Showing papers by "National University of Cuyo published in 2010"

Autophagosome Formation Depends on the Small GTPase Rab1 and Functional ER Exit Sites

Abstract: Autophagy is an important cellular degradation pathway present in all eukaryotic cells. Via this pathway, portions of the cytoplasm and/or organelles are sequestered in double-membrane structures called autophagosomes. In spite of the significant advance achieved in autophagy, the long-standing question about the source of the autophagic membrane remains unsolved. We have investigated the role of the secretory pathway in autophagosome biogenesis. Sar1 and Rab1b are monomeric GTPases that control traffic from the endoplasmic reticulum (ER) to the Golgi. We present evidence indicating that the activity of both proteins is required for autophagosome formation. Overexpression of dominant-negative mutants and the use of siRNAs impaired autophagosome generation as determined by LC3 puncta formation and light chain 3 (LC3)-II processing. In addition, our results indicate that the autophagic and secretory pathways intersect at a level preceding the brefeldin A blockage, suggesting that the transport from the cis/medial Golgi is not necessary for autophagosome biogenesis. Our present results highlight the role of transport from the ER in the initial events of the autophagic vacuole development.

Determination of melatonin in wine and plant extracts by capillary electrochromatography with immobilized carboxylic multi-walled carbon nanotubes as stationary phase.

Abstract: The finding of melatonin, the often called "hormone of darkness" in plants opens an interesting perspective associated to the plethora of health benefits related to the moderate consumption of red wine. In this study, the implementation of a new method for the determination of melatonin in complex food matrices by CEC with immobilized carboxylic multi-walled carbon nanotubes as stationary phase is demonstrated. The results indicated high electrochromatographic resolution, good capillary efficiencies and improved sensitivity respect to those obtained with conventional capillaries. In addition, it was demonstrated highly reproducible results between runs, days and columns. The LOD for melatonin was 0.01 ng/mL. The method was successfully applied to the determination of melatonin in red and white wine, grape skin and plant extracts of Salvia officinalis L .

Alpha-hemolysin is required for the activation of the autophagic pathway in Staphylococcus aureus-infected cells.

Abstract: Staphylococcus aureus is a pathogen that causes serious infectious diseases eventually leading to septic and toxic shock. Classically S. aureus has been considered an extracellular pathogen, but cumulative evidence indicates that it invades cells and replicates intracellularly leading to staphylococcal persistence and chronic disease. It has been previously shown that this pathogen localizes to LC3-labeled compartments and subverts the autophagy pathway. One of the key features of S. aureus infection is the production of a series of virulence factors, including secreted enzymes and toxins. In the present report we present evidence that the pore-forming toxin alpha-hemolysin (Hla) is a S. aureus secreted factor which participates in the activation of the autophagic pathway. In addition, our results indicate that although the toxin elicits an autophagic response this pathway is dysfunctional as indicated by the accumulation of the LC3-II form in cell lysates obtained from intoxicated cells. In addition, not only the purified Hla toxin but also the toxin-secreting pathogen prevented the maturation of autophagosomes. Interestingly, in cells infected with the wild-type strain of S. aureus the bacteria-containing compartments which recruited LC3 onto the limiting membrane did not accumulate the acidotropic probe LysoTracker. In contrast, those phagosomes containing the Hla(-) mutant (unable to produce the toxin) localized in an acidic compartment unlabeled by LC3. These results suggest that the LC3 protein is recruited only to those damaged vacuoles (i.e., perforated by the toxin), perhaps as an attempt to protect the cells. Furthermore, we have demonstrated that the toxin-dependent activation of autophagy (although it is regulated by calcium and requires Atg5) is independent of both PI3Kinase activity and Beclin 1 suggesting the involvement of a non-canonical autophagy pathway.

Transcriptome changes in grapevine ( Vitis vinifera L.) cv. Malbec leaves induced by ultraviolet-B radiation

Abstract: Ultraviolet-B radiation (UV-B, 280-315 nm) is a natural component of sunlight, which has numerous regulatory effects on plant physiology. The nature of the response to UV-B is dependent on fluence rate, dose, duration and wavelength of the UV-B treatment. Some reports have analyzed the changes in gene expression caused by UV-B light on several plant species using microarray technology. However, there is no information on the transcriptome response triggered by UV-B in grapevine. In this paper we investigate the gene expression responses of leaves from in vitro cultured Vitis vinifera cv. Malbec plants subjected to the same dose of biologically effective UV-B radiation (4.75 kJ m-2 d-1) administered at two different fluence rates (16 h at ≅ 8.25 μW cm-2, 4 h at ≅ 33 μW cm-2) using a new custom made GrapeGen Affymetrix GeneChip®. The number of genes modulated by high fluence rate UV-B doubled the number of genes modulated by low fluence UV-B. Their functional analyses revealed several functional categories commonly regulated by both UV-B treatments as well as categories more specifically modulated depending on UV-B fluence rate. General protective responses, namely the induction of pathways regulating synthesis of UV-B absorbing compounds such as the Phenylpropanoid pathway, the induction of different antioxidant defense systems and the activation of pathways commonly associated with pathogen defense and abiotic stress responses seem to play critical roles in grapevine responses against UV-B radiation. Furthermore, high fluence rate UV-B seemed to specifically modulate additional pathways and processes in order to protect grapevine plantlets against UV-B-induced oxidative stress, stop the cell cycle progression, and control protein degradation. On the other hand, low fluence rate UV-B regulated the expression of specific responses in the metabolism of auxin and abscisic acid as well as in the modification of cell walls that could be involved in UV-B acclimation-like processes. Our results show the UV-B radiation effects on the leaf transcriptome of grapevine (Vitis vinifera cv. Malbec) plantlets. Functional categories commonly modulated under both UV-B treatments as well as transcripts specifically regulated in an UV-B-intensity dependent way were identified. While high fluence rate UV-B had regulatory effects mainly on defense or general multiple-stress responses pathways, low fluence rate UV-B promoted the expression of genes that could be involved in UV-B protection or the amelioration of the UV-B-induced damage. This study also provides an extensive list of genes regulating multiple metabolic pathways involved in the response of grapevine to UV-B that can be used for future researches.

The 2010 Maule, Chile earthquake: Downdip rupture limit revealed by space geodesy

Abstract: Radar interferometry from the ALOS satellite captured the coseismic ground deformation associated with the 2010 Mw 8.8 Maule, Chile earthquake. The ALOS interferograms reveal a sharp transition in fringe pattern at ~150 km from the trench axis that is diagnostic of the downdip rupture limit of the Maule earthquake. An elastic dislocation model based on ascending and descending ALOS interferograms and 13 near-field 3-component GPS measurements reveals that the coseismic slip decreases more or less linearly from a maximum of 17 m (along-strike average of 6.5 m) at 18 km depth to near zero at 43–48 km depth, quantitatively indicating the downdip limit of the seismogenic zone. The depth at which slip drops to near zero appears to be at the intersection of the subducting plate with the continental Moho. Our model also suggests that the depth where coseismic slip vanishes is nearly uniform along the strike direction for a rupture length of ~600 km. The average coseismic slip vector and the interseismic velocity vector are not parallel, which can be interpreted as a deficit in strike-slip moment release.

Benefit and cost curves for typical pollination mutualisms

Abstract: Mutualisms provide benefits to interacting species, but they also involve costs. If costs come to exceed benefits as population density or the frequency of encounters between species increases, the interaction will no longer be mutualistic. Thus curves that represent benefits and costs as functions of interaction frequency are important tools for predicting when a mutualism will tip over into antagonism. Currently, most of what we know about benefit and cost curves in pollination mutualisms comes from highly specialized pollinating seed-consumer mutualisms, such as the yucca moth-yucca interaction. There, benefits to female reproduction saturate as the number of visits to a flower increases (because the amount of pollen needed to fertilize all the flower's ovules is finite), but costs continue to increase (because pollinator offspring consume developing seeds), leading to a peak in seed production at an intermediate number of visits. But for most plant-pollinator mutualisms, costs to the plant are more subtle than consumption of seeds, and how such costs scale with interaction frequency remains largely unknown. Here, we present reasonable benefit and cost curves that are appropriate for typical pollinator-plant interactions, and we show how they can result in a wide diversity of relationships between net benefit (benefit minus cost) and interaction frequency. We then use maximum-likelihood methods to fit net-benefit curves to measures of female reproductive success for three typical pollination mutualisms from two continents, and for each system we chose the most parsimonious model using information-criterion statistics. We discuss the implications of the shape of the net-benefit curve for the ecology and evolution of plant-pollinator mutualisms, as well as the challenges that lie ahead for disentangling the underlying benefit and cost curves for typical pollination mutualisms.

Hypercholesterolemia Impaired Sperm Functionality in Rabbits

Abstract: Hypercholesterolemia represents a high risk factor for frequent diseases and it has also been associated with poor semen quality that may lead to male infertility. The aim of this study was to analyze semen and sperm function in diet-induced hypercholesterolemic rabbits. Twelve adult White New Zealand male rabbits were fed ad libitum a control diet or a diet supplemented with 0.05% cholesterol. Rabbits under cholesterol-enriched diet significantly increased total cholesterol level in the serum. Semen examination revealed a significant reduction in semen volume and sperm motility in hypercholesterolemic rabbits (HCR). Sperm cell morphology was seriously affected, displaying primarily a “folded head”-head fold along the major axe-, and the presence of cytoplasmic droplet on sperm flagellum. Cholesterol was particularly increased in acrosomal region when detected by filipin probe. The rise in cholesterol concentration in sperm cells was determined quantitatively by Gas chromatographic-mass spectrometric analyses. We also found a reduction of protein tyrosine phosphorylation in sperm incubated under capacitating conditions from HCR. Interestingly, the addition of Protein Kinase A pathway activators -dibutyryl-cyclic AMP and iso-butylmethylxanthine- to the medium restored sperm capacitation. Finally, it was also reported a significant decrease in the percentage of reacted sperm in the presence of progesterone. In conclusion, our data showed that diet-induced hypercholesterolemia adversely affects semen quality and sperm motility, capacitation and acrosomal reaction in rabbits; probably due to an increase in cellular cholesterol content that alters membrane related events.

Emerging ionic liquid-based techniques for total-metal and metal-speciation analysis

Abstract: Ionic liquids (ILs) are generally considered to be more environmentally friendly than common organic solvents and have unique characteristics (e.g., effectively no vapor pressure, adjustable viscosity and miscibility in aqueous phases). They are also considered to be highly efficient extractant phases to improve analyte selectivity and sensitivity, so that they are important tools for chromatographic and spectrometric analysis. We review state-of-the-art applications of ILs in analytical chemistry with special emphasis on metal determination and speciation analysis. We discuss modern microextraction techniques based on ILs [e.g., dispersive liquid-liquid microextraction (DLLME), single-drop microextraction (SDME), and on-line LLME]. Also, we comment on potential applications and developments of solid-phase extraction (SPE) techniques involving IL-modified surface materials for metal retention and preconcentration. In all cases, we review crucial parameters and practical considerations of method optimization and application. Further, we critically compare IL-based methods in terms of analytical performance and environmental compatibility.

Sequence Diversity of Genes Encoding Botulinum Neurotoxin Type F

Abstract: Botulism due to type F botulinum neurotoxin (BoNT/F) is rare (<1% of cases), and only a limited number of clostridial strains producing this toxin type have been isolated. As a result, analysis of the diversity of genes encoding BoNT/F has been challenging. In this study, the entire bont/F nucleotide sequences were determined from 33 type F botulinum toxin-producing clostridial strains isolated from environmental sources and botulism outbreak investigations. We examined proteolytic and nonproteolytic Clostridium botulinum type F strains, bivalent strains, including Bf and Af, and Clostridium baratii type F strains. Phylogenetic analysis revealed that the bont/F genes examined formed 7 subtypes (F1 to F7) and that the nucleotide sequence identities of these subtypes differed by up to 25%. The genes from proteolytic (group I) C. botulinum strains formed subtypes F1 through F5, while the genes from nonproteolytic (group II) C. botulinum strains formed subtype F6. Subtype F7 was composed exclusively of bont/F genes from C. baratii strains. The region of the bont/F5 gene encoding the neurotoxin light chain was found to be highly divergent compared to the other subtypes. Although the bont/F5 nucleotide sequences were found to be identical in strains harboring this gene, the gene located directly upstream (ntnh/F) demonstrated sequence variation among representative strains of this subtype. These results demonstrate that extensive nucleotide diversity exists among genes encoding type F neurotoxins from strains with different phylogenetic backgrounds and from various geographical sources.

Exogenous ABA Increases Yield in Field-Grown Wheat with Moderate Water Restriction

Abstract: Water stress is one of the most important environmental factors that regulate a plant’s growth and development. In agronomic practice the effects of water stress are translated into low yield and/or reduced quality. Abscisic acid (ABA) sprays (1 mM) were applied to wheat plants at different phenological stages and the effects on several physiological variables and on yield were evaluated under field conditions at different water regimes. Studies were conducted in the field across three consecutive winter–spring seasons. ABA treatments were applied at the beginning of shoot enlargement and repeated at anthesis. Exogenous ABA increased shoot dry weight and maintained a high concentration of photosynthetic pigments for a longer period of time during grain growth and maturation. Although ABA applications increased stomatal closure immediately after its application, the longer-term effect was to allow for a greater ostiolar opening of the stomatal pore which resulted in increased conductance of gases and water vapor. ABA also improved the transport of photoassimilates from the leaves and stem to the developing grains, that is, it effectively increased the sink strength of the grains. This correlated with a yield increase without significantly changing the protein quality in the grains. Thus, elevated ABA levels from exogenous application or genetic selection could help improve agricultural production of grains in arid areas where irrigation is not possible.

Coxiella burnetii modulates Beclin 1 and Bcl-2, preventing host cell apoptosis to generate a persistent bacterial infection.

Abstract: Coxiella burnetii is the etiological agent of the human disease, Q fever, and is an obligate intracellular bacterium that invades and multiplies in a vacuole with lysosomal characteristics. We have previously shown that Coxiella interacts with the autophagic pathway as a strategy for its survival and replication. In addition, recent studies have shown that Coxiella exerts anti-apoptotic activity to maintain the host cell viability, thus generating a persistent infection. In the present report, we have explored the role of Beclin 1 and Bcl-2 in C. burnetii infection to elucidate how this bacterium modulates autophagy and apoptosis to its own benefit. Beclin 1, a Bcl-2 interacting protein, is required for autophagy. In this study, we show that Beclin 1 is recruited to the Coxiella-membrane vacuole, favoring its development and bacterial replication. In contrast, the anti-apoptotic protein Bcl-2 alters the normal development of the Coxiella-replicative compartment, in spite of also being recruited to the vacuole membrane. Furthermore, both vacuole development and the anti-apoptotic effect of C. burnetii are affected by Beclin 1 depletion and by the expression of a Beclin 1 mutant defective in Bcl-2 binding. Overall, these findings indicate that C. burnetii infection modulates autophagy and apoptotic pathways through Beclin 1/Bcl-2 interplay to establish a successful infection in the host cell.

Molecular Dynamics Simulation of Laser Melting of Nanocrystalline Au

Abstract: The mechanisms and kinetics of short pulse laser melting of single crystal and nanocrystalline Au films are investigated on the basis of the results of simulations performed with a model combining the molecular dynamics method with a continuum-level description of the laser excitation and subsequent relaxation of the conduction band electrons. A description of the thermophysical properties of Au that accounts for the contribution of the thermal excitation of d band electrons is incorporated into the model and is found to play a major role in defining the kinetics of the melting process. The effect of nanocrystalline structure on the melting process is investigated for a broad range of laser fluences. At high fluences, the grain boundary melting in nanocrystalline films results in a moderate decrease of the size of the crystalline grains at the initial stage of the laser heating and is followed by a rapid (within several picoseconds) collapse of the crystal structure in the remaining crystalline parts of t...

The strength of single crystal copper under uniaxial shock compression at 100 GPa

Abstract: In situ x-ray diffraction has been used to measure the shear strain (and thus strength) of single crystal copper shocked to 100 GPa pressures at strain rates over two orders of magnitude higher than those achieved previously. For shocks in the [001] direction there is a significant associated shear strain, while shocks in the [111] direction give negligible shear strain. We infer, using molecular dynamics simulations and VISAR (standing for 'velocity interferometer system for any reflector') measurements, that the strength of the material increases dramatically (to ~1 GPa) for these extreme strain rates.

Titania and titania–silver nanocomposite coatings grown by electrophoretic deposition from aqueous suspensions

Abstract: This study focused on the synthesis of titania–silver (TiO 2 –Ag) nanocomposite coatings with potential enhanced antibacterial properties for applications in orthopedic implants. Ag nanoparticles (4 nm) were grown directly on the surface of commercially available TiO 2 nanoparticles (23 nm) by nucleophilic reaction. The electrophoretic deposition (EPD) of TiO 2 and TiO 2 –Ag nanoparticles on titanium substrates using aqueous suspensions was investigated. Best results were achieved by using water and ethanol as co-solvents. The EPD process (voltage: 3 V, deposition time: 90 s) led to TiO 2 based coatings on titanium substrates exhibiting homogeneous and uniform microstructure. In vitro bioactivity tests in Kokubo's simulated body fluid (SBF) to evaluate the formation of hydroxyapatite (HA) on the coating surface were performed. The results showed that the extent of HA formation rapidly increased with increasing time in SBF and it decreased as silver amount in the coating increased. XRD, TEM and SEM-EDAX were used to investigate the microstructure of the nanomaterials and coatings. The high bioactivity of the TiO 2 based electrophoretic coatings indicates their potential for use as bioactive antibacterial layers in orthopedic implants.

Sediment records of highly variable mercury inputs to mountain lakes in Patagonia during the past millennium

Abstract: . High Hg levels in the pristine lacustrine ecosystems of the Nahuel Huapi National Park, a protected zone situated in the Andes of Northern Patagonia, Argentina, have initiated further investigations on Hg cycling and source identification. Here we report Hg records in sedimentary sequences to identify atmospheric sources during the past millennium. In addition to global transport and deposition, a potential atmospheric Hg source to be considered is the local emissions associated with volcanic activity, because the Park is situated in the Southern Volcanic Zone. Two sediment cores were extracted from Lake Toncek, a small, high-altitude system reflecting mainly direct inputs associated with atmospheric contributions, and Lake Moreno Oeste, a much larger and deeper lake having an extended watershed covered mostly by native forest. The sedimentary sequences were dated based on both 210Pb and 137Cs profiles. In addition, tephra layers were identified and geochemically characterized for chronological application and to investigate any association of volcanic eruptions with Hg records. Hg concentrations in sediments were measured along with 32 other elements, as well as organic matter, subfossil chironomids, and biogenic silica. Observed background Hg concentrations, determined from the sequence domains with lower values, ranged from 50 to 100 ng g−1 dry weight (DW), whereas the surficial layers reached 200 to 500 ng g−1 DW. In addition to this traditional pattern, however, two deep domains in both sequences showed dramatically increased Hg levels reaching 400 to 650 ng g−1 DW; the upper dated to the 18th to 19th centuries, and the lower around the 13th century. These concentrations are not only elevated in the present profiles but also many-fold above the background values determined in other fresh water sediments, as were also the Hg fluxes, reaching 120 to 150 μg m−2 y−1 in Lake Toncek . No correlation was observed between Hg concentrations and the contents of organic matter, subfossil chironomids, biogenic silica, or the other elements determined. However, distinctly increased Hg concentrations were observed immediately above some tephra layers, suggesting a link to volcanic events. Extended fires might be another potential atmospheric source because the earlier Hg peaks coincide with reported charcoal peaks, whereas the upper Hg peaks coincide with evidences of extended forest fires from tree-ring data and historical records.

Cold vapor ionic liquid-assisted headspace single-drop microextraction: a novel preconcentration technique for mercury species determination in complex matrix samples.

Abstract: A novel technique named cold vapor ionic liquid-assisted headspace single drop microextraction (CV-ILAHS-SDME) was developed for Hg species determination at trace levels. Inorganic (InHg) and organomercury (OrgHg) species separation, preconcentration and determination were performed by in situ cold vapor generation (CV) followed by headspace extraction with a suspended microdrop of a low cost RTIL, i.e. tetradecyl(trihexyl)phosphonium chloride (CYPHOS® IL 101), and direct injection in electrothermal atomic absorption spectrometry (ETAAS). Stannous chloride (SnCl2) was used to reduce Hg2+ to volatile Hg0, while oxidation of OrHg species permitted the determination of total Hg. OrHg species concentration was evaluated based on the difference between total Hg and InHg concentration. Different variables of CV-ILAHS-SDME technique, such as cold vapor generation conditions, temperature, sample and solvent volume, extraction time, and stirring rate were carefully studied. The analytical sensitivity was enhanced by a factor of 75. A low detection limit (10 ng L−1) and good precision (relative standard deviation of 4.6% at 0.25 μg L−1 Hg and n = 10) were achieved. Experimental results demonstrated that CV-ILAHS-SDME using CYPHOS® IL 101 is a rapid, cost-effective and green microextraction technique for Hg determination in samples with a complex matrix, such as sea water, fish tissues, hair and wine.

Short term hypothyroidism affects ovarian function in the cycling rat

Abstract: Background Rats made hypothyroid with propilthyouracil start showing abnormal cycling on the second cycle after the start of the treatment, with a high proportion of spontaneous pseudopregnancies and reduced fertility.

An artificial intelligence planning approach to manufacturing feature recognition

Abstract: Within manufacturing, features have been widely accepted as useful concepts, and in particular they are used as an interface between CAD and CAPP systems. Previous research on feature recognition focus on the issues of intersecting features and multiple interpretations, but do not address the problem of custom features representation. Representation of features is an important aspect for making feature recognition more applicable in practice. In this paper a hybrid procedural and knowledge-based approach based on artificial intelligence planning is presented, which addresses both classic feature interpretation and also feature representation problems. STEP designs are presented as case studies in order to demonstrate the effectiveness of the model.