Siderophore activity of myo-inositol hexakisphosphate in Pseudomonas aeruginosa

Myo-Inositol hexakisphosphate (InsP6), which is found in soil and most, if not all, plant and animal cells, has been estimated to have an affinity for Fe3+ in the range of 10(25) to 10(30) M-1. In this report, we demonstrate that the Fe-InsP6 complex has siderophore activity and is able to reverse the iron-restricted growth inhibition of Pseudomonas aeruginosa by ethylene diamine di(o-hydroxyphenyl)acetic acid. With 55Fe-InsP6 in transport studies, iron uptake is strongly iron regulated, being repressed after growth in iron-replete conditions and inhibited by treatment with potassium cyanide and carbonyl cyanide m-chlorophenylhydrazone. The kinetics of iron transport revealed a Km of 100 nM. Self-displacement of binding of [3H]InsP6 to isolated membranes by InsP6 revealed a single class of binding sites (Kd = 143 +/- 6 nM; Hill coefficient, 1.1 +/- 0.1). The binding of [3H]InsP6 to membranes was not dependent on whether cells had been grown under conditions of high or low iron concentrations. We believe that this is the first report of inositol polyphosphate activity in prokaryotic cells.

Glycogen, the preferred energy source for extracellular protein formation and growth of Aeromonas salmonicida

A study was made of the effect of supplementing a rich 3% (w/v) tryptone soya broth (TSB) medium and a poorer 1.7% (w/v) tryptone-based medium with glucose, maltose and glycogen, as carbon sources, on growth and exoprotein formation by Aeromonas salmonicida. In TSB, glucose inhibited growth and repressed exoprotein formation whilst maltose and glycogen had little effect, up to 20 h, when compared with an unsupplemented control. By contrast, in the poorer medium, over a 24-h incubation period, growth was stimulated three-fold by glycogen, and whilst exoprotein formation was low in comparison with that observed in TSB, the greatest production was observed in the presence of glycogen. Extracellular alpha-amylase was measured in the tryptone medium in the presence of the three carbon sources and the highest level, produced in the presence of glycogen, was 1.6 times that with added maltose whilst none was detectable with glucose present. This pattern was repeated in the case of the maltose-inducible porin, LamB, of the outer membrane.

Molecular regulation of iron uptake in pseudomonas aeruginosa

The microbial demand for iron is often met by the elaboration of siderophores into the surrounding medium and expression of cognate outer membrane receptors for the ferric siderophore complexes. Conditions of iron limitation, such as those encountered in vivo, cause Pseudomonas aeruginosa to express two high-affinity iron-uptake systems based on pyoverdin and pyochelin. These systems will operate both in the organism's natural habitat, soil and water, where the solubility of iron at neutral pH is extremely low, and in the human host where the availability of free iron is too low to sustain bacterial growth due to the iron-binding glycoproteins transferrin and lactoferrin. Cross-feeding and radiolabelled iron uptake experiments demonstrated that pyoverdin biosynthesis and uptake were highly heterogeneous amongst P.aeruginosa strains, that growth either in the presence of pyoverdin or pyochelin resulted in induction of specific IROMPs, and that induction of iron uptake is siderophore-specific. The P.aeruginosa Tn5 mutant PH1 is deficient in ferripyoverdin uptake and resistant to pyocin Sa, suggesting that the site of interaction of pyocin Sa is a ferripyoverdin receptor. Additional Tn5 mutants appeared to exploit different strategies to achieve pyocin Sa-resistance, involving modifications in expression of pyoverdin-mediated iron uptake, indicating that complex regulatory systems exist to enable these organisms to compete effectively for iron. Modulation of expression of IROMPs prompted a study of the mechanism of uptake of a semi-synthetic C(7) α-formamido substituted cephalosporin BRL 41897A. Sensitivity to this agent correlated with expression of the 75 kDa ferri-pyochelin receptor and demonstrated the potential of high-affinity iron uptake systems for targeting of novel antibiotics. Studies with ferri-pyoverdin uptake-deficient mutant PH1 indicated that expression of outer membrane protein G (OprG), which is usually expressed under iron-rich conditions and repressed under iron-deficient conditions, was perturbed. Attempts were made to clone the oprG gene using a degenerate probe based on the N-terminal amino acid sequence. A strongly hybridising HindIll restriction fragment was cloned and sequenced, but failed to reveal an open reading frame correspondmg to OprG. However, there appears to be good evidence that a part of the gene codmg for the hydrophilic membrane-associated ATP-binding component of a hitherto uncharacterised periplasmic- binding-protein-dependent transport system has been isolated. The full organisation and sequence of the operon, and substrate for this putative transport system, are yet: to be elucidated,

From therapeutic to political education: the centrality of affective sensibility in critical pedagogy

While the need for humanising education is pressing in neoliberal societies, the conditions for its possibility in formal institutions have become particularly cramped. A constellation of factors – the strength of neoliberal ideologies, the corporatisation of universities, the conflation of human freedom with consumer satisfaction, and a wider crisis of hope in the possibility or desirability of social change – make it difficult to apply classical theories of subject-transformation to new work in critical pedagogy. In particular, the growth of interest in pedagogies of comfort (as illustrated in certain forms of ‘therapeutic’ education and concerns about student ‘satisfaction’) and resistance to critical pedagogies suggest that subjectivty has become a primary site of political struggle in education. However, it can no longer be assumed that educators can (or should) liberate students’ repressed desires for ‘humanisation’ by politicising curricula, pedagogy or institutions. Rather, we must work to understand the new meanings and affective conditions of critical subjectivity itself. Bringing critical theories of subject transformation together with new work on ‘pedagogies of discomfort’, I suggest we can create new ways of opening up possibilities for critical education that respond to neoliberal subjectivities without corresponding to or affirming them.

Pichia pastoris regulates its gene-specific response to different carbon sources at the transcriptional, rather than the translational, level

Background: The methylotrophic, Crabtree-negative yeast Pichia pastoris is widely used as a heterologous protein production host. Strong inducible promoters derived from methanol utilization genes or constitutive glycolytic promoters are typically used to drive gene expression. Notably, genes involved in methanol utilization are not only repressed by the presence of glucose, but also by glycerol. This unusual regulatory behavior prompted us to study the regulation of carbon substrate utilization in different bioprocess conditions on a genome wide scale. Results: We performed microarray analysis on the total mRNA population as well as mRNA that had been fractionated according to ribosome occupancy. Translationally quiescent mRNAs were defined as being associated with single ribosomes (monosomes) and highly-translated mRNAs with multiple ribosomes (polysomes). We found that despite their lower growth rates, global translation was most active in methanol-grown P. pastoris cells, followed by excess glycerol- or glucose-grown cells. Transcript-specific translational responses were found to be minimal, while extensive transcriptional regulation was observed for cells grown on different carbon sources. Due to their respiratory metabolism, cells grown in excess glucose or glycerol had very similar expression profiles. Genes subject to glucose repression were mainly involved in the metabolism of alternative carbon sources including the control of glycerol uptake and metabolism. Peroxisomal and methanol utilization genes were confirmed to be subject to carbon substrate repression in excess glucose or glycerol, but were found to be strongly de-repressed in limiting glucose-conditions (as are often applied in fed batch cultivations) in addition to induction by methanol. Conclusions: P. pastoris cells grown in excess glycerol or glucose have similar transcript profiles in contrast to S. cerevisiae cells, in which the transcriptional response to these carbon sources is very different. The main response to different growth conditions in P. pastoris is transcriptional; translational regulation was not transcript-specific. The high proportion of mRNAs associated with polysomes in methanol-grown cells is a major finding of this study; it reveals that high productivity during methanol induction is directly linked to the growth condition and not only to promoter strength.

REST is a hypoxia-responsive transcriptional repressor

Cellular exposure to hypoxia results in altered gene expression in a range of physiologic and pathophysiologic states. Discrete cohorts of genes can be either up- or down-regulated in response to hypoxia. While the Hypoxia-Inducible Factor (HIF) is the primary driver of hypoxia-induced adaptive gene expression, less is known about the signalling mechanisms regulating hypoxiadependent gene repression. Using RNA-seq, we demonstrate that equivalent numbers of genes are induced and repressed in human embryonic kidney (HEK293) cells. We demonstrate that nuclear localization of the Repressor Element 1-Silencing Transcription factor (REST) is induced in hypoxia and that REST is responsible for regulating approximately 20% of the hypoxia-repressed genes. Using chromatin immunoprecipitation assays we demonstrate that REST-dependent gene repression is at least in part mediated by direct binding to the promoters of target genes. Based on these data, we propose that REST is a key mediator of gene repression in hypoxia.

The Blue Highway

The Blue Highway is a collection of eleven literary short stories and ten miniatures that depict men in trouble, searching for a code to live by. The miniatures are repressed memories, appearing suddenly like the tips of ice bergs and act as stepping stones (tension bridges) between the larger works. The stories begin at the end with "Time Out", the story of Frank, a down and out homeless vet at the end of his rope. Then we begin the journey along "The Blue Highway" with Danny and his gang of teenage bandits, taking themselves to Disney World to see if they can recapture their lost dream. On our journey we will meet Mark, the ex-killer, an old Cuban fisherman who will not give up his honor, a young man on a way to a war who discovers a fantastic treasure, a soldier on his way home again, two MP's who nearly kill the wrong man, we will spend a night on an African savannah with wild hyenas and finally, meet a grandfather who discovers the one gift which might save his family. The same gift which might save Frank as well. ^

Finding fact by using fiction

This study was to explore the psychoanalytic process that writers experience when they write memoirs. With psychoanalytic theory, the findings were that when writers compose memoirs which include repressed information, the writer's word choice or word block is heavily influenced by his/her own moral code. This idea led to the assertions that first, we are fragmented because of the discordance that arises between the structures of morality and language, the latter which includes good and evil; second, when we write memoirs, we must create a fictional identity that allows the different fragments of identity to operate under the illusion of continuity that language provides; and third, the language we use may transcend our repressed information into consciousness. The conclusion was that when past immoral truths are uncovered, the various fragments with their selfish aims and the fictional identity cease to exist in the wake of being. ^

La represión sexual como tema recurrente en la obra de Federico García Lorca

The purpose of this dissertation is to demonstrate that sexual repression is a recurring theme throughout a selection of works by Federico García Lorca. The introductory chapter focuses on the sexual theories of Sigmund Freud and Michel Foucault in setting the foundation for an analysis of sexually frustrated characters. In chapter two, an analysis of Lorca's rural trilogy reveals how marginalized female characters struggle to preserve societal customs that subjugate them to the patriarchal hegemony by limiting their free will and their sexual freedom. The subsequent chapter elaborates on the theme of repression in poems where the oppressed voice expresses and denounces the intolerance and persecution of those who condemn homosexuality through metaphors of darkness, impurity, and barren love. Chapter four analyzes two avant-garde plays where the underlying message is that of a society that hampers the expression of homosexual love by silencing desire and forcing individuals to mask their identities. The conclusion demonstrates once more the effects of society on sexual freedom, as well as explaining that the recurrence of repressed women is not a pretext in portraying the homosexual saga, but a pre-text in presenting them as an equally marginalized group.

Inducible Overexpression of Endothelin-3 in a Transgenic Mouse Leads to Hyperpigmentation That Decreases Over Time

Endothelin-3 (Edn3) has been shown to be an essential environmental cue in melanocyte development. Edn3 and its receptor, EdnrB, are allelic to mouse mutations occurring at the lethal spotting and piebald loci, respectively; these mutations result in hypopigmentation phenotypes. Mutations in the genes for both Edn3 and EdnrB are implicated in human pigmentation disorders such as Waardenburg-Shah syndrome, which is characterized by pigmentation defects, deafness, and megacolon. In this study, a tetracycline-inducible transgenic mouse model that overexpresses Edn3 under the control of the Keratin 5 promoter was shown to produce a hyperpigmentation phenotype that decreases over time. The expression pattern of transgenic Edn3 and its effects on the melanocyte population were examined in transgenic embryos, postnatal skin, and the skin of adult mice that exhibit faded hyperpigmentation. These studies suggest that overexpression of Edn3 in this model is restricted primarily to the roof plate of the neural tube and surface ectoderm in the developing embryo and to keratinocytes in the epidermis of postnatal mice. A decline in transgenic expression and a reduction in the dermal melanocytes and free melanin that characterize the phenotype in juvenile mice were shown to correlate with the fading of the hyperpigmentation phenotype. Transgenic mice in which transgenic expression was repressed (resulting in the disappearance of the hyperpigmentation phenotype) also exhibited a decrease in the dermal melanocyte population. The Edn3-overexpressing mice used in this study might be helpful m understanding human skin conditions characterized by dermal melanocytosis.

The blue highway

The Blue Highway is a collection of eleven literary short stories and ten miniature that depict men in trouble, searching for a code to live by. The miniatures are repressed memories, appearing suddenly like the tips of ice bergs and act as stepping stones (tension bridges) between the larger works. The stories begin at the end with "Time Out", the story of Frank, a down and out homeless vet at the end of his rope. Then we begin the journey along "The Blue Highway" with Danny and his gang of teenage bandits, taking themselves to Disney World to see if they can recapture their lost dream. On our journey we will meet Mark, the ex-killer, an old Cuban fisherman who will not give up his honor, a young man on a way to a war who discovers a fantastic treasure, a soldier on his way home again, two MP's who nearly kill the wrong man, we will spend a night on an African savannah with wild hyenas and finally, meet a grandfather who discovers the one gift which might save his family. The same gift which might save Frank as well.

Impact of high pCO2 and warmer temperatures on the process of silica biomineralization in the sponge Mycale grandis

Siliceous sponges have survived pre-historical mass extinction events caused by ocean acidification and recent studies suggest that siliceous sponges will continue to resist predicted increases in ocean acidity. In this study, we monitored silica biomineralization in the Hawaiian sponge Mycale grandis under predicted pCO2 and sea surface temperature scenarios for 2100. Our goal was to determine if spicule biomineralization was enhanced or repressed by ocean acidification and thermal stress by monitoring silica uptake rates during short-term (48 h) experiments and comparing biomineralized tissue ratios before and after a long-term (26 d) experiment. In the short-term experiment, we found that silica uptake rates were not impacted by high pCO2 (1050 µatm), warmer temperatures (27°C), or combined high pCO2 with warmer temperature (1119 µatm; 27°C) treatments. The long-term exposure experiments revealed no effect on survival or growth rates of M. grandis to high pCO2 (1198 µatm), warmer temperatures (25.6°C), or combined high pCO2 with warmer temperature (1225 µatm, 25.7°C) treatments, indicating that M. grandis will continue to prosper under predicted increases in pCO2 and sea surface temperature. However, ash-free dry weight to dry weight ratios, subtylostyle lengths, and silicified weight to dry weight ratios decreased under conditions of high pCO2 and combined pCO2 warmer temperature treatments. Our results show that rising ocean acidity and temperature have marginal negative effects on spicule biomineralization and will not affect sponge survival rates of M. grandis.

Physical, Geochemical, Geomechanical, Geophysical and Hydrodynamic Characterization and Anisotropy Assessment of Corvio Sandstone

Corvio sandstone is a ~20 m thick unit (Corvio Formation) that appears in the top section of the Frontada Formation (Campoó Group; Lower Cretaceous) located in Northern Spain in the southern margin of the Basque-Cantabrian Basin. Up to 228 plugs were cored from four 0.3 x 0.2 x 0.5 m blocks of Corvio sandstone, to perform a comprehensive characterization of the physical, mineralogical, geomechanical, geophysical and hydrodynamic properties of this geological formation, and the anisotropic assessment of the most relevant parameters. Here we present the first data set obtained on 53 plugs which covers (i) basic physical and chemical properties including density, porosity, specific surface area and elementary analysis (XRF - CHNS); (ii) the curves obtained during unconfined and confined strengths tests, the tensile strengths, the calculated static elastic moduli and the characteristic stress levels describing the brittle behaviour of the rock; (iii) P- and S-wave velocities (and dynamic elastic moduli) and their respective attenuation factors Qp and Qs, electrical resistivity for a wide range of confining stress; and (iv) permeability and transport tracer tests. Furthermore, the geophysical, permeability and transport tests were additionally performed along the three main orthogonal directions of the original blocks, in order to complete a preliminary anisotropic assessment of the Corvio sandstone.

Quando a indisciplina escolar é um sintoma? articulações psicanalíticas

From a clinical case reporting a severe “ scholarship indiscipline”, this work questioned how the indiscipline can be a symptom . To answer the question, it was analyzed the symptom concept from the Freudian - lacaneana perspective and theirs connections with “ scholarship indiscipline” subject . The research used a theoretical and clinical method , to show the connection between the case development and the psychoanalytic publications reviewing questions on the subject . It was undertook a historical analysis of the construction of disciplinary mechanism through the works of Foucault (1987, 1996), Deleuze (1988; 1992) and commentators. This historical analysis showed a dated and unnatural character of this discursive production named “ scholarship indiscipline”, revealing the indiscipline complaint comes from a social speech that imposes the idea that learn ing depends on the discipline. However , this idea type has a flaw , because always something escapes disciplining . The social answer to the escape is the complaint of indiscipline, which can be taken as a social symptom . Each child should find an answer to this speech and define the symptomatic character , or not , from itself . It was evaluated the symptom and its consequences in the clinic with the child in the Freud and Lacan teachings . The Freud view showed the symptom is the answer to a psychic work , replacing a repressed representation linked to an unbearable sexual dis satisfaction , providing a solution for the child to deal with castration and with the imposed social restrictions . A review of Freud's work undertaken by Lacan emphasizes the psycholog ical work characteristics undertaken by the speaker with its symptom by the link with the social aspect. To analyze, in each case, the position occupied by the patient of a complaint against the indiscipline can open the way to work with it. If the discipl ine is the answer of the subject towards the Other social, from psychoanalysis it is offering a help that allows reframe this response. Elucidating the symptomatic character, or not, from attitudes considered undisciplined, calls for the analysis of unique ness involved in the response of each child, their subjectivity.

Construção de bibliotecas de cDNAs subtraídos a partir das sementes de Mamona submetidas à estresse hídrico e análise in silico de transcritos potencialmente expressos sob condições de estresse: Metalotioneina, proteína reprimida por auxina de 12.5 kDa e Glutelina tipo-A3

Estresses ambientais abióticos são fatores que causam respostas ao nível molecular, fisiológico e morfológico em plantas, dependendo também de sua intensidade e duração. É visto que algumas espécies apresentam tolerância a condições estressantes e ao mesmo tempo são fontes naturais de matéria prima para indústria. Nesse contexto encontra-se a mamona (Ricinus comunnis L.), principal fonte de óleo de rícino valorizado por suas aplicações farmacêuticas e principalmente industriais, vem sendo usada como cultura em regiões onde a disponibilidade de água é reduzida, usada como fonte de renda para agricultura da região nordeste brasileira. Visto que pouco se sabe sobre as respostas moleculares que levam essa planta a tolerar regiões secas e como as sementes, principais foco de interesse, respondem a essa escassez, nesse trabalho foram construídas duas bibliotecas de cDNAs, onde a partir de uma abordagem subtrativa, continham RNAs diferencialmente expressos em sementes de plantas mamona submetidas ao estresse hídrico durante 5 dias (biblioteca L7), e a outra RNAs diferencialmente expressos em sementes controle (biblioteca L5). A biblioteca L7 apresentou a maior variedade de transcritos com um total de 182. A maior parte das funções estabelecidas pelo sistema Gene Ontology - GO, foram direcionadas aos “Processos Metabólicos” (526), em segundo “Respostas a estímulos” (57), o terceiro termo mais abundante foram referentes a “Desenvolvimento”(26). Já na biblioteca L5, foram encontrados 91 transcritos, com maior parte de suas funções referentes a “Processos Metabólicos”(413), em segundo “Respostas a estímulos” (8) e em terceiro Regulação (6). Alguns dos transcritos da biblioteca L7 foram escolhidos para análise por repetirem-se mais de 3x e não aparecerem na biblioteca L5, o que indica uma possível regulação positiva sobre estresse. As análises sobre Metalotioneína (4x), mostraram que a sequência de proteica apresentava os domínios conservados que a caracterizava como tipo II, onde são encontrados dois domínios funcionais ricos em cisteína com posições altamente conservadas, desempenhando a função de ligar-se a metais pesados, correlacionadas assim como a atividade de eliminação EROs e defesa contra o estresse oxidativo, além de apresentar homologia com a sequência de Bruguiera gymnorhiza, uma planta de mangue adaptada a ambientes salinos. Analisamos também os transcritos da referente a proteína AUXIN-REPRESSED 12.5 KDA (3x), apontada como sendo reprimida pelo hormônio auxina e associada ao processo de dormência da semente, é descrito em uma família gênica onde vários membros pertencem as vias de resposta ao estresse. Por último, analisamos a proteína GLUTELIN TYPE-A 3 (5x), uma importante proteína de armazenamento com caráter hidrofílico, possivelmente direcionada para o vacúolo. Em nosso trabalho foi possível observar um aumento de transcritos em relação a subtração controle, possivelmente reflexo do aumento do metabolismo da semente, tanto para resposta defensiva ao estresse hídrico quanto para o amadurecimento rápido da semente onde foram observados transcritos referentes a resposta oxidativa, controle hormonal, proteínas de reserva e produção de óleo.