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Cuitiño, J.I. - Scasso, R.A.
Rev. Asoc. Geol. Argent. 2010;66(3):406-417
2010

Descripción: Sedimentology and paleoenvironments of the Patagoniano and its transition to the Santa Cruz Formation south of Lago Argentino, southern Patagonia. Detailed sedimentological analysis of the Patagoniano and its transition to the Santa Cruz Formation has been carried out south of Lago Argentino, in southern Patagonia. The 180 meters thick succession of fossiliferous sandstones was subdivided into twelve lithofacies, grouped in three facies associations. From base to top we defined the facies association I, dominated by massive, bioturbated and fossiliferous sandstones; the facies association II, dominated by fining upward cycles of cross-stratified sandstones and heterolithic facies; and the facies association III dominated by thin conglomeratic levels interbedded with mudstones rich in plant remains and paleosoils. The facies association I is interpreted as the deposit of a transgressive, shallow sea with oyster rich biogenic accumulations, finally evolving to a coastal plain paleoenvironment. A new transgression originated the discontinuity underlying the facies association II, which was interpreted as a progradational estuarine system. Finally, the ongoing progradation produced the fluvial meandering system of facies association III, assigned to the Santa Cruz Formation. The new name "Estancia 25 de Mayo" is formalized here for the Patagoniano in the southernmost Cordillera Patagónica, instead of the problematic name Centinela Formation. The Estancia 25 de Mayo Formation was subdivided in two members: the lower Quién Sabe Member, equivalent to the facies association I, and the upper Bandurrias Member, equivalent to the (facies association II).
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Canepuccia, A.D. - Pérez, C.F. - Farina, J.L. - Alemany, D. - Iribarne, O.O.
Mar. Ecol. Prog. Ser. 2013;494:135-148
2013

Descripción: ABSTRACT: How species similarity changes between habitats along environmental gradients is still a central challenge in ecological studies. We assessed whether marsh plant characteristics are associated with geographic changes in environmental conditions and whether there are environmental factors associated with marsh-inland dissimilarity in species composition. Field samples of vegetation were collected at 6 sites along the SW Atlantic to determine plant characteristics (cover, tallness, richness and α-diversity), and marsh-inland dissimilarity (β-diversity) in species composition was calculated. PERMANOVA analysis showed that plant assemblage changes among sites. Forward stepwise multiple regression analysis showed that in lower marsh, plant cover increased in association with tidal range and decreased in association with salinity. In the high marsh, plant cover decreased in association with tidal range, salinity and with minimum temperatures. Plant richness increased in association with tidal range and with marsh area, while α-diversity decreased in association with precipitation and increased with salinity. Beta-diversity, estimated by SIMPER analysis, increased in association with precipitation and decreased with salinity and daily thermal amplitude. We present evidence that there is an increase in a-diversity but a decrease in β-diversity with environmental severity among co-specific marshes distributed along the SW Atlantic coast. Thus, communities developing in more benign conditions, regardless of their low local diversity, may increase biodiversity at a landscape scale by decreasing their similarities.© Inter-Research 2013. www.int-res.com.
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Piethé, R.D. - Palma, R.M.
Rev. Asoc. Geol. Argent. 2008;63(4):682-695
2008

Descripción: The study of the carbonate-ramp deposits of La Manga Formation has allowed us to recognize three facies associations. The lithofacies association A is composed by five lithofacies (A1-A5). This association represents part of the transgressive systems tract. It corresponds to external ramp deposits, accumulated below the storm-wave base. The A facies association appears in cycles of centimetric to decimetric thickness and belong to a retrograding parasequence set of 5th order. The lithofacies B association starts after an abrupt facies contact. It is characterized by six lithofacies (B1-B6). They are coarsening upward beds that form metric-scale cycles. These are interpreted as middle-ramp deposits dominated by storms. It corresponds to 4th order sequences. The lithofacies C includes three lithofacies (C1-C3). The small scale cycles were controlled by storm events, while the medium and large scale ones were likely originated by eustasy. The studied stratigraphic sections of La Manga Formation are included in two depostitional sequences SD-2 and SD-3. An abrupt discontinuity marks the beginning of the depositional sequence SD-2, which constitutes a transgressive system-tract, composed by a coarsening upward parasequence stacking set. Above a maximum flooding surface, manifested by a drastic change of facies, a low rate accommodation space period and a sea-level fall (characterized by a lowstand systems tract) are evidenced. The third depositional sequence SD-3 is composed by lagoon deposits. It starts after a paleokarstic surface, characterized by the development of a sheet-cake breccia and an intense vadose diagenesis.
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Mohana-Borges, R. - Silva, J.L. - Ruiz-Sanz, J. - De Prat-Gay, G.
Proc. Natl. Acad. Sci. U. S. A. 1999;96(14):7888-7893
1999

Descripción: The noncovalent complex formed by the association of two fragments of chymotrypsin inhibitor-2 is reversibly denatured by pressure in the absence of chemical denaturants. On pressure release, the complex returned to its original conformation through a biphasic reaction, with first-order rate constants of 0.012 and 0.002 s-1, respectively. The slowest phase arises from an interconversion of the pressure-denatured state, as revealed by double pressure-jump experiments. Below 5 μM, the process was concentration dependent with a second-order rate constant of 1,700 s-1 M-1. Fragment association at atmospheric pressure showed a similar break in the order of the reaction above 5 μM, but both first- and second-order folding/association rates are 2.5 times faster than those for the refolding of the pressure-denatured state. Although the folding rates of the intact protein and the association of the fragments displayed nonlinear Eyring behavior for the temperature dependence, refolding of the pressure-denatured complex showed a linear response. The negligible heat capacity of activation reflects a balance of minimal change in the burial of residues from the pressure-denatured state to the transition state. If we add the higher energy barrier in the refolding of the pressure-denatured state, the rate differences must lie in the structure of this state, which has to undergo a structural rearrangement. This clearly differs from the conformational flexibility of the isolated fragments or the largely unfolded denatured state of the intact protein in acid and provides insight into denatured states of proteins under folding conditions.
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Gil, M. - De Marco, R.J.
J. Exp. Biol. 2005;208(4):671-680
2005

Descripción: Early reports indicate that trophallaxis, i.e. the exchange of liquid food by mouth, may allow honeybees to assign nectar odours with predictive values to anticipate biological meaningful reward stimuli. Nevertheless, this type of learning has not been addressed directly. In the present study, pairs of animals were isolated to induce trophallaxis under controlled conditions and, afterwards, the honeybee proboscis extension reflex was used to investigate the possible role of trophallaxis in learning olfactory cues. The results demonstrate unambiguously that associative learning actually occurs by means of trophallaxis. Animals associate the odour (as the conditioned stimulus or CS) and the sucrose (as the unconditioned stimulus or US) present in the solution they receive through trophallaxis. Moreover, this particular kind of learning leads to long-term olfactory memories after a single learning trial, even when trophallaxis is brief. In addition, we found that the strength of association is clearly affected by CS and US intensity as well as the recent previous foraging experiences of the animals. Comparisons are presented among several features of the learning during trophallaxis and the classical conditioning of the proboscis extension reflex with restrained subjects. Finally, the relevance of learning through trophallaxis in the task of successful foraging is discussed.
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Garberoglio, R.M. - Lazo, D.G.
Rev. Bras. Paleontol. 2011;14(3):215-228
2011

Descripción: One morphotype of sabellids (Sabellida, Sabellidae) and two of serpulids (Sabellida, Serpulidae), found as encrusters on scleractinian ramose corals of the species Stereocaenia triboleti (Koby) and Columastrea antiqua Gerth, from the Agrio Formation (early Hauterivian) from Neuquén Basin, Argentina, are described. The identified morphotypes, Glomerula lombricus (Defrance), Mucroserpula mucroserpula Regenhardt and Propomatoceros sulcicarinatus Ware, have been previously recorded from the Early Cretaceous of the northern Tethys. Two different type of sabellid and serpulidcoral associations have been recognized. The first and more abundant association corresponds to post-mortem encrustation on corals branches. The second one corresponds to a symbiotic association between the serpulid P. sulcicarinatus and both species of corals. The serpulid tubes are recorded parallel to the coral branches reaching the upper tip of them and they were bioimmured within the coral as they grew upwards. The studied symbiotic relationship between serpulids and corals may be regarded as a mutualism as both members probably benefited each other. This type of association has similarities with recent cases of symbiosis between serpulids and corals, but had no fossil record until now. © 2011 by the Sociedade Brasileira de Paleontologia.
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Fusari, C.M. - Lia, V.V. - Hopp, H.E. - Heinz, R.A. - Paniego, N.B.
BMC Plant Biol. 2008;8
2008

Descripción: Background. Association analysis is a powerful tool to identify gene loci that may contribute to phenotypic variation. This includes the estimation of nucleotide diversity, the assessment of linkage disequilibrium structure (LD) and the evaluation of selection processes. Trait mapping by allele association requires a high-density map, which could be obtained by the addition of Single Nucleotide Polymorphisms (SNPs) and short insertion and/or deletions (indels) to SSR and AFLP genetic maps. Nucleotide diversity analysis of randomly selected candidate regions is a promising approach for the success of association analysis and fine mapping in the sunflower genome. Moreover, knowledge of the distance over which LD persists, in agronomically meaningful sunflower accessions, is important to establish the density of markers and the experimental design for association analysis. Results. A set of 28 candidate genes related to biotic and abiotic stresses were studied in 19 sunflower inbred lines. A total of 14,348 bp of sequence alignment was analyzed per individual. In average, 1 SNP was found per 69 nucleotides and 38 indels were identified in the complete data set. The mean nucleotide polymorphism was moderate ( = 0.0056), as expected for inbred materials. The number of haplotypes per region ranged from 1 to 9 (mean = 3.54 1.88). Model-based population structure analysis allowed detection of admixed individuals within the set of accessions examined. Two putative gene pools were identified (G1 and G2), with a large proportion of the inbred lines being assigned to one of them (G1). Consistent with the absence of population sub-structuring, LD for G1 decayed more rapidly (r 2= 0.48 at 643 bp; trend line, pooled data) than the LD trend line for the entire set of 19 individuals (r2= 0.64 for the same distance). Conclusion. Knowledge about the patterns of diversity and the genetic relationships between breeding materials could be an invaluable aid in crop improvement strategies. The relatively high frequency of SNPs within the elite inbred lines studied here, along with the predicted extent of LD over distances of 100 kbp (r2∼0.1) suggest that high resolution association mapping in sunflower could be achieved with marker densities lower than those usually reported in the literature.
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Penas-Steinhardt, A. - Barcos, L.S. - Belforte, F.S. - de Sereday, M. - Vilariño, J. - Gonzalez, C.D. - Martínez-Larrad, M.T. - Tellechea, M.L. - Serrano-Ríos, M. - Poskus, E. - Frechtel, G.D. - Leskow, F.C.
PLoS ONE 2012;7(12)
2012

Descripción: Subclinical low-grade systemic inflammation has been associated with obesity, insulin resistance and metabolic syndrome (MS). Recent studies have highlighted the role of gut microbiota in these disorders. The toll-like receptor 4 (TLR4) plays a key role in the innate immune response activation. We studied two polymorphisms (+3725G/C and 11350G/C) in the 3′ untranslated region (3′UTR) of the TLR4 gene that may alter its expression and their association with metabolic disorders related to systemic inflammation. We cloned the 3′UTR into a luciferase reporter system and compared wild-type 3′UTR (WT) and +3725C variant (MUT) constructs luciferase activities. MUT construct reduced the reporter gene activity by 30% compared to WT (P = 0.0001). To evaluate the association between these polymorphisms with biochemical and clinical overweight related variables, we conducted a population cross-sectional study in 966 men of Argentine general population. Considering smoking as a confounding variable that causes systemic inflammation, we studied these possible effects in both, smokers and nonsmokers. The 11350G/C polymorphism was not detected in our sample whereas the CC genotype of +3725 polymorphism was associated with lean subjects (p = 0.011) and higher Adiponectin levels (p = 0.021). Subjects without any NCEP/ATP III MS component were associated with this genotype as well (p = 0.001). These results were strengthened in nonsmokers, in which CC genotype was associated with lean subjects (p = 0.003) and compared with G carriers showed significantly lower BMI (25.53 vs. 28.60 kg/m2; p = 0.023) and waist circumference (89.27 vs. 97.51 cm; p = 0.025). None of these associations were found in smokers. These results showed that +3725C variant has a functional effect down-regulating gene expression and it could be considered as a predictive factor against overweight, particularly in nonsmokers. Considering the role of TLR4 in inflammation, these findings would suggest that the presence of +3725C variant could predict a lower prevalence of chronic metabolic disorders. © 2012 Penas-Steinhardt et al.
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Echeverría, P.C. - Mazaira, G. - Erlejman, A. - Gomez-Sanchez, C. - Pilipuk, G.P. - Galigniana, M.D.
Mol. Cell. Biol. 2009;29(17):4788-4797
2009

Descripción: Glucocorticoid receptor (GR) is cytoplasmic in the absence of ligand and localizes to the nucleus after steroid binding. Previous evidence demonstrated that the hsp90-based heterocomplex bound to GR is required for the efficient retrotransport of the receptor to the nuclear compartment. We examined the putative association of GR and its associated chaperone heterocomplex with structures of the nuclear pore. We found that importin β and the integral nuclear pore glycoprotein Nup62 interact with hsp90, hsp70, p23, and the TPR domain proteins FKBP52 and PP5. Nup62 and GR were able to interact in a more efficient manner when chaperoned by the hsp90-based heterocomplex. Interestingly, the binding of hsp70 and p23 to Nup62 does not require the presence of hsp90, whereas the association of FKBP52 and PP5 is hsp90 dependent, as indicated by the results of experiments where the hsp90 function was disrupted with radicicol. The ability of both FKBP52 and PP5 to interact with Nup62 was abrogated in cells overexpressing the TPR peptide. Importantly, GR cross-linked to the hsp90 heterocomplex was able to translocate to the nucleus in digitonin-permeabilized cells treated with steroid, suggesting that GR could pass through the pore in its untransformed state. Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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Soto, I.M. - Soto, E.M. - Carreira, V.P. - Hurtado, J. - Fanara, J.J. - Hasson, E.
J. Insect Sci. 2010;10
2010

Descripción: The inversion polymorphisms of the cactophilic Drosophila buzzatti Patterson and Wheeler (Diptera: Drosophilidae) were studied in new areas of its distribution in Argentina. A total of thirty-eight natural populations, including 29 from previous studies, were analyzed using multiple regression analyses. The results showed that about 23% of total variation was accounted for by a multiple regression model in which only altitude contributed significantly to population variation, despite the fact that latitude and longitude were also included in the model. Also, inversion frequencies exhibited significant associations with mean annual temperature, precipitation, and atmospheric pressure. In addition, expected heterozygosity exhibited a negative association with temperature and precipitation and a positive association with atmospheric pressure. The close similarity of the patterns detected in this larger dataset to previous reports is an indication of the stability of the clines. Also, the concurrence of the clines detected in Argentina with those reported for colonizing populations of Australia suggests the involvement of natural selection as the main mechanism shaping inversion frequencies in D. buzzatii.
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Ruiz, J.A. - López, N.I. - Fernández, R.O. - Méndez, B.S.
Appl. Environ. Microbiol. 2001;67(1):225-230
2001

Descripción: Pseudomonas oleovorans GPo1 and its polyhydroxyalkanoic acid (PHA) depolymerization-minus mutant, GPo500 phaZ, residing in natural water microcosms, were utilized to asses the effect of PHA availability on survival and resistance to stress agents. The wild-type strain showed increased survival compared to the PHA depolymerase-minus strain. The appearance of a round cellular shape, characteristic of bacteria growing under starvation conditions, was delayed in the wild type in comparison to the mutant strain. Percent survival at the end of ethanol and heat challenges was always higher in GPo1 than in GPo500. Based on these results and on early experiments (H. Hippe, Arch. Mikrobiol. 56:248-277, 1967) that suggested an association of PHA utilization with respiration and oxidative phosphorylation, we investigated the association between PHA degradation and nucleotide accumulation. ATP and guanosine tetraphosphate (ppGpp) production was analyzed under culture conditions leading to PHA depolymerization. A rise in the ATP and ppGpp levels appeared concomitant with PHA degradation, while this phenomenon was not observed in the mutant strain unable to degrade the polymer. Complementation of the phaZ mutation restored the wild-type phenotype.
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Del C. Batlle, A.M. - Stella, A.M. - Ferramola, A.M. - Sopena, Y. - De Xifra, E.A.W. - Sancovich, H.A.
Int. J. Biochem. 1978;9(6):401-406
1978

Descripción: 1. 1. Evidence for dissociation, renaturation, re-association and re-hybridization of bovine liver aminolaevulinate dehydratase attached to Sepharose 4B is reported. 2. 2. When insolubilized enzyme was treated with 3 and 6 M urea, non covalently bound subunits were dissociated and detected in the eluate; these subunits can be re-associated into a soluble functioning enzyme with a specific activity close to that of the original pure soluble dehydratase preparation. 3. 3. After being washed with a renaturing buffer mixture, the matrix-bound subunits recovered a level of enzymatic activity equal to 50 and 20% of that of the immobilized native aminolaevulinate dehydratase. 4. 4. The reversibility of the dissociation process was investigated. Bound-subunits dehydratase can associate with nascent soluble bovine liver aminolaevulinate dehydratase subunits in situ. The product of such treatment, bound-re-associated enzyme, has the same activity as that of the original bound-dehydratase. The matrix-bound-dissociated bovine liver enzyme was also re-hybridized with soluble dehydratase subunits from E. gracilis. 5. 5. The apparent Km and optimum pH of the immobilized subunits were the same as those of the bound-octameric enzyme. 6. 6. A scheme is proposed, explaining the sequence of reactions leading from the bound-octameric dehydratase to the possible different derivatives, formed during the dissociation and re-association experiments. © 1978.
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Penna, I.M. - Hermanns, R.L. - Folguera, A.
Rev. Asoc. Geol. Argent. 2007;62(3):460-466
2007

Descripción: Considering the high concentration of rock avalanches in the northern part of the Neuquén province of Argentina (36°-38°S and 70°-71°W) and their association with lacustrine basins forming natural dams, their catastrophic collapse seems not to be such a common process as could be expected for these cases. The present work depicts the area immediately affected by the outburst flood corresponding to the Navarrete dam catastrophic collapse (175 × 106 m3), through a sedimentological analysis, generated since the arroyo Colorado was blocked due to a rock avalanche deposit during not well constrained postglacial times. The outburst flood dispersion is initially inferred based on morphological criteria and then determined from a sedimentological analysis of its matrix. Furthermore, the matrix is compared to that of the fluvial terraces, which have been sampled along the arroyo Colorado. This comparison shows that even though differences among them are not so significant through most of the study area, matrix frequency starts to be distinctive from the most distant point of sampling.
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Scasso, R.A. - Concheyro, A.
Rev. Asoc. Geol. Argent. 1999;54(3):290-297
1999

Descripción: Calcareouos nannofosils, duration and origin of limestone-marl cycles (Late Jurassic, Neuquén Basin). A nannofossil association corresponding to Zone NJ20b was defined in the rhythmically interbedded limestones and marls of the Los Catutos Member, Vaca Muerta Formation. It confirmed the Late Middle Tithonian age, previously assigned on the basis of ammonites, to the beds which crop out in the locality of Zapala, Neuquén Province, Neuquén Basin. The stratigraphical resolution of the nannoflora was no better than that of the ammonite fauna, but a good correlation between zones can be established. This represents a powerful and useful tool for future biostratigraphical studies in the Neuquén Basin. On the basis of the ammonite and nannofossil zones, a period of 1 my was estimated for the deposition of the 25 m thick sequence of limestones and marls. From these data a high sedimentation rate (5 m/ka on average) was calculated. As a consequence, the duration of the individual limestone-marl cycles is about 20 ka on average, in good agreement with the precession period of the earth axis. Thus, rhythmic changes in sedimentation would be related to climatic changes driven by orbital forcing. © 1999 Asociación Geológica Argentina.
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Scasso, R.A. - Concheyro, A.
Rev. Asoc. Geol. Argent. 1997;52(4):290-297
1997

Descripción: A nan-nofossil association corresponding to Zone NJ20b was defined in the rhythmically interbedded limestones and marls of the Los Catutos Member, Vaca Muerta Formation. It confirmed the Late Middle Tithonian age, previously assigned on the basis of ammonites, to the beds which crop out in the locality of Zapala, Neuquén Province, Neuquén Basin. The stratigraphical resolution of the nannoflora was no better than that of the ammonite fauna, but a good correlation between zones can be established. This represents a powerful and useful tool for future biostratigraphical studies in the Neuquén Basin. On the basis of the ammonite and nannofossil zones, a period of 1 my was estimated for the deposition of the 25 m thick sequence of limestones and marls. From these data a high sedimentation rate (5 m/ka on average) was calculated. As a consequence, the duration of the individual limestone-marl cycles is about 20 ka on average, in good agreement with the precession period of the earth axis. Thus, rhythmic changes in sedimentation would be related to climatic changes driven by orbital forcing. © 1999 Asociacián Gcológica.
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Ramos, A.M. - Tadic, L.F. - Cinto, I. - Carmona, M. - Gally, M.
Mycotaxon 2013;123:457-465
2013

Descripción: Twenty-six isolates obtained from soybean crops (Glycine max) with typical anthracnose symptoms were identified as Colletotrichum truncatum (73 %) and C. destructivum (26 %). Their genetic relationships were studied using the AFLP method. A UPGMA phenogram divided the strains into two clusters corresponding with the two species. Genetic distances based on association coefficient were 0.71-0.89 among the 18 C. truncatum strains and 0.67-1 among the eight C. destructivum strains. Genetic variability within species, measured in terms of percentage of polymorphic loci, was high (<90%). Only two isolates showed 100% similarity, suggesting high intraspecific variability. © 2013. Mycotaxon, Ltd.
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Ardila, F.J. - Tandecarz, J.S.
Plant Physiol. 1992;99(4):1342-1347
1992

Descripción: Potato (Solanum tuberosum L.) tuber UDP-glucose:protein transglucosylase (UPTG) (EC 2.4.1.112) is involved in the first of a two-step mechanism proposed for protein-bound α-glucan synthesis by catalyzing the covalent attachment of a single glucose residue to an acceptor protein. The resulting glucosylated 38-kilodalton polypeptide would then serve as a primer for enzymic glucan chain elongation during the second step. In the present report, we describe the fast protein liquid chromatography purification of UPTG from a membrane pellet of potato tuber. An apparently close association of UPTG, phosphorylase, and starch synthase was observed under native conditions during different purification steps. Enrichment of a 38-kilodalton polypeptide was found throughout enzyme purification. It is now shown that the purified UPTG, with an apparent molecular mass of 38 kilodaltons, undergoes self-glucosylation in a UDP-glucose- and Mn2+-dependent reaction. Therefore, it is concluded that UPTG is the enzyme and at the same time the priming protein required for the biogenesis of protein-bound α-glucan in potato tuber.
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Dekanty, A. - Romero, N.M. - Bertolin, A.P. - Thomas, M.G. - Leishman, C.C. - Perez-Perri, J.I. - Boccaccio, G.L. - Wappner, P.
PLoS Genet. 2010;6(6):1-10
2010

Descripción: Hypoxia-inducible factors (HIFs) are a family of evolutionary conserved alpha-beta heterodimeric transcription factors that induce a wide range of genes in response to low oxygen tension. Molecular mechanisms that mediate oxygen-dependent HIF regulation operate at the level of the alpha subunit, controlling protein stability, subcellular localization, and transcriptional coactivator recruitment. We have conducted an unbiased genome-wide RNA interference (RNAi) screen in Drosophila cells aimed to the identification of genes required for HIF activity. After 3 rounds of selection, 30 genes emerged as critical HIF regulators in hypoxia, most of which had not been previously associated with HIF biology. The list of genes includes components of chromatin remodeling complexes, transcription elongation factors, and translational regulators. One remarkable hit was the argonaute 1 (ago1) gene, a central element of the microRNA (miRNA) translational silencing machinery. Further studies confirmed the physiological role of the miRNA machinery in HIF-dependent transcription. This study reveals the occurrence of novel mechanisms of HIF regulation, which might contribute to developing novel strategies for therapeutic intervention of HIF-related pathologies, including heart attack, cancer, and stroke. © 2010 Dekanty et al.
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Roberti, M.J. - Jovin, T.M. - Jares-Erijman, E.
PLoS ONE 2011;6(8)
2011

Descripción: We assessed the intracellular association states of the Parkinson's disease related protein α-synuclein (AS) in living cells by transfection with a functional recombinant mutant protein (AS-C4) bearing a tetracysteine tag binding the fluorogenic biarsenical ligands FlAsH and ReAsH, The aggregation states of AS-C4 were assessed by in situ microscopy of molecular translational mobility with FRAP (fluorescence recovery after photobleaching) and of local molecular density with confocal fluorescence anisotropy (CFA). FRAP recovery was quantitative and rapid in regions of free protein, whereas AS in larger aggregates was>80% immobile. A small 16% recovery characterized by an apparent diffusion constant of 0.03-0.04 μm 2/s was attributed to the dynamics of smaller, associated forms of AS-C4 and the exchange of mobile species with the larger immobile aggregates. By CFA, the larger aggregates exhibited high brightness and very low anisotropy, consistent with homoFRET between closely packed AS, for which a Förster distance (R o) of 5.3 nm was calculated. Other bright regions had high anisotropy values, close to that of monomeric AS, and indicative of membrane-associated protein with both low mobility and low degree of association. The anisotropy-fluorescence intensity correlations also revealed regions of free protein or of small aggregates, undetectable by conventional fluorescence imaging alone. The combined strategy (FRAP+CFA) provides a highly sensitive means for elucidating both the dynamics and structural features of protein aggregates and other intracellular complexes in living cells, and can be extended to other amyloid systems and to drug screening protocols. © 2011 Roberti et al.
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