880 research outputs found
Poly-l/dl-lactic acid films functionalized with collagen IV as carrier substrata for corneal epithelial stem cells
Limbal epithelial stem cells (LESCs) are responsible for the renewal of corneal epithelium. Cultivated limbal epithelial transplantation is the current treatment of choice for restoring the loss or dysfunction of LESCs. To perform this procedure, a substratum is necessary for in vitro culturing of limbal epithelial cells and their subsequent transplantation onto the ocular surface. In this work, we evaluated poly-L/DL-lactic acid 70:30 (PLA) films functionalized with type IV collagen (col IV) as potential in vitro carrier substrata for LESCs. We first demonstrated that PLA-col IV films were biocompatible and suitable for the proliferation of human corneal epithelial cells. Subsequently, limbal epithelial cell suspensions, isolated from human limbal rings, were cultivated using culture medium that did not contain animal components. The cells adhered significantly faster to PLA-col IV films than to tissue culture plastic (TCP). The mRNA expression levels for the LESC specific markers, K15, P63α and ABCG2 were similar or greater (significantly in the case of K15) in limbal epithelial cells cultured on PLA-col IV films than limbal epithelial cells cultured on TCP. The percentage of cells expressing the corneal (K3, K12) and the LESC (P63α, ABCG2) specific markers was similar for both substrata. These results suggest that the PLA-col IV films promoted LESC attachment and helped to maintain their undifferentiated stem cell phenotype. Consequently, these substrata offer an alternative for the transplantation of limbal cells onto the ocular surface.This work was supported by the Carlos III National Institute of Health, Spain (CIBER-BBN and Spanish Network on Cell Therapy, (TerCel RD12/0019/0036), MINECO/FEDER, EU), and the Castilla y León Regional Government, Spain (Regional Center for Regenerative Medicine and Cell Therapy, SAN673/VA/28/08 and SAN126/VA11/09)
Un estudio histórico del problema de las piezas prismáticas rectas sometidas a compresión. Parte II
The first part of this article is complemented with a short historical revision of some of the theoretical models and numerical methods employed in the resolution of the outlined problems when its analytical description has not been possible.Some of the positions that they have been used to approach the equations that govern the models are mentioned in a first paragraph: analytical methods with closed solutions, approximate methods, matrix methods, finite elements and generalized splines. A second paragraph lists different numerical techniques employed for the determination of the deformed, section forces, paths, limit and critical points.Se complementa la primera parte de este trabajo con un breve repaso histórico de algunos de los modelos teóricos y métodos numéricos empleados en la resolución de los problemas planteados en el artículo anterior, cuando no ha sido posible su descripción analítica. En un primer apartado se mencionan algunos de los métodos que se han utilizado para abordar las ecuaciones que rigen los modelos: analíticos con soluciones cerradas, aproximados, matriciales, elementos finitos y splines generalizados. En un segundo apartado se enumeran distintas técnicas numéricas empleadas para la determinación de los aspectos que describen los problemas en relación con las vigas-columna: deformadas, esfuerzos, trayectorias, puntos límite y críticos, etc
Structural and functional characterization of phosphomimetic mutants of cytochrome c at threonine 28 and serine 47
Protein function is frequently modulated by post-translational modifications of specific residues. Cytochrome c, in particular, is phosphorylated in vivo at threonine 28 and serine 47. However, the effect of such modifications on the physiological functions of cytochrome c – namely, the transfer of electrons in the respiratory electron transport chain and the triggering of programmed cell death – is still unknown. Here we replace each of these two residues by aspartate, in order to mimic phosphorylation, and report the structural and functional changes in the resulting cytochrome c variants. We find that the T28D mutant causes a 30-mV decrease on the midpoint redox potential and lowers the affinity for the distal site of Arabidopsis thaliana cytochrome c1 in complex III. Both the T28D and S47D variants display a higher efficiency as electron donors for the cytochrome c oxidase activity of complex IV. In both protein mutants, the peroxidase activity is significantly higher, which is related to the ability of cytochrome c to leave the mitochondria and reach the cytoplasm. We also find that both mutations at serine 47 (S47D and S47A) impair the ability of cytoplasmic cytochrome c to activate the caspases cascade, which is essential for triggering programmed cell death.Peer reviewe
Oxidative stress is tightly regulated by cytochrome c phosphorylation and respirasome factors in mitochondria
Respiratory cytochrome c has been found to be phosphorylated at tyrosine 97 in the postischemic brain upon neuroprotective insulin treatment, but how such posttranslational modification affects mitochondrial metabolism is unclear. Here, we report the structural features and functional behavior of a phosphomimetic cytochrome c mutant, which was generated by site-specific incorporation at position 97 of p-carboxymethyl-l-phenylalanine using the evolved tRNA synthetase method. We found that the point mutation does not alter the overall folding and heme environment of cytochrome c, but significantly affects the entire oxidative phosphorylation process. In fact, the electron donation rate of the mutant heme protein to cytochrome c oxidase, or complex IV, within respiratory supercomplexes was higher than that of the wild-type species, in agreement with the observed decrease in reactive oxygen species production. Direct contact of cytochrome c with the respiratory supercomplex factor HIGD1A (hypoxia-inducible domain family member 1A) is reported here, with the mutant heme protein exhibiting a lower affinity than the wild-type species. Interestingly, phosphomimetic cytochrome c also exhibited a lower caspase-3 activation activity. Altogether, these findings yield a better understanding of the molecular basis for mitochondrial metabolism in acute diseases, such as brain ischemia, and thus could allow the use of phosphomimetic cytochrome c as a neuroprotector with therapeutic applications.España, Junta de Andalucía BIO-198España MINECO BFU2015-71017/BM
Intravitreal injection of proinsulin-loaded microspheres delays photoreceptor cell death and vision loss in the rd10 mouse model of retinitis pigmentosa
9 p.-6 fig.PURPOSE. The induction of proinsulin expression by transgenesis or intramuscular gene therapy has been shown previously to retard retinal degeneration in mouse and rat models of retinitis pigmentosa (RP), a group of inherited conditions that result in visual impairment. We investigated whether intraocular treatment with biodegradable poly (lactic-co-glycolic) acid microspheres (PLGA-MS) loaded with proinsulin has cellular and functional neuroprotective effects in the retinaMETHODS. Experiments were performed using the Pde6brd10 mouse model of RP.
Methionylated human recombinant proinsulin (hPI) was formulated in PLGA-MS, which
were administered by intravitreal injection on postnatal days (P) 14 to 15. Retinal
neuroprotection was assessed at P25 by electroretinography, and by evaluating outer nuclear layer (ONL) cellular preservation. The attenuation of photoreceptor cell death by hPI was determined by TUNEL assay in cultured P22 retinas, as well as Akt phosphorylation by immunoblottingRESULTS. We successfully formulated hPI PLGA-MS to deliver the active molecule for several weeks in vitro. The amplitude of b-cone and mixed b-waves in electroretinographic recording was significantly higher in eyes injected with hPI-PLGA-MS compared to control eyes.Treatment with hPI-PLGA-MS attenuated photoreceptor cell loss, as revealed by comparing ONL thickness and the number of cell rows in this layer in treated versus untreated retinas. Finally, hPI prevented photoreceptor cell death and increased AktThr308 phosphorylation in organotypic cultured retinas.CONCLUSIONS. Retinal degeneration in the rd10 mouse was slowed by a single intravitreal injection of hPI-PLGA-MS. Human recombinant proinsulin elicited a rapid and effective neuroprotective effect when administered in biodegradable microspheres, which may constitute a future potentially feasible delivery method for proinsulin-based treatment of RP.Supported by Grants from the Spanish Ministerio de Ciencia e
Innovacion (MICINN) and Spanish Ministerio de EconomÍa y
Competitividad (MINECO), SAF2010-21879 (EJdlR and PdlV),
SAF2013-41059-R (FdP and EJdlR), and technical personnel
support from CIBERDEM, ISCIII, Madrid, SpainPeer reviewe
Biodegradable Polyester Films from Renewable Aleuritic Acid: Surface Modifications Induced by Melt-polycondensation in Air.
Good water barrier properties and biocompatibility of long-chain biopolyesters like
cutin and suberin have inspired the design of synthetic mimetic materials. Most of these
biopolymers are made from esterified mid-chain functionalized ω-long chain hydroxyacids.
Aleuritic (9,10,16-trihydroxypalmitic) acid is such a polyhydroxylated fatty acid and
is also the major constituent of natural lac resin, a relatively abundant and renewable
resource. Insoluble and thermostable films have been prepared from aleuritic acid by meltcondensation
polymerization in air without catalysts, an easy and attractive procedure for
large scale production. Intended to be used as a protective coating, the barrier's performance
is expected to be conditioned by physical and chemical modifications induced by oxygen
on the air-exposed side. Hence, the chemical composition, texture, mechanical behavior,
hydrophobicity, chemical resistance and biodegradation of the film surface have been studied
by attenuated total reflection–Fourier transform infrared spectroscopy (ATR–FTIR), atomic
force microscopy (AFM), nanoindentation and water contact angle (WCA). It has been
demonstrated that the occurrence of side oxidation reactions conditions the surface physical
and chemical properties of these polyhydroxyester films. Additionally, the addition of palmitic
acid to reduce the presence of hydrophilic free hydroxyl groups was found to have a strong
influence on these parametersPeer reviewe
The cytochrome f–plastocyanin complex as a model to study transient interactions between redox proteins
AbstractTransient complexes, with a lifetime ranging between microseconds and seconds, are essential for biochemical reactions requiring a fast turnover. That is the case of the interactions between proteins engaged in electron transfer reactions, which are involved in relevant physiological processes such as respiration and photosynthesis. In the latter, the copper protein plastocyanin acts as a soluble carrier transferring electrons between the two membrane-embedded complexes cytochrome b6f and photosystem I. Here we review the combination of experimental efforts in the literature to unveil the functional and structural features of the complex between cytochrome f and plastocyanin, which have widely been used as a suitable model for analyzing transient redox interactions
Structural basis for inhibition of the histone chaperone activity of SET/TAF-Iß by cytochrome c
Chromatin is pivotal for regulation of the DNA damage process insofar as it influences access to DNA and serves as a DNA repair docking site. Recent works identify histone chaperones as key re- gulators of damaged chromatin’s transcriptional activity. However, understanding how chaperones are modulated during DNA damage response is still challenging. This study reveals that the histone chap- erone SET/TAF-Iß interacts with cytochrome c following DNA damage. Specifically, cytochrome c is shown to be translocated into cell nuclei upon induction of DNA damage, but not upon stimulation of the death receptor or stress-induced pathways. Cytochrome c was found to competitively hinder binding of SET/TAF-Iß to core histones, thereby locking its histone-binding domains and inhibiting its nucle- osome assembly activity. In addition, we have used NMR spectros- copy, calorimetry, mutagenesis, and molecular docking to provide an insight into the structural features of the formation of the complex between cytochrome c and SET/TAF-Iß. Overall, these findings estab- lish a framework for understanding the molecular basis of cyto- chrome c-mediated blocking of SET/TAF-Iß, which subsequently may facilitate the development of new drugs to silence the oncogenic effect of SET/TAF-Iß’s histone chaperone activity
An Embedded System in Smart Inverters for Power Quality and Safety Functionality
The electricity sector is undergoing an evolution that demands the development of a
network model with a high level of intelligence, known as a Smart Grid. One of the factors accelerating
these changes is the development and implementation of renewable energy. In particular, increased
photovoltaic generation can affect the network’s stability. One line of action is to provide inverters
with a management capacity that enables them to act upon the grid in order to compensate for these
problems. This paper describes the design and development of a prototype embedded system able to
integrate with a photovoltaic inverter and provide it with multifunctional ability in order to analyze
power quality and operate with protection. The most important subsystems of this prototype are
described, indicating their operating fundamentals. This prototype has been tested with class A
protocols according to IEC 61000-4-30 and IEC 62586-2. Tests have also been carried out to validate
the response time in generating orders and alarm signals for protections. The highlights of these
experimental results are discussed. Some descriptive aspects of the integration of the prototype in an
experimental smart inverter are also commented upon
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