analyzes the work of poets Tomaz Salamun and Lyn Hejinian, in relation to theorists Theodor Adorno and Maurice Merleau-Ponty, to articulate a poetics specific to the poems in the collection. The poetics is described via the literary and anthropological uses of metaphor, which are employed to unify text, writer, and reader. The phenomenology of ritual and ritual theory address these contingents to conclude the preface. The collection of poems is divided into three sections, each a distinct, interrelated collection of poetic modes.