The in vivo and in vitro formation of sticky DNA and the GAA.TTC trinucleotide repeat associated with Friedreich's ataxia

Friedreich's ataxia is caused by the expansion of the GAA•TTC trinucleotide repeat sequence located in intron 1 of the frataxin gene. The long GAA•TTC repeats are known to form several non-B DNA structures including hairpins, triplexes, parallel DNA and sticky DNA. Therefore it is believed that alternative DNA structures play a role in the loss of mRNA transcript and functional frataxin protein in FRDA patients. We wanted to further elucidate the characteristics for formation and stability of sticky DNA by evaluating the structure in a plasmid based system in vitro and in vivo in Escherichia coli. The negative supercoil density of plasmids harboring different lengths of GAA•TTC repeats, as well as either one or two repeat tracts were studied in E. coli to determine if plasmids containing two long tracts (≥60 repeats) in a direct repeat orientation would have a different topological effect in vivo compared to plasmids that harbored only one GAA•TTC tract or two tracts of < 60 repeats. The experiments revealed that, in fact, sticky DNA forming plasmids had a lower average negative supercoil density (-σ) compared to all other control plasmids used that had the potential to form other non-B DNA structures such as triplexes or Z-DNA. Also, the requirements for in vitro dissociation and reconstitution of the DNA•DNA associated region of sticky DNA were evaluated. Results conclude that the two repeat tracts associate in the presence of negative supercoiling and MgCl 2 or MnCl2 in a time and concentration-dependent manner. Interaction of the repeat sequences was not observed in the absence of negative supercoiling and/or MgCl2 or in the presence of other monovalent or divalent cations, indicating that supercoiling and quite specific cations are needed for the association of sticky DNA. These are the first experiments studying a more specific role of supercoiling and cation influence on this DNA conformation. To support our model of the topological effects of sticky DNA in plasmids, changes in sticky DNA band migration was measured with reference to the linear DNA after treatment with increasing concentrations of ethidium bromide (EtBr). The presence of independent negative supercoil domains was confirmed by this method and found to be segregated by the DNA-DNA associated region. Sequence-specific polyamide molecules were used to test the effect of binding of the ligands to the GAA•TTC repeats on the inhibition of sticky DNA. The destabilization of the sticky DNA conformation in vitro through this binding of the polyamides demonstrated the first conceptual therapeutic approach for the treatment of FRDA at the DNA molecular level. ^ Thus, examining the properties of sticky DNA formed by these long repeat tracts is important in the elucidation of the possible role of sticky DNA in Friedreich's ataxia. ^

PREPARATION AND CHARACTERIZATION OF FAD DEPENDENT NADPH CYTOCHROME P-450 REDUCTASE (FLAVOPROTEINS, APOFLAVOPROTEIN)

NADPH cytochrome P-450 reductase releases FMN and FAD upon dilution into slightly acidic potassium bromide. The flavins are released with positive cooperativity. Dithiothreitol protects the FAD dependent cytochrome c reductase activity against inactivation by free radicals. Behavior in potassium bromide is sensitive to changes in the pH. High performance hydroxylapatite resolved the FAD dependent reductase from holoreductase. For 96% FAD dependent reductase, the overall yield was 12%.^ High FAD dependence was matched by a low FAD content, with FAD/FMN as low as 0.015. There were three molecules of FMN for every four molecules of reductase. The aporeductase had negligible activity towards cytochrome c, ferricyanide, menadione, dichlorophenolindophenol, nitro blue tetrazolium, oxygen and acetyl pyridine adenine dinucleotide phosphate. A four minute incubation in FAD reconstituted one half to all of the specific activity, per milligram protein, of untreated reductase, depending upon the substrate. After a two hour reconstitution, the reductase eluted from hydroxylapatite at the location of holoreductase. It had little flavin dependence, was equimolar in FMN and FAD, and had nearly the specific activity (per mole flavin) of untreated reductase.^ The lack of activity and the ability of FMN to also reconstitute suggest that the redox center of FAD is essential for catalysis, rather than for structure. Dependence upon FAD is consistent with existing hypotheses for the catalytic cycle of the reductase. ^

IDENTIFICATION AND CHARACTERIZATION OF DISTINCT POPULATIONS OF CLONOGENIC BONE MARROW STROMAL CELLS CAPABLE OF TRANSFERRING THE HEMATOPOIETIC MICROENVIRONMENT IN VIVO AND SUPPORTING LT-HSCs IN VITRO

Bone marrow (BM) stromal cells are ascribed two key functions, 1) stem cells for non-hematopoietic tissues (MSC) and 2) as components of the hematopoietic stem cell niche. Current approaches studying the stromal cell system in the mouse are complicated by the low yield of clonogenic progenitors (CFU-F). Given the perivascular location of MSC in BM, we developed an alternative methodology to isolate MSC from mBM. An intact ‘plug’ of bone marrow is expelled from bones and enzymatically disaggregated to yield a single cell suspension. The recovery of CFU-F (1917.95+199) reproducibly exceeds that obtained using the standard BM flushing technique (14.32+1.9) by at least 2 orders of magnitude (P<0.001; N = 8) with an accompanying 196-fold enrichment of CFU-F frequency. Purified BM stromal and vascular endothelial cell populations are readily obtained by FACS. A detailed immunophenotypic analysis of lineage depleted BM identified PDGFRαβPOS stromal cell subpopulations distinguished by their expression of CD105. Both subpopulations retained their original phenotype of CD105 expression in culture and demonstrate MSC properties of multi-lineage differentiation and the ability to transfer the hematopoietic microenvironment in vivo. To determine the capacity of either subpopulation to support long-term multi-lineage reconstituting HSCs, we fractionated BM stromal cells into either the LinNEGPDGFRαβPOSCD105POS and LINNEGPDGFRαβPOSCD105LOW/- populations and tested their capacity to support LT-HSC by co-culturing each population with either 1 or 10 HSCs for 10 days. Following the 10 day co-culture period, both populations supported transplantable HSCs from 10 cells/well co-cultures demonstrating high levels of donor repopulation with an average of 65+23.6% chimerism from CD105POS co-cultures and 49.3+19.5% chimerism from the CD105NEG co-cultures. However, we observed a significant difference when mice were transplanted with the progeny of a single co-cultured HSC. In these experiments, CD105POS co-cultures (100%) demonstrated long-term multi- lineage reconstitution, while only 4 of 8 mice (50%) from CD105NEG -single HSC co-cultures demonstrated long-term reconstitution, suggesting a more limited expansion of functional stem cells. Taken together, these results demonstrate that the PDGFRαβCD105POS stromal cell subpopulation is distinguished by a unique capacity to support the expansion of long-term reconstituting HSCs in vitro.

Binding of the HIV-1 gp120 -V3 to cellular glycosphingolipids is necessary for CXCR4 recruitment and infection

Infection by human immunodeficiency virus type 1 (HIV-1) is a multi-step process, and detailed analyses of the various events critical for productive infection are necessary to clearly understanding the infection process and identifying novel targets for therapeutic interventions. Evidence from this study reveals binding of the viral envelope protein to host cell glycosphingolipids (GSLs) as a novel event necessary for the orderly progression of the host cell-entry and productive infection by HIV-1. Data obtained from co-immunoprecipitation analyses and confocal microscopy showed that the ability of viral envelope to interact with the co-receptor CXCR4 and productive infection of HIV-1 were inhibited in cells rendered GSL-deficient, while both these activities were restored after reconstitution of the cells with specific GSLs like GM3. Furthermore, evidence was obtained using peptide-inhibitors of HIV-1 infection to show that binding of a specific region within the V3-loop of the envelope protein gp120 to the host cell GSLs is the trigger necessary for the CD4-bound gp120 to recruit the CXCR4 co-receptor. Infection-inhibitory activity of the V3 peptides was compromised in GSL-deficient cells, but could be restored by reconstitution of GSLs. Based on these findings, a revised model for HIV-1 infection is proposed that accounts for the established interactions between the viral envelope and host cell receptors while enumerating the importance of the new findings that fill the gap in the current knowledge of the sequential events for the HIV-1 entry. According to this model, post-CD4 binding of the HIV-1 envelope surface protein gp120 to host cell GSLs, mediated by the gp120-V3 region, enables formation of the gp120-CD4-GSL-CXCR4 immune-complex and productive infection. The identification of cellular GSLs as an additional class of co-factors necessary for HIV-1 infection is important for enhancing the basic knowledge of the HIV-1 entry that can be exploited for developing novel antiviral therapeutic strategies. ^

The distribution of sulfur in oceanic crust with respect to oxidative diagenesis (Table 1)

A detailed geochemical-petrological examination of layer 2 basalts recovered during Leg 37 of the DSDP has revealed that the original distribution, form and abundance of igneous sulfide have been profoundly altered during low-grade oxidative diagenesis. The net result appears to have been a rather pervasive remobilization of igneous sulfide to form secondary pyrite accompanied by a bulk loss of sulfur equivalent to about 50-60% of the original igneous value, assuming initial saturation. It is suggested that during infiltration of seawater into the massive crystalline rock, igneous sulfide has experienced pervasive oxidation, under conditions of limited oxidation potential, to form a series of unstable, soluble sulfur species, primarily in the form of SO3[2-] and S2O3[2-]. Spontaneous decomposition of these intermediate compounds through disproportionation has resulted in partial reconstitution of the sulfur as secondary pyrite and the generation of SO4[2-] ion, which, due to its kinetic stability, has been lost from the basalt system and ultimately transferred to the ocean. This model not only satisfies the geochemical and petrological observations but also provides a suitable explanation for the highly variable delta34S values which characterize secondary sulfides in deep ocean floor basalts.

Ordenamento territorial em Recife-pe : Os impactos urbanísticos e sociais da realocação de comunidades através do projeto Via Mangue

The logic of territorial ordainment in Recife has been developed mainly through the seclusion of the unwanted and the removal of stilt houses and slums in order to make room and prepare the space for new private enterprises. As an example of ordainment we took the 'Via Mangue' project, which is part of Recife's mobility plan and is one of the main projects aimed at the city's preparation for the 2014 FIFA World Cup Brazil. We believe that the project makes use of the 'great social benefits' discourse in order to cover its actual and practical consequences which, taken as whole, lead to the favoring of private sector over the public interest. That being so, the main goal of the present work is to make an analysis of territorial ordainment in Recife through the execution of the Via Mangue project; observing at the same time the urban and social impacts caused by the relocation of communities to the Via Mangue III housing complex, and verifying whether this policy actually promoted substantial improvement of habitability or only a precarious social inclusion of these populations. Our research was conducted and operated at three levels. First, the conceptual reconstitution of territorial ordainment; second, documental and cartographic research on the Via Mangue project; and last, fieldwork with observation of the constructed space and personal interviews with members of the families relocated to the Via Mangue III housing complex. We hope the present work could be a valuable contribution to the comprehension of the complexities involved in the relocation of families to housing complexes built by the government

Tres versiones contemporáneas de la comunidad : Hacia una teoría política post-fundacionalista

Ante el embate del particularismo, la emergencia de los nacionalismos reaccionarios en el mundo contemporáneo, y la difusión del comunitarismo en la filosofía política, es necesario que la teoría política asuma un rol crítico ante estos procesos teóricos y políticos. Esa es la tarea que desde el postfundacionalismo se plantean la obra de Jean-Luc Nancy, Roberto Espósito y Ernesto Laclau. En este trabajo intentamos escrutar la visión que estos autores postulan sobre la comunidad. Se verá que, mas allá de sus distintas estrategias argumentativas y de perspectiva, se desarrolla en estos tres autores una idea de la comunidad fundamentalmente anti-esencialista, una comunidad improductiva (Nancy), estructurada en torno a una nada (Espósito) y cuya única manera de reconstitución es a través de la articulación política (Laclau)

Tres versiones contemporáneas de la comunidad : Hacia una teoría política post-fundacionalista

Ante el embate del particularismo, la emergencia de los nacionalismos reaccionarios en el mundo contemporáneo, y la difusión del comunitarismo en la filosofía política, es necesario que la teoría política asuma un rol crítico ante estos procesos teóricos y políticos. Esa es la tarea que desde el postfundacionalismo se plantean la obra de Jean-Luc Nancy, Roberto Espósito y Ernesto Laclau. En este trabajo intentamos escrutar la visión que estos autores postulan sobre la comunidad. Se verá que, mas allá de sus distintas estrategias argumentativas y de perspectiva, se desarrolla en estos tres autores una idea de la comunidad fundamentalmente anti-esencialista, una comunidad improductiva (Nancy), estructurada en torno a una nada (Espósito) y cuya única manera de reconstitución es a través de la articulación política (Laclau)

Ordenamento territorial em Recife-pe : Os impactos urbanísticos e sociais da realocação de comunidades através do projeto Via Mangue

The logic of territorial ordainment in Recife has been developed mainly through the seclusion of the unwanted and the removal of stilt houses and slums in order to make room and prepare the space for new private enterprises. As an example of ordainment we took the 'Via Mangue' project, which is part of Recife's mobility plan and is one of the main projects aimed at the city's preparation for the 2014 FIFA World Cup Brazil. We believe that the project makes use of the 'great social benefits' discourse in order to cover its actual and practical consequences which, taken as whole, lead to the favoring of private sector over the public interest. That being so, the main goal of the present work is to make an analysis of territorial ordainment in Recife through the execution of the Via Mangue project; observing at the same time the urban and social impacts caused by the relocation of communities to the Via Mangue III housing complex, and verifying whether this policy actually promoted substantial improvement of habitability or only a precarious social inclusion of these populations. Our research was conducted and operated at three levels. First, the conceptual reconstitution of territorial ordainment; second, documental and cartographic research on the Via Mangue project; and last, fieldwork with observation of the constructed space and personal interviews with members of the families relocated to the Via Mangue III housing complex. We hope the present work could be a valuable contribution to the comprehension of the complexities involved in the relocation of families to housing complexes built by the government

Ordenamento territorial em Recife-pe : Os impactos urbanísticos e sociais da realocação de comunidades através do projeto Via Mangue

The logic of territorial ordainment in Recife has been developed mainly through the seclusion of the unwanted and the removal of stilt houses and slums in order to make room and prepare the space for new private enterprises. As an example of ordainment we took the 'Via Mangue' project, which is part of Recife's mobility plan and is one of the main projects aimed at the city's preparation for the 2014 FIFA World Cup Brazil. We believe that the project makes use of the 'great social benefits' discourse in order to cover its actual and practical consequences which, taken as whole, lead to the favoring of private sector over the public interest. That being so, the main goal of the present work is to make an analysis of territorial ordainment in Recife through the execution of the Via Mangue project; observing at the same time the urban and social impacts caused by the relocation of communities to the Via Mangue III housing complex, and verifying whether this policy actually promoted substantial improvement of habitability or only a precarious social inclusion of these populations. Our research was conducted and operated at three levels. First, the conceptual reconstitution of territorial ordainment; second, documental and cartographic research on the Via Mangue project; and last, fieldwork with observation of the constructed space and personal interviews with members of the families relocated to the Via Mangue III housing complex. We hope the present work could be a valuable contribution to the comprehension of the complexities involved in the relocation of families to housing complexes built by the government

Tres versiones contemporáneas de la comunidad : Hacia una teoría política post-fundacionalista

Ante el embate del particularismo, la emergencia de los nacionalismos reaccionarios en el mundo contemporáneo, y la difusión del comunitarismo en la filosofía política, es necesario que la teoría política asuma un rol crítico ante estos procesos teóricos y políticos. Esa es la tarea que desde el postfundacionalismo se plantean la obra de Jean-Luc Nancy, Roberto Espósito y Ernesto Laclau. En este trabajo intentamos escrutar la visión que estos autores postulan sobre la comunidad. Se verá que, mas allá de sus distintas estrategias argumentativas y de perspectiva, se desarrolla en estos tres autores una idea de la comunidad fundamentalmente anti-esencialista, una comunidad improductiva (Nancy), estructurada en torno a una nada (Espósito) y cuya única manera de reconstitución es a través de la articulación política (Laclau)