The effects of cholesterol on the time-resolved emission anisotropy of 12-(9-anthroyloxy)stearic acid in dipalmitoylphosphatidylcholine bilayers

The time-resolved fluorescence emission anisotropy of 12-(9-anthroyloxy)stearic acid (12-AS) and 1,6-diphenyl-1,3,5-hexatriene (DPH) have been measured in dipalmitoylphosphatidylcholine liposomes in the presence and absence of 40 mol% cholesterol at temperatures above and below the phase transition temperature (41°C). By using a synchronously-pumped mode-locked frequency-doubled dye laser and single photon counting detection with an excitation response function of 300 picosecond, rotational correlation times down to less than 1 nanosecond could be resolved. Whereas DPH showed only small changes in the limiting anisotropy on the addition of cholesterol, 12-AS showed significant increases in this parameter with the effect being potentiated at higher temperatures. This difference in behaviour has been attributed to a fluorophore-cholesterol interaction that resulted in a change in the fluorophore geometry. Not only do DPH and 12-AS sense different depolarizing rotations due to the different directions of their emission dipoles but also differ in their lipid interactions which alter their limiting anisotropies. The implication is that the comparison of steady-state anisotropy measurements between chemically identical fluorophores in different lipid environments may be complicated by molecular distortions that change the motions to which the steady-state fluorescence parameters will be sensitive.     

Isolation and Characterization of the Putative Photoreceptor for Phototaxis in Amoebae of the Cellular Slime Mold,Dictyostelium discoideum

Amoebae of the cellular slime mold Dictyostelium discoideum (strain AX2) produce a pigment with an absorption spectrum that closely resembles the action spectrum for phototaxis. The protein-pigment complex was isolated and purified by sucrose gradient centrifugation, fast protein liquid chromatography (FPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). It is tightly membrane-bound and the bulk of it is located in the mitochondrial membrane fraction, while a small part is located in the cytoplasmic membrane fraction, as indicated by marker enzyme tests (succinate dehydrogenase for mitochondria and alkaline phosphatase for the cytoplasmic membrane). It is speculated that the pigment bound to the cytoplasmic membrane acts as photoreceptor and that bound to the mitochondria operates as a shading pigment in the light direction perception mechanism of Dictyostelium amoebae.     

Insect UV-, and green-photoreceptor membranes studied by the freeze-fracture technique

Summary The membranes of the microvilli of UV- and green-photoreceptors of the ant Myrmecia gulosa have been studied with the freeze-fracture technique. Both inner fracture faces, the cytoplasmic P-face and the extracellular E-face, are covered by globular particles. The P-face particles appear to be randomly distributed, occasionally forming clusters. Their density is about 7,000/m², and their mean diameter is 8.5 nm. The E-face particles, however, are arranged in an ordered square pattern with a center-to-center spacing of 9 nm. The density and distribution of P- and E-face particles are the same in both the UV- and the green-photoreceptor membranes. No differences were found in the ultrastructural organization of photoreceptor membranes after dark or light adaptation. It is suggested that the P-face particles represent rhodopsin molecules.     

The use of isochronal reference standards in phase and modulation fluorescence lifetime measurements

Errors in phase and modulation lifetime measurements observed with the only commercially available instrument are readily apparent when the Debye-Sears modulation tank is not perfectly tuned. Unfortunately, we have found that exact tuning was extremely difficult to achieve and maintain. We report that these errors could be reduced by using single-lifetime (homogeneous) reference standards whose fluorescence lifetime approximated that of the unknown sample (isochronal standards). A number of useful standards are suggested. In the proposed method, the phase shift and relative modulation of the sample emission are measured using the isochronal standard as a reference to determine the effective characteristics of the sinusoidal excitation. The importance of the improvement in accuracy accomplished by the proposed methods is illustrated by the accurate resolution of fluorescence lifetime heterogeneity for two known heterogeneous samples.     

Protection against oxidative stress by vitamin D in cone cells

Photoreceptor degeneration (PD) refers to a group of heterogeneous outer retinal dystrophies characterized by the death of photoreceptors. Both oxidative stress and inflammation are involved in the pathogenesis of PD. We investigate whether vitamin D has a potential for the treatment of PD by evaluating the anti‐oxidative stress and anti‐inflammatory properties of the active form of vitamin D₃, 1,α, 25‐dihydroxyvitamin D₃, in a mouse cone cell line, 661W. Mouse cone cells were treated with H₂O₂ or a mixture of H₂O₂ and vitamin D; cell viability was determined. The production of reactive oxygen species (ROS) in treated and untreated cells was measured. The expression of key anti‐oxidative stress and inflammatory genes in treated and untreated cells was determined. Treatment with vitamin D significantly increased cell viability and decreased ROS production in 661W cells under oxidative stress induced by H₂O₂. H₂O₂ treatment in 661W cells can significantly down‐regulate the expression of antioxidant genes and up‐regulate the expression of neurotoxic cytokines. Vitamin D treatment significantly reversed these effects and restored the expression of antioxidant genes. Vitamin D treatment also can block H₂O₂ induced oxidative damages. The data suggested that vitamin D may offer a therapeutic potential for patients with PD.     

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.     

Environmental sustainability of orthopedic devices produced with powder bed fusion

Additive manufacturing consists in melting metallic powders to produce objects from 3D data, layer upon layer. Its industrial applications range from automotive, biomedical (e.g., prosthetic implants for dentistry and orthopedics), aeronautics and others. This study uses life cycle assessment to evaluate the possible improvement in environmental performance of laser‐based powder bed fusion additive manufacturing systems on prosthetic device production. Environmental impacts due to manufacturing, use, and end of life of the designed solution were assessed. In addition, two powder production technologies, gas atomization (GA) and plasma atomization (PA), were compared in order to establish the most sustainable one. Production via traditional subtractive technologies and the additive manufacturing production were also compared. 3D building was found to have a significant environmental advantage compared to the traditional technology. The powder production process considerably influences on a damage point of view the additive manufacturing process; however, its impact can be mitigated if GA powders are employed.     

A non‐canonical rhodopsin‐mediated insulin receptor signaling pathway in retinal photoreceptor neurons

We previously reported a ligand‐independent and rhodopsin‐dependent insulin receptor (IR) neuroprotective signaling pathway in both rod and cone photoreceptor cells, which is activated through protein–protein interaction. Our previous studies were performed with either retina or isolated rod or cone outer segment preparations and the expression of IR signaling proteins were examined. The isolation of outer segments with large portions of the attached inner segments is a technical challenge. Optiprep? density gradient medium has been used to isolate the cells and subcellular organelles, Optiprep? is a non‐ionic iodixanol‐based medium with a density of 1.320 g/mL. We employed this method to examine the expression of IR and its signaling proteins, and activation of one of the downstream effectors of the IR in isolated photoreceptor cells. Identification of the signaling complexes will be helpful for therapeutic targeting in disease conditions.     

Risposte Fotomorfogeniche Indotte Dalla Radiazione UV-B in Brassica Oleracea var. Capitata

Abstract

Photomorphogenic responses induced by UV-B radiation in Brassica oleracea var. capitata.—Ultraviolet radiation can induce a plethora of “damaging” and “non damaging” effects in higher plants. We analyzed two possible photomorphogenic responses to UV-B radiation, the anthocyanin accumulation and the inhibition of hypocotyl elongation by modifying the UV spectral range with specific cut-off filters, under two levels of photon fluence rate. Experimental results suggest that detrimental effects are due to shorter wavelenghts of UV-B region (less than 305 nm); in contrast some adaptative responses may be induced by longer wavelenghts of UV-B region (between 305 and 320 nm). We attempted to determine the involvment of endogenous anthocyanin content in the UV-B photoprotection. Our experiments suggest a secondary role of anthocyanin accumulation in UV-B plant adaptation.     

Highly luminescent CdTe/CdS/ZnO core/shell/shell quantum dots fabricated using an aqueous strategy

To create core/shell/shell quantum dots (QDs) with high stability against a harmful chemical environment, CdTe/CdS QDs were coated with a ZnO shell in an aqueous solution. An interfaced CdS layer sandwiched between a CdTe core and ZnO shell provided relaxation of the strain at the core/shell interface since lattice parameters of CdS are intermediate between those of CdTe and ZnO. The photoluminescence (PL) peak wavelength of the core/shell/shell QDs was shifted from 569 to 615 nm by adjusting the size of CdTe cores and thickness of CdS and ZnO shells, along with the highest PL quantum yield of the core/shell/shell QDs reaching 80%, which implies promising applications in the field of biomedical labeling. Due to the decrease of surface defects, it was observed that PL lifetimes significantly increased at room temperature as follows: 29.6 34.2, and 47.5 ns for CdTe (537 nm), CdTe/CdS (555 nm) and CdTe/CdS/ZnO (581 nm) QDs, respectively. Copyright © 2012 John Wiley & Sons, Ltd.