Interferon β transduction of peripheral blood lymphocytes from HIV-infected donors increases Th1-type cytokine production and improves the proliferative response to recall antigens

We are developing a gene therapy method of HIV infection based on the constitutive low production of interferon (IFN) β. Peripheral blood lymphocytes (PBL) from HIV-infected patients at different clinical stages of infection were efficiently transduced with the HMB-HbHuIFNβ retroviral vector. The constitutive low production of IFN-β in cultured PBL from HIV-infected patients resulted in a decreased viral production and an enhanced survival of CD4+ cells, and this protective effect was observed only in the PBL derived from donors having a CD4+ cell count above 200 per mm3. In IFN-β-transduced PBL from healthy and from HIV-infected donors, the production of the Th1-type cytokines IFN-γ and interleukin (IL)-12 was enhanced. In IFN-β-transduced PBL from HIV-infected donors, the production of IL-4, IL-6, IL-10, and tumor necrosis factor α was maintained at normal levels, contrary to the increased levels produced by the untransduced PBL. The proliferative response to recall antigens was partially restored in IFN-β-transduced PBL from donors with an impaired antigen response. Thus, in addition to inhibiting HIV replication, IFN-β transduction of PBL from HIV-infected donors improves several parameters of immune function.

Localization and activity of lysyl oxidase within nuclei of fibrogenic cells

Lysyl oxidase (EC 1.4.3.13) oxidizes peptidyl lysine to peptidyl aldehyde residues within collagen and elastin, thus initiating formation of the covalent cross-linkages that insolubilize these extracellular proteins. Recent findings raise the possibility that this enzyme may also function intracellularly. The present study provides evidence by immunocytochemical confocal microscopy, Western blot analysis, enzyme assays, and chemical analyses for lysyl oxidase reaction products that this enzyme is present and active within rat vascular smooth muscle cell nuclei. Confocal microscopy indicates its presence within nuclei of 3T3 fibroblasts, as well.

Activation of a Frizzled-2/β-adrenergic receptor chimera promotes Wnt signaling and differentiation of mouse F9 teratocarcinoma cells via Gαo and Gαt

The frizzled gene family of putative Wnt receptors encodes proteins that have a seven-transmembrane-spanning motif characteristic of G protein-linked receptors, though no loss-of-function studies have demonstrated a requirement for G proteins for Frizzled signaling. We engineered a Frizzled-2 chimera responsive to β-adrenergic agonist by using the ligand-binding domains of the β2-adrenergic receptor. The expectation was that the chimera would be sensitive both to drug-mediated activation and blockade, thereby circumventing the problem of purifying soluble and active Wnt ligand to activate Frizzled. Expression of the chimera in zebrafish embryos demonstrated isoproterenol (ISO)-stimulated, propranolol-sensitive calcium transients, thereby confirming the β-adrenergic nature of Wnt signaling by the chimeric receptor. Because F9 embryonic teratocarcinoma cells form primitive endoderm after stable transfection of Frizzled-2 chimera and stimulation with ISO, they were subject to depletion of G protein subunits. ISO stimulation of endoderm formation of F9 stem cells expressing the chimeric receptor was blocked by pertussis toxin and by oligodeoxynucleotide antisense to Gαo, Gαt2, and Gβ2. Our results demonstrate the requirement of two pertussis toxin-sensitive G proteins, Gαo and Gαt, for signaling by the Frizzled-2 receptor.

Mapping the intrinsic curvature and flexibility along the DNA chain

The energy of DNA deformation plays a crucial and active role in its packaging and its function in the cell. Considerable effort has gone into developing methodologies capable of evaluating the local sequence-directed curvature and flexibility of a DNA chain. These studies thus far have focused on DNA constructs expressly tailored either with anomalous flexibility or curvature tracts. Here we demonstrate that these two structural properties can be mapped also along the chain of a “natural” DNA with any sequence on the basis of its scanning force microscope (SFM) images. To know the orientation of the sequence of the investigated DNA molecules in their SFM images, we prepared a palindromic dimer of the long DNA molecule under study. The palindromic symmetry also acted as an internal gauge of the statistical significance of the analysis carried out on the SFM images of the dimer molecules. It was found that although the curvature modulus is not efficient in separating static and dynamic contributions to the curvature of the population of molecules, the curvature taken with its direction (its sign in two dimensions) permits the direct separation of the intrinsic curvature from the flexibility contributions. The sequence-dependent flexibility seems to vary monotonically with the chain's intrinsic curvature; the chain rigidity was found to modulate as its local thermodynamic stability and does not correlate with the dinucleotide chain rigidities evaluation made from x-ray data by other authors.

Vaccination for protection of retinal ganglion cells against death from glutamate cytotoxicity and ocular hypertension: Implications for glaucoma

Our group recently demonstrated that autoimmune T cells directed against central nervous system-associated myelin antigens protect neurons from secondary degeneration. We further showed that the synthetic peptide copolymer 1 (Cop-1), known to suppress experimental autoimmune encephalomyelitis, can be safely substituted for the natural myelin antigen in both passive and active immunization for neuroprotection of the injured optic nerve. Here we attempted to determine whether similar immunizations are protective from retinal ganglion cell loss resulting from a direct biochemical insult caused, for example, by glutamate (a major mediator of degeneration in acute and chronic optic nerve insults) and in a rat model of ocular hypertension. Passive immunization with T cells reactive to myelin basic protein or active immunization with myelin oligodendrocyte glycoprotein-derived peptide, although neuroprotective after optic nerve injury, was ineffective against glutamate toxicity in mice and rats. In contrast, the number of surviving retinal ganglion cells per square millimeter in glutamate-injected retinas was significantly larger in mice immunized 10 days previously with Cop-1 emulsified in complete Freund's adjuvant than in mice injected with PBS in the same adjuvant (2,133 ± 270 and 1,329 ± 121, respectively, mean ± SEM; P < 0.02). A similar pattern was observed when mice were immunized on the day of glutamate injection (1,777 ± 101 compared with 1,414 ± 36; P < 0.05), but not when they were immunized 48 h later. These findings suggest that protection from glutamate toxicity requires reinforcement of the immune system by antigens that are different from those associated with myelin. The use of Cop-1 apparently circumvents this antigen specificity barrier. In the rat ocular hypertension model, which simulates glaucoma, immunization with Cop-1 significantly reduced the retinal ganglion cell loss from 27.8% ± 6.8% to 4.3% ± 1.6%, without affecting the intraocular pressure. This study may point the way to a therapy for glaucoma, a neurodegenerative disease of the optic nerve often associated with increased intraocular pressure, as well as for acute and chronic degenerative disorders in which glutamate is a prominent participant.

Gardenification of tropical conserved wildlands: Multitasking, multicropping, and multiusers

Tropical wildlands and their biodiversity will survive in perpetuity only through their integration into human society. One protocol for integration is to explicitly recognize conserved tropical wildlands as wildland gardens. A major way to facilitate the generation of goods and services by a wildland garden is to generate a public-domain Yellow Pages for its organisms. Such a Yellow Pages is part and parcel of high-quality search-and-delivery from wildland gardens. And, as they and their organisms become better understood, they become higher quality biodiversity storage devices than are large freezers. One obstacle to wildland garden survival is that specific goods and services, such as biodiversity prospecting, lack development protocols that automatically shunt the profits back to the source. Other obstacles are that environmental services contracts have the unappealing trait of asking for the payment of environmental credit card bills and implying delegation of centralized governmental authority to decentralized social structures. Many of the potential conflicts associated with wildland gardens may be reduced by recognizing two sets of social rules for perpetuating biodiversity and ecosystems, one set for the wildland garden and one set for the agroscape. In the former, maintaining wildland biodiversity and ecosystem survival in perpetuity through minimally damaging use is paramount, while in the agroscape, wild biodiversity and ecosystems are tools for a healthy and productive agroecosystem, and the loss of much of the original is acceptable.

Structural aspects of activation pathways of aspartic protease zymogens and viral 3C protease precursors

The three-dimensional structures of the inactive protein precursors (zymogens) of the serine, cysteine, aspartic, and metalloprotease classes of proteolytic enzymes are known. Comparisons of these structures with those of the mature, active proteases reveal that, in general, the preformed, active conformations of the residues involved in catalysis are rendered sterically inaccessible to substrates by the residues of the zymogens’ N-terminal extensions or prosegments. The prosegments interact in nonsubstrate-like fashions with the residues of the active sites in most of the cases. The gastric aspartic proteases have a well-characterized zymogen conversion pathway. Structures of human progastricsin, the inactive intermediate 2, and active human pepsin are known and have been used to define the conversion pathway. The structure of the zymogen precursor of plasmepsin II, the malarial aspartic protease, shows a new twist on the mode of inactivation used by the gastric zymogens. The prosegment of proplasmepsin disrupts the active conformation of the two catalytic aspartic acid residues by inducing a major reorientation of the two domains of the mature protease. The picornaviral 2A and 3C proteases have a chymotrypsin-like tertiary structure but with a cysteine nucleophile. These enzymes cleave themselves from the viral polyprotein in cis (intramolecular cleavage) and carry out trans cleavages of other scissile peptides important for the virus life cycle. Although the structure of the precursor viral polyprotein is unknown, it probably resembles the organization of the proenzymes of the bacterial serine proteases, subtilisin, and α-lytic protease. Cleavage of the prosegment is known to occur in cis for these precursor molecules.

Comparison of Binding Properties and Early Biological Effects of Elicitins in Tobacco Cells1

Elicitins are a family of small proteins secreted by Phytophthora species that have a high degree of homology and elicit defense reactions in tobacco (Nicotiana tabacum). They display acidic or basic characteristics, the acidic elicitins being less efficient in inducing plant necrosis. In this study we compared the binding properties of four elicitins (two basic and two acidic) and early-induced signal transduction events (Ca2+ influx, extracellular medium alkalinization, and active oxygen species production). The affinity for tobacco plasma membrane-binding sites and the number of binding sites were similar for all four elicitins. Furthermore, elicitins compete with one another for binding sites, suggesting that they interact with the same receptor. The four elicitins induced Ca2+ influx, extracellular medium alkalinization, and the production of active oxygen species in tobacco cell suspensions, but the intensity and kinetics of these effects were different from one elicitin to another. As a general observation the concentrations that induce similar levels of biological activities were lower for basic elicitins (with the exception of cinnamomin-induced Ca2+ uptake). The qualitative similarity of early events induced by elicitins indicates a common transduction scheme, whereas fine signal transduction tuning is different in each elicitin.

Membrane healing and restoration of contractility after mechanical injury in isolated skeletal muscle fibers of the frog.

In single isolated skeletal muscle fibers of the frog, we studied (i) the recovery from large sarcolemmal mechanical injuries of the response to electric stimulation and (ii) the integrity of the sarcolemma under the light microscope. In Ringer's solution, the damaged cells stopped contracting and deteriorated completely within 1 hr. In the presence of phosphatidylcholine (0.025 g/ml in Ringer's solution), the injured cells initially responded with local twitches. Within 0.5 hr, contractility and membrane integrity started to recover and both were back to control levels within 3 hr. When these cells were placed back in normal Ringer's solution, they remained viable and active for several hours. Our results suggest that phosphatidylcholine can protect muscle fibers from the effects of sarcolemmal injury.

Treinamento de habilidades sociais com universitários da área de fonoaudiologia: análise de conteúdo

Além do aprendizado de conhecimentos básicos e técnicos, promover competência social e habilidades interpessoais de universitários da área da Saúde torna-se fundamental para o desenvolvimento de relações saudáveis e produtivas no contexto acadêmico e na linha do cuidado. O campo das Habilidades Sociais, uma área da Psicologia, tem fornecido fundamentação desde a metodologia de avaliação de repertório social, até programas de Treinamento de Habilidades Sociais (THS) em contextos educacionais. Atualmente, é crescente o interesse em pesquisas qualitativas na Saúde por englobar o universo de significados, motivos, crenças, valores e atitudes, correspondendo a um espaço mais profundo das relações humanas. Objetivos: Investigar do ponto de vista qualitativo o programa de THS com universitários, oferecido pela disciplina de Psicologia III no Curso de Fonoaudiologia; descrever as potencialidades e dificuldades do repertório de HS conforme a autopercepção dos universitários; identificar os ganhos percebidos pelos universitários ao longo do programa de THS; analisar a relação entre o THS com o autoconhecimento nas relações interpessoais e na relação profissional-paciente. Metodologia: Participaram desta pesquisa 22 universitários, com faixa etária entre 19 e 21 anos de ambos os sexos, que cursavam o segundo ano do Curso de Fonoaudiologia de uma Instituição de Ensino Superior do interior paulista. Empregaram-se abordagem qualitativa e análise de conteúdo dos relatos produzidos pelos universitários no 1o, 8o e 15o encontros do THS e das sínteses (8o e 15o). Resultados: Foram identificadas três temas para análise: \"A importância do conhecimento teórico/técnico para a formação do fonoaudiólogo\", \"A importância do THS no contexto acadêmico e para a prática clínica\" e \"Avaliação dos universitários sobre os efeitos do THS\". No que se refere ao conhecimento teórico/técnico, os relatos apontaram a importância de que este seja desenvolvido de maneira contínua, valorizando as metodologias ativas empregadas. Quanto ao treinamento, os participantes descreveram maior prevalência de potencialidades e dificuldades das habilidades assertivas nos relacionamentos interpessoais do contexto acadêmico. Também foi apontada maior dificuldade de empatia com colegas e na comunicação com professores. No contexto profissional, as dificuldades permearam a ansiedade de falar em público e ser assertivo ao trabalhar em grupo. Quanto aos ganhos identificados, prevaleceu a assertividade com os dois interlocutores envolvidos nesta pesquisa; porém, todas as habilidades sociais tiveram resultados considerados positivos nos dois contextos, destacando a comunicação empática com paciente como facilitadora do processo terapêutico. O THS foi avaliado positivamente, pois permitiu a identificação de potencialidades, dificuldades e ganhos no repertório de HS no contexto acadêmico, sendo apontado como uma forma de preparação para a prática clínica, promovendo o autoconhecimento, o desenvolvimento de comportamentos socialmente habilidosos e a generalização para outros contextos. Conclusão: A inserção do THS na formação profissional de universitários na Fonoaudiologia mostrou-se bastante promissora ao ampliar o repertório de habilidades e competências para além do conhecimento teórico/técnico, tendo o autoconhecimento papel central neste processo, culminando com mudanças de comportamento na vida pessoal e profissional. Salienta-se a relevância do THS em cursos de graduação na área da Saúde, pelo contexto da relação profissional-paciente.

Looking Forward to a Dynamic Third Age - Comparative Survey Report

BALL (Be Active Through Lifelong Learning) is an Erasmus + project of the European Union with the chief objective of analysing the degree of Preparation for Retirement among European citizens. The team working for this project, funded by the EU, is composed by organizations with broad experience at work with the third age from three European countries, Poland, Iceland and Spain, and the results obtained from these three contexts aim to provide a clear picture about the state of the art in preparation for retirement nowadays. The main objective of the project is to develop innovative guidelines and recommendations for use at lifelong learning centres; universities; companies; unions; associations; local and regional authorities who need and wish to prepare and encourage individuals under their auspices to prepare for the third age. The project defines the age group of 50 to 70 (the “baby boomer” generation) as the target group for such early preparations. The project and its outcomes will be used to raise awareness of these important issues and disseminate the results throughout the European Educational Area and the worldwide U3A network.

Ageing Modernity: Growing old in the EU in the 21st Century. EU Centre in Singapore Background Brief No. 7, December 2012

How do urban growth and an ageing population affect or inform planning at the national and regional level in the European Union? At a time when economic stagnation, environmental concerns and demographic changes are affecting social and political discourses, they are also influencing urban form and development. Furthermore, as people are living longer, they still have much to contribute in their later years to their communities and to the economy. As a result, the policy and urban design paradigm is shifting as the relative importance of older people grows. By looking at the connections between ageing, design and the environment, and the role of policy and planning strategies, this brief seeks to understand how the EU plans for an ageing population and attempts to reshape communities in an increasingly challenging socioeconomic context. This brief looks at some of the regional initiatives and guidelines and highlights the role the EU is playing in influencing some of the development trends, particularly those in urban centres.

Iniciativas de participação cidadã de idosos em Portugal: um estudo exploratório

A perspetiva participativa tem ganho destaque na área do envelhecimento, impulsionada em parte pela divulgação da noção de envelhecimento ativo. Tal ênfase não foi contudo acompanhado por equivalente aprofundamento do conceito e das práticas, assistindo-se à perda de especificidade do termo, em particular na aceção de envolvimento cívico e político dos cidadãos idosos. Como se pode afinal definir participação cidadã sénior? Que tipo de atividades inclui? E o que se está concretamente a fazer em Portugal neste âmbito? Estas são questões a que o artigo procura responder, com base num estudo exploratório de levantamento de iniciativas locais de “participação sénior”.

Extending working life in Belgium. CEPS Working Document No. 386, 22 November 2013

This report aims at understanding how persons aged 50 years and older are and can be integrated into the working society in Belgium. We are interested in how people in this age group can be induced to engage in various forms of employment and lifelong learning. Based on secondary literature, descriptive databases as well as interviews with experts and focus groups, we find that the discussion on active ageing in Belgium is well advanced with numerous contributions by academics, stakeholders, social partners, the public administration and interest groups. The wish to retire at 60 is widely shared, but at the same time the majority of Belgium’s elderly are able and would be willing to work under specific conditions. Therefore, we recommend that Belgium should invest in more flexible systems including a revision of the tax scheme, such as the part-time retirement system proposed by the insurance company Delta Lloyd. An equally relevant recommendation would be to ensure that public employment agencies, employers and agencies that provide training encourage all workers to work and learn regardless of their age.

Extending working life in Finland. CEPS Working Document No. 387, 22 November 2013

This report reviews national and private initiatives to allow the elderly to continue their participation in the Finnish labour market and provides an analysis of the labour market and living conditions of seniors. We are interested in how those over 50 can be engaged in various forms of employment and lifelong learning. We find strong evidence that Finland generally provides good institutional conditions for active ageing. The quick and early ageing process was tackled by the fundamental pension reform that already prolonged retirement substantially and will probably facilitate later retirement as the attitudes concerning retirement change. On the other hand, Finland still seems to lag behind the other Nordic welfare states, has considerable problems in providing the same health conditions to low educated people in physically demanding occupations and could - – with respect to family pension in particular – invest further efforts in reforming the pension system. While many of the reforms Finland has conducted seem to be favourable and transferable to other European countries that still face the steepest phases of ageing in their societies, a reluctance towards changing attitudes that we observe in Finland, shows that organizing active ageing is a long-term project.