Effect of homocysteinylation of low density lipoproteins on lipid peroxidation of human endothelial cells

Homocysteine-thiolactone (HcyT) is a toxic product whose synthesis is directly proportional to plasma homocysteine (Hcy) levels. Previous studies demonstrated that the interaction between HcyT and low density lipoproteins (LDL) induces the formation of homocystamide-LDL adducts (Hcy-LDL). Structural and functional alterations of Hcy-LDL have been described and it has been suggested that homocysteinylation could increase atherogenicity of LDL. Oxidative damage of endothelial cells (EC) is considered to be a critical aspect of the atherosclerotic process. To further investigate the molecular mechanisms involved in the atherogenicity of homocysteinylated LDL, we studied the effect of interaction between Hcy-LDL and EC on cell oxidative damage, using human aortic endothelial cells (HAEC) as experimental model. Homocysteinylation of LDL was carried out by incubation of LDL, isolated from plasma of healthy normolipemic subjects, with HcyT (10-100 microM). In our experimental conditions, homocysteinylation treatment was not accompanied by oxidative damage of LDL. No modifications of apoprotein structure and physico-chemical properties were observed in Hcy-LDL with respect to control LDL (c-LDL), as evaluated using the intrinsic fluorescence of tryptophan and the probe Laurdan incorporated in lipoproteins. Our results demonstrated that Hcy-LDL incubated at 37 degrees C for 3 h with HAEC, induced an oxidative damage on human EC with a significant increase of lipid hydroperoxides in cells incubated with Hcy-LDL with respect to cell incubated with c-LDL. The compositional changes were associated with a significant decrease viability in cells treated with Hcy-LDL. The relationship between the levels of -SH groups of LDL and the oxidative damage of HAEC has been demonstrated. These results suggest that Hcy-LDL exert a cytotoxic effect that is likely related to an increase in lipid peroxidation and oxidative damage of EC.     

Curcumin Modulates the NMDA Receptor Subunit Composition Through a Mechanism Involving CaMKII and Ser/Thr Protein Phosphatases

Curcumin is one of the major compounds contained in turmeric, the powdered rhizome of Curcuma longa. Results obtained in various experimental models indicate that curcumin has the potential to treat a large variety of neuronal diseases. Excitotoxicity, the toxicity due to pathological glutamate receptors stimulation, has been considered to be involved in several ocular pathologies including ischemia, glaucoma, and diabetic retinopathy. The NMDA receptor (NMDAR), a heteromeric ligand-gated ion channel, is composed of GluN1 and GluN2 subunits. There are four GluN2 subunits (GluN2A-D), which are major determinants of the functional properties of NMDARs. It is widely accepted that GluN2B has a pivotal role in excitotoxicity while the role of GluN2A remains controversial. We previously demonstrated that curcumin is neuroprotective against NMDA-induced excitotoxicity with a mechanism involving an increase of GluN2A subunit activity. In this paper, we investigate the mechanisms involved in curcumin-induced GluN2A increase in retinal cultures. Our results show that curcumin treatment activated CaMKII with a time-course that paralleled those of GluN2A increase. Moreover, KN-93, a CaMKII inhibitor, was able to block the effect of curcumin on GluN2A expression. Finally, in our experimental model, curcumin reduced ser/thr phosphatases activity. Using okadaic acid, a specific PP1 and PP2A blocker, we observed an increase in GluN2A levels in cultures. The ability of okadaic acid to mimic the effect of curcumin on GluN2A expression suggests that curcumin might regulate GluN2A expression through a phosphatase-dependent mechanism. In conclusion, our findings indicate curcumin modulation of CaMKII and/or ser/thr phosphatases activities as a mechanism involved in GluN2A expression and neuroprotection against excitotoxicity.     

Imaging of Endocytic Trafficking and Extracellular Vesicles Released Under Neratinib Treatment in ERBB2+ Breast Cancer Cells

Breast cancers (BCa) with ERBB2 amplification show rapid tumor growth, increased disease progression, and lower survival rate. Deregulated intracellular trafficking and extracellular vesicle (EVs) release are mechanisms that support cancer progression and resistance to treatments. Neratinib (NE) is a Food and Drug Administration–approved pan-ERBB inhibitor employed for the treatment of ERBB2⁺ BCa that blocks signaling and causes survival inhibition. However, the effects of NE on ERBB2 internalization, its trafficking to multivesicular bodies (MVBs), and the release of EVs that originate from these organelles remain poorly studied. By confocal and electron microscopy, we observed that low nanomolar doses of NE induced a modest ERBB2 internalization along with an increase of clathrin-mediated endocytosis and of the CD63+ MVB compartment in SKBR-3 cells. Furthermore, we showed in the culture supernatant two distinct EV subsets, based on their size and ERBB2 positivity: small (30–100 nm) ERBB2⁻ EVs and large (>100 nm) ERBB2⁺ EVs. In particular, we found that NE increased the overall release of EVs, which displayed a reduced ERBB2 positivity compared with controls. Taken together, these results provide novel insight into the effects of NE on ERBB2⁺ BCa cells that may lead to a reduction of ERBB2 potentially transferred to distant target cells by EVs:     

Antarctic ecosystems in transition – life between stresses and opportunities

Important findings from the second decade of the 21st century on the impact of environmental change on biological processes in the Antarctic were synthesised by 26 international experts. Ten key messages emerged that have stakeholder-relevance and/or a high impact for the scientific community. They address (i) altered biogeochemical cycles, (ii) ocean acidification, (iii) climate change hotspots, (iv) unexpected dynamism in seabed-dwelling populations, (v) spatial range shifts, (vi) adaptation and thermal resilience, (vii) sea ice related biological fluctuations, (viii) pollution, (ix) endangered terrestrial endemism and (x) the discovery of unknown habitats. Most Antarctic biotas are exposed to multiple stresses and considered vulnerable to environmental change due to narrow tolerance ranges, rapid change, projected circumpolar impacts, low potential for timely genetic adaptation, and migration barriers. Important ecosystem functions, such as primary production and energy transfer between trophic levels, have already changed, and biodiversity patterns have shifted. A confidence assessment of the degree of ‘scientific understanding’ revealed an intermediate level for most of the more detailed sub-messages, indicating that process-oriented research has been successful in the past decade. Additional efforts are necessary, however, to achieve the level of robustness in scientific knowledge that is required to inform protection measures of the unique Antarctic terrestrial and marine ecosystems, and their contributions to global biodiversity and ecosystem services.     

An innovative approach to molecularly imprinted capillaries for polar templates by grafting polymerization

Molecularly imprinted polymers have been successfully used as selective stationary phases in capillary electrophoresis. Notwithstanding, this technique suffers from several drawbacks as the loss of molecular recognition properties in aqueous media and the lack of feasibility for imprinted systems directed towards highly polar templates soluble in aqueous environments only. Thus, the preparation of imprinted polymers for highly polar, water-soluble analytes, represents a challenge. In this work, we present an innovative approach to overcome these drawbacks. It is based on a surface molecular imprinting technique that uses preformed macromonomers as both functional recognition elements and cross-linking agents. A poly-2-hydroxyethyl-co-methacrylic acid linear polymer was grafted from the surface of silica capillaries. The grafted polymer was exhaustively esterified with methacrylic anhydride to obtain polyethylendimethacrylate-co-methacrylic acid linear chains. Then, as a proof of concept, an adequate amount of a very polar template like penicillin V was added in a hydro-organic mixture, and a thin layer of imprinted polymer was obtained by cross-linking the polymer linear chains. The binding behaviour of the imprinted and non-imprinted capillaries was evaluated in different separation conditions in order to assess the presence of template selectivity and molecular recognition effects. The experimental results clearly show that this innovative kind of imprinted material can be easily obtained in very polar polymerization environments and that it is characterized by enhanced molecular recognition properties in aqueous buffers and good selectivity towards the template and strictly related molecules.     

Genetic analysis of the φC31 -specific phage growth limitation (Pgl) system of Streptomyces coelicolor A3(2)

The phage growth limitation (Pgl) system of Streptomyces coelicolor A3(2) was shown to be specific to φC31 homo-immune phages, and to be absent from the closely related strain Streptomyces Iividans. A 16 kb fragment of S. coelicolor A3(2) DNA was isolated which complemented the Pgl^? phenotype of J1501, a pgl mutant derivative of the Pgl^tsS. coelicolor strain M130. The cloned DNA complemented only half of the available pgl mutants, which therefore represented at least two groups, designated Pgl class A and class B strains. It follows that more than one kind of high-frequency genetic event can lead to the Pgl^? phenotype. Crosses between class A and class B strains yielded high frequencies of Pgl⁺ recombinants. Crosses between strains of the same class gave no Pgl⁺ recombinants. The cloned DNA was altered by deletion or apparent point mutation upon passage through the two class B strains tested, such that it was no longer capable of complementing class A strains. This accumulation of mutations might suggest that the expression of the cloned DNA is toxic to at least some class B strains. The nature of the genetic instability associated with the Pgl system was not detectable by Southern blot analysis.     

Sun‐induced chlorophyll fluorescence from high‐resolution imaging spectroscopy data to quantify spatio‐temporal patterns of photosynthetic function in crop canopies

Passive detection of sun‐induced chlorophyll fluorescence (SIF) using spectroscopy has been proposed as a proxy to quantify changes in photochemical efficiency at canopy level under natural light conditions. In this study, we explored the use of imaging spectroscopy to quantify spatio‐temporal dynamics of SIF within crop canopies and its sensitivity to track patterns of photosynthetic activity originating from the interaction between vegetation structure and incoming radiation as well as variations in plant function. SIF was retrieved using the Fraunhofer Line Depth (FLD) principle from imaging spectroscopy data acquired at different time scales a few metres above several crop canopies growing under natural illumination. We report the first maps of canopy SIF in high spatial resolution. Changes of SIF were monitored at different time scales ranging from quick variations under induced stress conditions to seasonal dynamics. Natural changes were primarily determined by varying levels and distribution of photosynthetic active radiation (PAR). However, this relationship changed throughout the day demonstrating an additional physiological component modulating spatio‐temporal patterns of SIF emission. We successfully used detailed SIF maps to track changes in the canopy's photochemical activity under field conditions, providing a new tool to evaluate complex patterns of photosynthesis within the canopy.     

Role of PKC-delta activity in glutathione-depleted neuroblastoma cells

Protein kinases C (PKCs) are a family of isoenzymes sensitive to oxidative modifications and involved in the transduction signal pathways that regulate cell growth. As such, they can act as cellular sensors able to intercept intracellular redox changes and promote the primary adaptive cell response. In this study, we have demonstrated that PKC isoforms are specifically influenced by the amount of intracellular glutathione (GSH). The greatest GSH depletion is associated with a maximal reactive oxygen species (ROS) production and accompanied by an increase in the activity of the delta isoform and a concomitant inactivation of alpha. ROS generation induced early morphological changes in GSH-depleted neuroblastoma cells characterized, at the intracellular level, by the modulation of PKC-delta activity that was involved in the pathway leading to apoptosis. When cells were pretreated with rottlerin, their survival was improved by the ability of this compound to inhibit the activity of PKC-delta and to counteract ROS production. These results define a novel role of PKC-delta in the cell signaling pathway triggered by GSH loss normally associated with many neurodegenerative diseases and clinically employed in the treatment of neuroblastoma.