Descripción: The polysaccharides from cystocarpic Iridaea undulosa, soluble and insoluble in 2M potassium chloride, Cs and Ci, respectively, were treated with alkali and fractionated by precipitation with increasing concentrations of KCl. They were later separated by ion-exchange chromatography, to yield fractions enriched in an α-(1→6)-glucan, agaroids and carrageenans.

Descripción: The dielectric breakdown model to describe the dielectric breakdown patterns in both conductor-loaded and insulator-loaded composites was generalized. With a variable concentration p, particles were distributed at random in a matrix. According to particle characteristics, the generalized model assigned different probabilities to breakdown channel formation. The fractal dimension D and the parameters of the Weibull distribution was used to characterize the dielectric breakdown patterns.

Descripción: A systematic study is presented of the effects of trehalose on the physical properties of extruded DPPC-cholesterol unilamellar vesicles. Particular emphasis is placed on examining how the interactions present in the hydrated state translate into those in the dehydrated state. Observations from HSDSC and DSC are used to examine the phase behavior of hydrated and dehydrated vesicles, respectively. The concentration of trehalose inside and outside the vesicles is manipulated, and is shown to affect the relative stability of the membranes. Our results show for the first time that a combination of high inner and low outer trehalose concentration is able to decrease the gel-to-liquid crystalline phase temperature (Tm), while any other combination will not. Upon dehydration, the Tm of all lipid mixtures increases. The extent of the increase depends on the trehalose distribution across the bilayer. The Tm changes in the same direction with trehalose concentration for both freeze-dried and fully hydrated samples, suggesting that the trehalose distribution across the vesicle membrane, as well as the trehalose-phospholipid interaction, is maintained upon lyophilization. The results presented in this work may aid in the formulation of systems to be used in the lyophilization of liposomes for drug delivery applications. © 2006 Elsevier B.V. All rights reserved.

Descripción: The effect of chronic exposure to Escherichia coli on morphometrical parameters, different antioxidant defenses, lipid peroxidation and protein oxidation has been studied in digestive gland of the freshwater mussel Diplodon chilensis in the laboratory during 6 weeks. In, a parallel field study, we evaluated morphometrical and oxidative stress parameters in digestive glands of mussels collected from a sewage polluted area (SMA) and from a pristine area (control). Both sites were characterized by analyzing bacteriological and physic-chemical parameters. In the laboratory D. chilensis was able to filter bacteria at high concentrations with a clearing rate of 0.510 ± 0.036. L/h per gram of dry soft tissue mass. The digestive gland mass to shell length ratio (DGM/SL), reduced glutathione (GSH), lipid peroxidation, as concentration of thiobartituric acid reactive substances (TBARS), and glutathione-S-transferase (GST) activity of mussels fed with bacteria were significantly higher than those of control mussels after the fourth week. Fecal bacteria in lake water samples were undetectable in the control, and higher than 24,000. MPN (most probable number)/100. mL in SMA. DGM/SL was higher in SMA. No differences between sites were observed in total lipid and protein content, neither in superoxide dismutase activity. GSH content was higher in SMA, with no difference in the oxidized form. GST activity and MDA were significantly higher in SMA but protein oxidation was not affected. © 2011 Elsevier GmbH.

Descripción: Photoactive materials based on dye molecules incorporated into thin films or bulk solids are useful for applications as photosensitization, photocatalysis, solar cell sensitization and fluorescent labeling, among others. In most cases, high concentrations of dyes are desirable to maximize light absorption. Under these circumstances, the proximity of dye molecules leads to the formation of aggregates and statistical traps, which dissipate the excitation energy and lower the population of excited states. The search for enhancement of light collection, avoiding energy wasting requires accounting the photophysical parameters quantitatively, including the determination of quantum yields, complicated by the presence of light scattering when particulate materials are considered. In this work we summarize recent advances on the photophysics of dyes in light-scattering materials, with particular focus on the effect of dye concentration. We show how experimental reflectance, fluorescence and laser-induced optoacoustic spectroscopy data can be used together with theoretical models for the quantitative evaluation of inner filter effects, fluorescence and triplet formation quantum yields and energy transfer efficiencies. © 2013 The American Society of Photobiology.

Descripción: In this work we studied the effect of NaCl on the thermodynamic and dynamic properties of supercooled water, for salt concentrations between 0.19 and 1.33mol kg-1, using molecular dynamic simulations for TIP5PE water model and ion parameters specially designed to be used in combination with this potential. We studied the isobaric heat capacity (Cp) temperature dependence and observed a maximum in Cp, occurring at Tm, that moves to lower temperature values with increasing salt concentration. Many characteristic changes were observed at scaled temperature TTm ∼ 0.96, namely a minimum in the density of the system, a reduction of the slope of the number of hydrogen bonds vs. temperature, and a crossover from Vogel-Tamman-Fulcher to Arrhenius dynamics. Finally, at low temperatures we observed that water dynamics become heterogeneous with an apparently common relationship between the fraction of immobile molecules and T/Tm for all studied systems. © 2011 American Institute of Physics.

Descripción: 1. 1.|Porphobilinogenase has been isolated and purified from cow liver and its components, porphobilinogen deaminase and uroporphyrinogen isomerase, have been separated from each other and purified. 2. 2.|The effect of NH4+ was studied. The deaminase exhibited classical Michaelis-Menten kinetics in the absence or presence of NH4+, which at high concentrations behaved as a noncompetitive inhibitor of the deaminase. As expected from Hill plots, n = 1 both in the absence or presence of NH4+. Instead, when activity of porpho bilinogenase is plotted versus porphobilinogen concentration, sigmoid curves are obtained; but the presence of NH4+ at different concentrations altered the kinetic parameters of this enzymic system, again showing normal kinetics. In addition, n values were found to be 2 for porphobilinogen per porphobilinogenase molecule and 1 in the presence of NH4+ which behaves as a competitive inhibitor of the isomerase. Results are discussed in relation to the allosteric theories of monod et al.1,2 and liver porphobilinogenase is proposed to be an allosteric protein. 3. 3.|The presence of an ultrafiltrable factor which stimulates uroporphyrinogen formation from porphobilinogen has been revealed. © 1969.

Descripción: VjbR is a LuxR homolog that regulates transcription of many genes including important virulence determinants of the facultative intracellular pathogen Brucella abortus. This transcription factor belongs to a family of regulators that participate in a cell-cell communication process called quorum sensing, which enables bacteria to respond to changes in cell population density by monitoring concentration of self produced autoinducer molecules. Unlike almost all other LuxR-type proteins, VjbR binds to DNA and activates transcription in the absence of any autoinducer signal. To investigate the mechanisms by which Brucella induces VjbR-mediated transcriptional activation, and to determine how inappropriate spatio-temporal expression of the VjbR target genes is prevented, we focused on the study of expression of vjbR itself. By assaying different parameters related to the intracellular lifestyle of Brucella, we identified a restricted set of conditions that triggers VjbR protein expression. Such conditions required the convergence of two signals of different nature: a specific pH value of 5.5 and the presence of urocanic acid, a metabolite involved in the connection between virulence and metabolism of Brucella. In addition, we also observed an urocanic acid, pH-dependent expression of RibH2 and VirB7, two additional intracellular survival-related proteins of Brucella. Analysis of promoter activities and determination of mRNA levels demonstrated that the urocanic acid-dependent mechanisms that induced expression of VjbR, RibH2, and VirB7 act at the post-transcriptional level. Taken together, our findings support a model whereby Brucella induces VjbR-mediated transcription by modulating expression of VjbR in response to specific signals related to the changing environment encountered within the host. © 2012 Arocena et al.

Descripción: Trypanosoma cruzi, etiological agent of Chagas' disease, has a complex life cycle which involves the invasion of mammalian host cells, differentiation and intracellular replication. Here we report the first insights into the biological role of a poly(ADP-ribose) glycohydrolase in a trypanosomatid (TcPARG). In silico analysis of the TcPARG gene pointed out the conservation of key residues involved in the catalytic process and, by Western blot, we demonstrated that it is expressed in a life stage-dependant manner. Indirect immunofluorescence assays and electron microscopy using an anti-TcPARG antibody showed that this enzyme is localized in the nucleus independently of the presence of DNA damage or cell cycle stage. The addition of poly(ADP-ribose) glycohydrolase inhibitors ADP-HPD (adenosine diphosphate (hydroxymethyl) pyrrolidinediol) or DEA (6,9-diamino-2-ethoxyacridine lactate monohydrate) to the culture media, both at a 1 μM concentration, reduced in vitro epimastigote growth by 35% and 37% respectively, when compared to control cultures. We also showed that ADP-HPD 1 μM can lead to an alteration in the progression of the cell cycle in hydroxyurea synchronized cultures of T. cruzi epimastigotes. Outstandingly, here we demonstrate that the lack of poly(ADP-ribose) glycohydrolase activity in Vero and A549 host cells, achieved by chemical inhibition or iRNA, produces the reduction of the percentage of infected cells as well as the number of amastigotes per cell and trypomastigotes released, leading to a nearly complete abrogation of the infection process. We conclude that both, T. cruzi and the host, poly(ADP-ribose) glycohydrolase activities are important players in the life cycle of Trypanosoma cruzi, emerging as a promising therapeutic target for the treatment of Chagas' disease. © 2013 Vilchez Larrea et al.

Descripción: Dimethyl sulfoxide (DMSO) has been known to enhance cell membrane permeability of drugs or DNA. Molecular dynamics (MD) simulations with single-component lipid bilayers predicted the existence of three regimes of action of DMSO: membrane loosening, pore formation and bilayer collapse. We show here that these modes of action are also reproduced in the presence of cholesterol in the bilayer, and we provide a description at the atomic detail of the DMSO-mediated process of pore formation in cholesterol-containing lipid membranes. We also successfully explore the applicability of DMSO to promote plasma membrane permeability to water, calcium ions (Ca2+) and Yo-Pro-1 iodide (Yo-Pro-1) in living cell membranes. The experimental results on cells in culture can be easily explained according to the three expected regimes: in the presence of low doses of DMSO, the membrane of the cells exhibits undulations but no permeability increase can be detected, while at intermediate DMSO concentrations cells are permeabilized to water and calcium but not to larger molecules as Yo-Pro-1. These two behaviors can be associated to the MD-predicted consequences of the effects of the DMSO at low and intermediate DMSO concentrations. At larger DMSO concentrations, permeabilization is larger, as even Yo-Pro-1 can enter the cells as predicted by the DMSO-induced membrane-destructuring effects described in the MD simulations. © 2012 de Ménorval et al.

Descripción: Nauplii of the branchiopod crustacean Artemia spp. are widely used as live food in aquaculture. Their nutritional value depends essentially on the fatty acid profile of their lipids, which can be very variable. Study of this variability (often species- and/or ecology-related) proposes interesting theoretical and applied questions. In the present study, cysts (dehydrated dormant eggs) from Argentinean Artemia spp. populations were analysed for their fatty acid profile. Most of the cyst samples analysed (n = 16) were obtained from Artemia persimilis populations, but 3 belonged to A. franciscana. Previous research indicated that A. persimilis populations are confined to inland biotopes, but in this study we report the presence of A. persimilis populations in coastal ecosystems. Cysts from all populations exhibited fatty acid profiles typical of either marine or freshwater, irrespective of the inland or coastal origin of their biotope. This study represents the first time that a significant amount of eicosapentaenoic acid (EPA, 20:5n-3) was detected in A. persimilis cyst samples. We hypothesize on some factors that presumably influence the fatty acid profiles of Artemia spp. cysts: (1) food resources from different habitats, which in turn can be influenced by environmental parameters, (2) the genetic make-up of each population, and/or (3) mechanisms of selective feeding. © Inter-Research 2007.

Descripción: Calcium signals are involved in a large variety of physiological processes. Their versatility relies on the diversity of spatiotemporal behaviors that the calcium concentration can display. Calcium entry through inositol 1,4,5-trisphosphate (IP3) receptors (IP3R's) is a key component that participates in both local signals such as "puffs" and in global waves. IP3R's are usually organized in clusters on the membrane of the endoplasmic reticulum and their spatial distribution has important effects on the resulting signal. Recent high resolution observations [1] of Ca2+ puffs offer a window to study intra-cluster organization. The experiments give the distribution of the number of IP3R's that open during each puff without much processing. Here we present a simple model with which we interpret the experimental distribution in terms of two stochastic processes: IP3 binding and unbinding and Ca2+-mediated inter-channel coupling. Depending on the parameters of the system, the distribution may be dominated by one or the other process. The transition between both extreme cases is similar to a percolation process. We show how, from an analysis of the experimental distribution, information can be obtained on the relative weight of the two processes. The largest distance over which Ca2+mediated coupling acts and the density of IP3-bound IP3R's of the cluster can also be estimated. The approach allows us to infer properties of the interactions among the channels of the cluster from statistical information on their emergent collective behavior. © 2010 Solovey, Dawson.

Descripción: The importance of the content of anionic phospholipids [cardiolipin (CL) and phosphatidylglycerol (PG)] in the osmotic adaptation and in the membrane structure of Bacillus subtilis cultures was investigated. Insertion mutations in the three putative cardiolipin synthase genes (ywiE, ywnE and ywjE) were obtained. Only the ywnE mutation resulted in a complete deficiency in cardiolipin and thus corresponds to a true clsA gene. The osmotolerance of a clsA mutant was impaired: although at NaCl concentrations lower than 1.2 M the growth curves were similar to those of its wild-type control, at 1 .5 M NaCl (LBN medium) the lag period increased and the maximal optical density reached was lower. The membrane of the clsA mutant strain showed an increased PG content, at both exponential and stationary phase, but no trace of CL in either LB or LBN medium. As well as the deficiency in CL synthesis, the clsA mutant showed other differences in lipid and fatty acids content compared to the wild-type, suggesting a cross-regulation in membrane lipid pathways, crucial for the maintenance of membrane functionality and integrity. The biophysical characteristics of membranes and large unilamellar vesicles from the wild-type and clsA mutant strains were studied by Laurdan's steady-state fluorescence spectroscopy. At physiological temperature, the clsA mutant showed a decreased lateral lipid packing in the protein-free vesicles and isolated membranes compared with the wild-type strain. Interestingly, the lateral lipid packing of the membranes of both the wild-type and clsA mutant strains increased when they were grown in LBN. In a conditional IPTG-controlled pgsA mutant, unable to synthesize PG and CL in the absence of IPTG, the osmoresistance of the cultures correlated with their content of anionic phospholipids. The transcriptional activity of the clsA and pgsA genes was similar and increased twofold upon entry to stationary phase or under osmotic upshift. Overall, these results support the involvement of the anionic phospholipids in the growth of B. subtilis in media containing elevated NaCl concentrations. © 2006 SGM.

Descripción: Background: Self-assembly is a common theme in proteins of unrelated sequences or functions. The human papillomavirus E7 oncoprotein is an extended dimer with an intrinsically disordered domain, that can form large spherical oligomers. These are the major species in the cytosol of HPV transformed and cancerous cells. E7 binds to a large number of targets, some of which lead to cell transformation. Thus, the assembly process not only is of biological relevance, but represents a model system to investigate a widely distributed mechanism. Methodology/Principal Findings: Using various techniques, we monitored changes in secondary, tertiary and quaternary structure in a time course manner. By applying a robust kinetic model developed by Zlotnik, we determined the slow formation of a monomeric "Z-nucleus" after zinc removal, followed by an elongation phase consisting of sequential second-order events whereby one monomer is added at a time. This elongation process takes place at a strikingly slow overall average rate of one monomer added every 28 seconds at 20 μM protein concentration, strongly suggesting either a rearrangement of the growing complex after binding of each monomer or the existence of a "conformation editing" mechanism through which the monomer binds and releases until the appropriate conformation is adopted. The oligomerization determinant lies within its small 5 kDa C-terminal globular domain and, remarkably, the E7 N-terminal intrinsically disordered domain stabilizes the oligomer, preventing an insoluble amyloid route. Conclusion: We described a controlled ordered mechanism with features in common with soluble amyloid precursors, chaperones, and other spherical oligomers, thus sharing determining factors for symmetry, size and shape. In addition, such a controlled and discrete polymerization reaction provides a valuable tool for nanotechnological applications. Finally, its increased immunogenicity related to its supramolecular structure is the basis for the development of a promising therapeutic vaccine candidate for treating HPV cancerous lesions. © 2012 Smal et al.

Descripción: The p53 tumor suppressor protein is an important regulator of cell proliferation and apoptosis. p53 can be found in the nucleus and in the cytosol, and the subcellular location is key to control p53 function. In this work, we found that a widely used monoclonal antibody against p53, termed Pab 1801 (Pan antibody 1801) yields a remarkable punctate signal in the cytoplasm of several cell lines of human origin. Surprisingly, these puncta were also observed in two independent p53-null cell lines. Moreover, the foci stained with the Pab 1801 were present in rat cells, although Pab 1801 recognizes an epitope that is not conserved in rodent p53. In contrast, the Pab 1801 nuclear staining corresponded to genuine p53, as it was upregulated by p53-stimulating drugs and absent in p53-null cells. We identified the Pab 1801 cytoplasmic puncta as P Bodies (PBs), which are involved in mRNA regulation. We found that, in several cell lines, including U2OS, WI38, SK-N-SH and HCT116, the Pab 1801 puncta strictly colocalize with PBs identified with specific antibodies against the PB components Hedls, Dcp1a, Xrn1 or Rck/p54. PBs are highly dynamic and accordingly, the Pab 1801 puncta vanished when PBs dissolved upon treatment with cycloheximide, a drug that causes polysome stabilization and PB disruption. In addition, the knockdown of specific PB components that affect PB integrity simultaneously caused PB dissolution and the disappearance of the Pab 1801 puncta. Our results reveal a strong cross-reactivity of the Pab 1801 with unknown PB component(s). This was observed upon distinct immunostaining protocols, thus meaning a major limitation on the use of this antibody for p53 imaging in the cytoplasm of most cell types of human or rodent origin. © 2012 Thomas et al.