Mechanisms of internalization and recycling of the chemokine receptor, CCR5

CCR5 is a G protein-coupled receptor that binds several natural chemokines but it is also a coreceptor for the entry of M tropic strains of HIV-1 into cells. Levels of CCR5 on the cell surface are important for the rate of HIV-1 infection and are determined by a number of factors including the rates of CCR5 internalization and recycling. Here we investigated the involvement of the actin cytoskeleton in the control of ligand-induced internalization and recycling of CCR5. Cytochalasin D, an actin depolymerizing agent, inhibited chemokine-induced internalization of CCR5 and recycling of the receptor in stably transfected CHO cells and in the monocytic cell line, THP-1. CCR5 internalization and recycling were inhibited by Toxin B and C-3 exoenzyme treatment in CHO and THP-1 cells, confirming activation of members of the RhoGTPase family by CCR5. The specific Rho kinase inhibitor Y27632, however, had no effect on CCR5 internalization or recycling. Ligand-induced activation of CCR5 leads to Rho kinase-dependent formation of focal adhesion complexes. These data indicate that CCR5 internalization and recycling are regulated by actin polymerization and activation of small G proteins in a Rho-dependent manner.

The chemokine receptor, CCR5

The chemokine receptor, CCR5, is a G protein coupled receptor responsible for some of the effects of the chemokines CCL3, CCL4 and CCL5. It is also one of the co-receptors for the entry of human immunodeficiency virus-1 (HIV-1) into cells. Regulation of CCR5 number on cells is, therefore, important for determining the infection rate by HIV-1. (C) 2003 Elsevier Ltd. All rights reserved.

Diverse signalling by different chemokines through the chemokine receptor CCR5

We have investigated the signalling properties of the chemokine receptor, CCR5, using several assays for agonism: stimulation of changes in intracellular Ca(2+) or CCR5 internalisation in CHO cells expressing CCR5 or stimulation of [(35)S]GTPgammaS binding in membranes of CHO cells expressing CCR5. Four isoforms of the chemokine CCL3 with different amino termini (CCL3, CCL3(2-70), CCL3(5-70), CCL3L1) were tested in these assays in order to probe structure/activity relationships. Each isoform exhibited agonism. The pattern of agonism (potency, maximal effect) was different in the three assays, although the rank order was the same with CCL3L1 being the most potent and efficacious. The data show that the amino terminus of the chemokine is important for signalling. A proline at position 2 (CCL3L1) provides for high potency and efficacy but the isoform with a serine at position 2 (CCL3(2-70)) is as efficacious in some assays showing that the proline is not the only determinant of high efficacy. We also increased the sensitivity of CCR5 signalling by treating cells with sodium butyrate, thus increasing the receptor/G protein ratio. This allowed the detection of a change in intracellular Ca(2+) after treatment with CCL7 and Met-RANTES showing that these ligands possess measurable but low efficacy. This study therefore shows that sodium butyrate treatment increases the sensitivity of signalling assays and enables the detection of efficacy in ligands previously considered as antagonists. The use of different assay systems, therefore, provides different estimates of efficacy for some ligands at this receptor.

Analysis of second messenger pathways stimulated by different chemokines acting at the chemokine receptor

The chemokine receptor, CCR5, responds to several chemokines leading to changes in activity in several signalling pathways. Here, we investigated the ability of different chemokines to provide differential activation of pathways. The effects of five CC chemokines acting at CCR5 were investigated for their ability to inhibit forskolin- stimulated 3'-5'-cyclic adenosine monophosphate (cAMP) accumulation and to stimulate Ca2+ mobilisation. in Chinese hamster ovary (CHO) cells expressing CCR5. Macrophage inflammatory protein 1 alpha (D26A) (MIP-1 alpha (D26A), CCL3 (D26A)), regulated on activation, normal T-cell expressed and secreted (RANTES, CCLS), MIP-1 beta (CCL4) and monocyte chemoattractant protein 2 (MCP-2, CCL8) were able to inhibit forskolin -stimulated CAMP accumulation, whilst MCP-4 (CCL13) could not elicit a response. CCL3 (D26A), CCL4, CCLS, CCL8 and CCL13 were able to stimulate Ca2+ mobilisation. through CCRS, although CCL3 (D26A) and CCL5 exhibited biphasic concentration-response curves. The Ca2+ responses induced by CCL4, CCL5, CCL8 and CCL13 were abolished by pertussis toxin, whereas the response to CCL3 (D26A) was only partially inhibited by pertussis toxin, indicating G(i/o)-independent signalling induced by this chemokine. Although the rank order of potency of chemokines was similar between the two assays, certain chemokines displayed different pharmacological profiles in cAMP inhibition and Ca2+ mobilisation assays. For instance, whilst CCL13 could not inhibit forskolin-stimulated cAMP accumulation, this chemokine was able to induce Ca2+ mobilisation via CCR5. It is concluded that different chemokines acting at CCR5 can induce different pharmacological responses, which may account for the broad spectrum of chemokines that can act at CCRS. (C) 2007 Elsevier Inc. All rights reserved.

Allosteric effects of antagonists on signalling by the chemokine receptor CCR5

Antagonists of the chemokine receptor, CCRS, may provide important new drugs for the treatment of HIV-1. In this study we have examined the mechanism of action of two functional antagonists of the chemokine receptor CCRS (UK-396,794, UK-438,235) in signalling and internalisation assays using CHO cells expressing CCR5. Both compounds were potent inverse agonists versus agonist-independent [S-3]GTP gamma S binding to membranes of CHO cells expressing CCR5. Both compounds also acted as allosteric inhibitors of CCL5 (RANTES) and CCL8 (MCP-2) -stimulated [S-35]GTP gamma S binding to CHO-CCR5 membranes, reducing the potency and maximal effects of the two chemokines. The data are consistent with effects of the allosteric inhibitors on both the binding and signalling of the chemokines. Both compounds inhibited CCR5 internalisation triggered by chemokines. When CHO-CCR5 cells were treated with either of the two compounds for prolonged periods of time (24 h) an increase (similar to 15%) in cell surface CCRS was detected. (C) 2007 Elsevier Inc. All rights reserved

An assessment of upper ocean salinity content from the ocean reanalyses inter-comparison project (ORA-IP)

Many institutions worldwide have developed ocean reanalyses systems (ORAs) utilizing a variety of ocean models and assimilation techniques. However, the quality of salinity reanalyses arising from the various ORAs has not yet been comprehensively assessed. In this study, we assess the upper ocean salinity content (depth-averaged over 0–700 m) from 14 ORAs and 3 objective ocean analysis systems (OOAs) as part of the Ocean Reanalyses Intercomparison Project. Our results show that the best agreement between estimates of salinity from different ORAs is obtained in the tropical Pacific, likely due to relatively abundant atmospheric and oceanic observations in this region. The largest disagreement in salinity reanalyses is in the Southern Ocean along the Antarctic circumpolar current as a consequence of the sparseness of both atmospheric and oceanic observations in this region. The West Pacific warm pool is the largest region where the signal to noise ratio of reanalysed salinity anomalies is >1. Therefore, the current salinity reanalyses in the tropical Pacific Ocean may be more reliable than those in the Southern Ocean and regions along the western boundary currents. Moreover, we found that the assimilation of salinity in ocean regions with relatively strong ocean fronts is still a common problem as seen in most ORAs. The impact of the Argo data on the salinity reanalyses is visible, especially within the upper 500m, where the interannual variability is large. The increasing trend in global-averaged salinity anomalies can only be found within the top 0–300m layer, but with quite large diversity among different ORAs. Beneath the 300m depth, the global-averaged salinity anomalies from most ORAs switch their trends from a slightly growing trend before 2002 to a decreasing trend after 2002. The rapid switch in the trend is most likely an artefact of the dramatic change in the observing system due to the implementation of Argo.

The Ocean Reanalysis Intercomparison project (ORA-IP)

Uncertainty in ocean analysis methods and deficiencies in the observing system are major obstacles for the reliable reconstruction of the past ocean climate. The variety of existing ocean reanalyses is exploited in a multi-reanalysis ensemble to improve the ocean state estimation and to gauge uncertainty levels. The ensemble-based analysis of signal-to-noise ratio allows the identification of ocean characteristics for which the estimation is robust (such as tropical mixed-layer-depth, upper ocean heat content), and where large uncertainty exists (deep ocean, Southern Ocean, sea ice thickness, salinity), providing guidance for future enhancement of the observing and data assimilation systems.

Intercomparison of the Arctic sea ice cover in global ocean–sea ice reanalyses from the ORA-IP project

Ocean–sea ice reanalyses are crucial for assessing the variability and recent trends in the Arctic sea ice cover. This is especially true for sea ice volume, as long-term and large scale sea ice thickness observations are inexistent. Results from the Ocean ReAnalyses Intercomparison Project (ORA-IP) are presented, with a focus on Arctic sea ice fields reconstructed by state-of-the-art global ocean reanalyses. Differences between the various reanalyses are explored in terms of the effects of data assimilation, model physics and atmospheric forcing on properties of the sea ice cover, including concentration, thickness, velocity and snow. Amongst the 14 reanalyses studied here, 9 assimilate sea ice concentration, and none assimilate sea ice thickness data. The comparison reveals an overall agreement in the reconstructed concentration fields, mainly because of the constraints in surface temperature imposed by direct assimilation of ocean observations, prescribed or assimilated atmospheric forcing and assimilation of sea ice concentration. However, some spread still exists amongst the reanalyses, due to a variety of factors. In particular, a large spread in sea ice thickness is found within the ensemble of reanalyses, partially caused by the biases inherited from their sea ice model components. Biases are also affected by the assimilation of sea ice concentration and the treatment of sea ice thickness in the data assimilation process. An important outcome of this study is that the spatial distribution of ice volume varies widely between products, with no reanalysis standing out as clearly superior as compared to altimetry estimates. The ice thickness from systems without assimilation of sea ice concentration is not worse than that from systems constrained with sea ice observations. An evaluation of the sea ice velocity fields reveals that ice drifts too fast in most systems. As an ensemble, the ORA-IP reanalyses capture trends in Arctic sea ice area and extent relatively well. However, the ensemble can not be used to get a robust estimate of recent trends in the Arctic sea ice volume. Biases in the reanalyses certainly impact the simulated air–sea fluxes in the polar regions, and questions the suitability of current sea ice reanalyses to initialize seasonal forecasts.

Melatonin and IP(3)-induced Ca(2+) Release from Intracellular Stores in the Malaria Parasite Plasmodium falciparum within Infected Red Blood Cells

IP(3)-dependent Ca(2+) signaling controls a myriad of cellular processes in higher eukaryotes and similar signaling pathways are evolutionarily conserved in Plasmodium, the intracellular parasite that causes malaria. We have reported that isolated, permeabilized Plasmodium chabaudi, releases Ca(2+) upon addition of exogenous IP(3). In the present study, we investigated whether the IP(3) signaling pathway operates in intact Plasmodium falciparum, the major disease-causing human malaria parasite. P. falciparum-infected red blood cells (RBCs) in the trophozoite stage were simultaneously loaded with the Ca(2+) indicator Fluo-4/AM and caged-IP(3). Photolytic release of IP(3) elicited a transient Ca(2+) increase in the cytosol of the intact parasite within the RBC. The intracellular Ca(2+) pools of the parasite were selectively discharged, using thapsigargin to deplete endoplasmic reticulum (ER) Ca(2+) and the antimalarial chloroquine to deplete Ca(2+) from acidocalcisomes. These data show that the ER is the major IP(3)-sensitive Ca(2+) store. Previous work has shown that the human host hormone melatonin regulates P. falciparum cell cycle via a Ca(2+)-dependent pathway. In the present study, we demonstrate that melatonin increases inositol-polyphosphate production in intact intraerythrocytic parasite. Moreover, the Ca(2+) responses to melatonin and uncaging of IP(3) were mutually exclusive in infected RBCs. Taken together these data provide evidence that melatonin activates PLC to generate IP(3) and open ER-localized IP(3)-sensitive Ca(2+) channels in P. falciparum. This receptor signaling pathway is likely to be involved in the regulation and synchronization of parasite cell cycle progression.

Differential expression of cytokines, chemokines and chemokine receptors in patients with coronary artery disease

Monocytes/macrophages and lymphocytes have a key role in the pathogenesis of atherosclerosis through the production of inflammatory and anti-inflammatory cytokines. We evaluated mRNA expression and protein production of CCL2, CXCL8, CXCL9, CXCL10, IFN-gamma and IL-10 in vitro as well as the expression of the CCR2 and CXCR3 receptors in peripheral blood mononuclear cells (PBMCs) of patients with coronary artery disease (CAD) and healthy controls in the presence or absence of oxidized LDL (oxLDL). Patients with CAD showed higher constitutive expression of CCL2, CXCL8, CXCL9, CXCL10 and IFN-gamma mRNA and, after stimulation with oxLDL, higher expression of CCL2 and CXCL8 mRNA than the control group. We also detected higher levels of CCL2 and CXCL8 in supernatants of oxLDL-stimulated PBMCs from CAD patients than in corresponding supernatants from controls. Patients with CAD had a higher percentage of constitutive CCR2(+) and CXCR3(+) cells after stimulation with oxLDL. Among CAD patients, the main differences between the stable (SA) and unstable angina (UA) groups were lower IL-10 mRNA production in the latter group. Altogether, our data suggest that PBMCs from CAD patients are able to produce higher concentrations of chemokines and cytokines involved in the regulation of monocyte and lymphocyte migration and retention in atherosclerotic lesions. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

IP network usage accounting

De modo a manter políticas de utilização aceitável dos seus serviços de Internet, a NOS Madeira tem usado um sistema de fabrico próprio onde os clientes são catalogados de acordo com o tráfego que realizam. Contudo, esse sistema tornou-se demasiado antigo para as necessidades atuais da empresa. Usava tecnologias descontinuadas, não tinha interfaces de integração, faltava modularidade e não tinha a flexibilidade necessária para expandir as regras de negócio. Este projeto centra-se na implementação de um dos três subsistemas que substituem o sistema antigo: o subsistema controlador. O objetivo é modernizar, facilitar a manutenção e garantir maior flexibilidade. Tudo isto com recurso a linguagens de programação atuais como o PHP, ferramentas como a Zend Framework e mantendo em mente as melhores práticas de programação. São apresentados a especificação e modelação do sistema, assim como todos os detalhes da implementação em conjunto com as decisões e problemas encontrados. Os testes e resultados, incluindo a entrada com sucesso em produção do sistema, juntamente com sugestões de melhorias futuras concluem este trabalho.

IP network usage accounting, parte 2: accounting system

O controlo de banda larga é um conceito importante quando lidamos com redes de larga escala. Os ISPs precisam de garantir disponibilidade e qualidade de serviço a todos os clientes, enquanto garantem que a rede como um todo não fica mais lenta. Para garantir isto, é necessário que os ISPs recolham dados de tráfego, analisem-nos e usem-nos para definir a velocidade de banda larga de cada cliente. A NOS Madeira implementou, durante vários anos, um sistema semelhante. No entanto, este sistema encontrava-se obsoleto, sendo necessário construir um novo, totalmente de raíz. Entre as limitações encontrava-se a impossibilidade de alterar os algoritmos de análise de tráfego, fraca integração com os serviços de gestão de rede da NOS Madeira e reduzida escalabilidade e modularidade. O sistema IP Network Usage Accounting é a resposta a estes problemas. Este projeto foca-se no desenvolvimento do subsistema Accounting System, o segundo dos três subsistemas que compõem o sistema IP Network Usage Accounting. Este subsistema, implementado com sucesso e atualmente em produção na NOS Madeira, é responsável por analisar os dados referidos acima e usar os resultados dessa análise para direcionar a disponibilidade de banda larga, de acordo com o uso da rede de cada cliente.

Compostos orgânicos como substratos na formação de mudas de ipê-amarelo (Tabebuia Chrysotricha (Mart. Ex. Dc.) Standl) irrigadas com água residuária

A necessidade de estudar a utilização dos resíduos de podas de árvores é de grande importância ambiental para solucionar os problemas de resíduos sólidos existentes nas áreas urbanas junto com os resíduos de lixos domésticos. O estudo destes materiais foi avaliado com o desenvolvimento de mudas de ipê-amarelo (Tabebuia Chrysotricha (Mart. Ex. Dc.) sandl) em diferentes misturas de substratos e tipos de água para irrigação. O experimento foi instalado no Departamento de Engenharia Rural da Faculdade de Ciências Agrárias e Veterinárias - UNESP, Câmpus de Jaboticabal. Foram realizados dois experimentos, avaliados juntamente com delineamento experimental em blocos casualizados, de oito substratos, duas qualidades de águas e quatro repetições, totalizando 64 parcelas. Cada parcela foi composta por 30 plantas (cinco linhas de seis plantas), sendo consideradas como úteis as três linhas de quatro plantas centrais da parcela. Foram testados oito substratos, resultantes da combinação de substrato comercial, composto de lixo e composto de poda de árvores com dois tipos de água de irrigação (água potável e residuária) e quatro repetições. Para acompanhar o desenvolvimento das mudas de ipê-amarelo, foram avaliados a altura da parte aérea das plantas (H) e o diâmetro do colmo (D). As características foram avaliadas aos 21; 42; 63 e 84 dias após a emergência. da análise dos resultados, possibilitou-se concluir que os substratos estudados promoveram diferenças significativas para a altura média das plantas e o diâmetro de colmo, em todos os períodos de avaliação. Os substratos 4 e 5 e a água residuária apresentaram os melhores resultados no desenvolvimento das mudas de ipê-amarelo.

Vermiculita como substrato para o teste de germinação de sementes de ipê-amarelo

Vermiculite is a substrate used in seedling production of forest species and can be an option for the germination test of ipe seeds. The objective of this research was to evaluate the use of vermiculite in the germination test of Tabebuia chrysotricha seeds and to stablish appropriate particle size and moisture for this substrate. Four replications of 20 seeds were sown in soil, sand, paper rolls, and vermiculite with different particle sizes: micron (0.15-0.20 mm), superthin (0.21-0.30 mm), thin (0.30-0.50 mm) and medium (0.50-1.19 mm) moistened with water equivalent to 0.5, 1.0, 1.5 and 2.0 times the weight of the dry subtrate. Germination test was carried out at 30 degrees C. The percentage of normal seedlings was obtained seven days after sowing (first count) and then weekly until 28 days, when abnormal seedlings and dead seeds were also counted. It was concluded that the germination test of ipe seeds can be carried out with thin or medium vermiculite moistened with water equivalent to 1.5 times the dry weight of the substrate. These treatments resulted in higher and faster germination (21 days).

Efeito do tamanho de tubetes na qualidade de mudas de Jatobá (Hymenaea courbaril L. var. stilbocarpa (Hayne) Lee et Lang.), Ipê-amarelo (Tabebuia chrysotricha (Mart. ex DC.) Sandl.) e Guarucaia (Parapiptadenia rigida (Benth.) Brenan)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)