Titration of cardiolipin by either 10-<Emphasis Type="Italic">N</Emphasis>-nonyl acridine orange or acridine orange sensitizes the adenine nucleotide carrier to permeability transition

Under the action of carboxyatractyloside or fatty acids, adenine nucleotide translocase switches its function from nucleotide carrier to modulator of the opening of a non-specific pore. In addition to the effect of these agents, this modification in activity is, in some way, dependent on the influence of the lipid milieu of the membrane. Cardiolipin is, among other membrane phospholipids, the one that interacts the most with the translocase. This work shows that 10-N-nonyl acridine orange and acridine orange, probes for this phospholipid, modify the sensitivity of the translocase to carboxyatractyloside, oleate, and palmitate to induce permeability transition. The results also show that these probes stimulate the release of mitochondrial cytochrome c, and increase labeling of the carrier by eosin 5-maleimide. Based on the aforementioned it is proposed that the increase in sensitivity is due to a conformational change in the translocase, induced by the binding of the probe to cardiolipin.     

Proteomic characterization of human proinflammatory M1 and anti‐inflammatory M2 macrophages and their response to Candida albicans

In response to different stimuli, macrophages can differentiate into either a pro‐inflammatory subtype (M1, classically activated macrophages) or acquire an anti‐inflammatory phenotype (M2, alternatively activated macrophages). Candida albicans is the most important opportunistic fungus in nosocomial infections, and it is contended by neutrophils and macrophages during the first steps of the invasive infection. Murine macrophages responses to C. albicans have been widely studied, whereas the responses of human‐polarized macrophages remain less characterized. In this study, we have characterized the proteomic differences between human M1‐ and M2‐polarized macrophages, both in basal conditions and in response to C. albicans, by quantitative proteomics (2DE). This proteomic approach allowed us to identify metabolic routes and cytoskeletal rearrangement components that are the most relevant differences between M1 and M2 macrophages. The analysis has revealed fructose‐1,6‐bisphosphatase 1, a critical enzyme in gluconeogenesis, up‐regulated in M1, as a novel protein marker for macrophage polarization. Regarding the response to C. albicans, an M1‐to‐M2 switch in polarization was observed. This M1‐to‐M2 switch might contribute to Candida pathogenicity by decreasing the generation of specific immune responses, thus enhancing fungal survival and colonization, or instead, may be part of the host attempt to reduce the inflammation and limit the damage of the infection.     

The structure of an α-D-glucan from Cyttaria harioti Fischer

A homogeneous glucan has been isolated from the fruiting bodies of Cyttaria harioti Fischer. Partial acid hydrolysis produced major amounts of isomaltose, whereas acetolysis gave maltose and maltotriose. Enzymic hydrolysis with amylo-glucosidase and pullulanase indicated a structure based on maltotriose residues connected by (1→6)-α-D linkages. This conclusion was supported by periodate-oxidation data which also showed that 3–7% of the glucose resisted oxidation. Methylation analysis confirmed the presence of (1→6) and (1→4) linkages in the ratio 1:2.4.     

The permeability transition pore as a pathway for the release of mitochondrial DNA

This study shows that under oxidative stress DNA from liver mitochondria (mtDNA) can be released through the non-specific permeability transition pore. Pore opening was induced after the addition of Fe2+ and hydrogen peroxide, in the presence of calcium ions. Under these conditions mitochondria undergo large extent swelling, accompanied by the generation of thiobarbituric acid-reactive substances. It was observed that mtDNA was hydrolyzed after the oxidative stress, and it is proposed that some of the fragments were released from the matrix, in such a way that approximately 12% of the total mtDNA remained in the mitochondria. The remaining genetic material was analyzed, after its extraction in an agarose gel. The fragments released were smaller that 1000 bp, by analysis in a native 8% polyacrilamide gel. The presence of cyclosporin A, that inhibited permeability transition, also inhibited mtDNA release by roughly 52%.     

The determination of the asymmetrical stereochemical distribution of fatty acids in triacylglycerols

The fatty acid distribution in triacylglycerols (TAGs) is a factor that contributes to the intrinsic properties of oils from different species variants. Many hypotheses have been proposed to explain the specific distribution of fatty acids in the different naturally occurring oils. Currently, the 1,3-random-2-random theory is more or less accepted, but it has been widely shown that most vegetable oils do not behave randomly in the sn-1 and sn-3 stereochemical positions. For this reason, complex methodologies have been developed to analyze the fatty acid composition of the three stereochemical positions in TAGs. In this article, we propose that by calculating the asymmetric alpha coefficient, the stereochemical asymmetry of fatty acids in TAG molecular species can be defined. This coefficient reflects the relative content of fatty acids at the sn-1 and sn-3 positions and may overcome the problems found mainly with complex sn-1, sn-2, and sn-3 stereochemical analysis of fatty acids in TAG. The alpha coefficient is calculated from the fatty acid, sn-2 fatty acid, and TAG composition of the oil. Indeed, through this coefficient, it has been possible to show that, despite having the same overall content, the stearic acid distribution in the sn-1 and sn-3 positions is not random in some oils.     

Agaric acid induces mitochondrial permeability transition through its interaction with the adenine nucleotide translocase. Its dependence on membrane fluidity

The effect of agaric acid as inducer of mitochondrial permeability transition was studied. It was found that: (i) agaric acid (AA) promoted efflux of accumulated Ca2+, collapse of transmembrane potential, and mitochondrial swelling; (ii) these effects depend on membrane fluidity; (iii) ADP inhibited the effect of AA on Ca2+ efflux, and (iv) AA blocked binding of the sulfhydryl reagent, eosin-5-maleimide, to the adenine nucleotide translocase. It is proposed that AA induces pore opening through binding of the citrate moiety to the ADP/ATP carrier; this interaction must be stabilized by insertion of the alkyl chain in the lipid milieu of the membrane.     

3D Imaging Detection of HER2 Based in the Use of Novel Affibody-Quantum Dots Probes and Ratiometric Analysis

Patients with breast cancer (BC) overexpressing HER2 (HER2+) are selected for Trastuzumab treatment, which blocks HER2 and improves cancer prognosis. However, HER2+ diagnosis, by the gold standard, immunohistochemistry, could lead to errors, associated to: a) variability in sample manipulation (thin 2D sections), b) use of subjective algorithms, and c) heterogeneity of HER2 expression within the tissue. Therefore, we explored HER2 3D detection by multiplexed imaging of Affibody-Quantum Dots conjugates (Aff-QD), ratiometric analysis (RMA_FI) and thresholding, using BC multicellular tumor spheroids (BC-MTS) (~120 μm of diameter) as 3D model of BC. HER2+, HER2– and hybrid HER2+/? BC-MTS (mimicking heterogeneous tissue) were incubated simultaneously with two Aff-QD probes (anti-HER2 and negative control (NC), respectively, (1:1)). Confocal XY sections were recorded along the Z distance, and processed by automatized RMA_FI (anti-HER2 Aff-QD/ NC). Quantifying the NC fluorescence allowed to predict the fraction of non-specific accumulation of the anti-HER2 probe within the thick sample, and resolve the specific HER2 level. HER2 was detected up to 30 μm within intact BC-MTS, however, permeabilization improved detection up to 70 μm. Specific HER2 signal was objectively quantified, and HER2 3D-density of 9.2, 48.3 and 30.8% were obtained in HER2?, HER2+ and hybrid HER2+/? permeabilized BC-MTS, respectively. Therefore, by combining the multiplexing capacity of Aff-QD probes and RMA_FI, we overcame the challenge of non-specific probe accumulation in 3D samples with minimal processing, yielding a fast, specific spatial HER2 detection and objective quantification.     

A catalytically inactive gelatinase B/MMP-9 mutant impairs homing of chronic lymphocytic leukemia cells by altering migration regulatory pathways

We previously showed that MMP-9 overexpression impairs migration of primary CLL cells and MEC-1 cells transfected with MMP-9. To determine the contribution of non-proteolytic activities to this effect we generated MEC-1 transfectants stably expressing catalytically inactive MMP-9MutE (MMP-9MutE-cells). In xenograft models in mice, MMP-9MutE-cells showed impaired homing to spleen and bone marrow, compared to cells transfected with empty vector (Mock-cells). In vitro transendothelial and random migration of MMP-9MutE-cells were also reduced. Biochemical analyses indicated that RhoAGTPase and p-Akt were not downregulated by MMP-9MutE, at difference with MMP-9. However, MMP-9MutE-cells or primary cells incubated with MMP-9MutE had significantly reduced p-ERK and increased PTEN, accounting for the impaired migration. Our results emphasize the role of non-proteolytic MMP-9 functions contributing to CLL progression.     

Environmental Niche Overlap between Common and Dusky Dolphins in North Patagonia,Argentina

Research on the ecology of sympatric dolphins has increased worldwide in recent decades. However, many dolphin associations such as that between common dolphins (Delphinus delphis) and dusky dolphins (Lagenorhynchus obscurus) are poorly understood. The present study was conducted in the San Matías Gulf (SMG) ecosystem (North Patagonia, Argentina) where a high diet overlap among both species was found. The main objective of the present work was to explore the niche overlap of common and dusky dolphins in the habitat and temporal dimensions. The specific aims were (a) to evaluate the habitat use strategies of both species through a comparison of their group attributes (social composition, size and activity), and (b) to evaluate their habitat preferences and habitat overlap through Environmental Niche modeling considering two oceanographic seasons. To accomplish these aims, we used a historic database of opportunistic and systematic records collected from 1983 to 2011. Common and dusky dolphins exhibited similar patterns of group size (from less than 10 to more than 100 individuals), activity (both species use the area to feed, nurse, and copulate), and composition (adults, juveniles, and mothers with calves were observed for both species). Also, both species were observed travelling and feeding in mixed-species groups. Specific overlap indices were higher for common dolphins than for dusky dolphins, but all indices were low, suggesting that they are mainly segregated in the habitat dimension. In the case of common dolphins, the best habitats were located in the northwest of the gulf far from the coast. In the warm season they prefer areas with temperate sea surface and in the cold season they prefer areas with relatively high variability of sea surface temperature. Meanwhile, dusky dolphins prefer areas with steep slopes close to the coast in the southwestern sector of the gulf in both seasons.