Unusual Robustness of Neurotransmitter Vesicle Membranes against Serotonin-Induced Perturbations.

Nature confines hundreds of millimolar of amphiphilic neurotransmitters, such as serotonin, in synaptic vesicles. This appears to be a puzzle, as the mechanical properties of lipid bilayer membranes of individual major polar lipid constituents of synaptic vesicles [phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS)] are significantly affected by serotonin, sometimes even at few millimolar concentrations. These properties are measured by atomic force microscopy, and their results are corroborated by molecular dynamics simulations. Complementary 2H solid-state NMR measurements also show that the lipid acyl chain order parameters are strongly affected by serotonin. The resolution of the puzzle lies in the remarkably different properties displayed by the mixture of these lipids, at molar ratios mimicking those of natural vesicles (PC:PE:PS:Cholesterol = 3:5:2:5). Bilayers constituting of these lipids are minimally perturbed by serotonin, and show only a graded response at physiological concentrations (>100 mM). Significantly, the cholesterol (up to 33% molar ratio) plays only a minor role in dictating these mechanical perturbations, with PC:PE:PS:Cholesterol = 3:5:2:5 and 3:5:2:0 showing similar perturbations. We infer that nature uses an emergent mechanical property of a specific mixture of lipids, all individually vulnerable to serotonin, to appropriately respond to physiological serotonin levels.

Interaction of guanidinium and ammonium cations with phosphatidylcholine and phosphatidylserine lipid bilayers - Calorimetric, spectroscopic and molecular dynamics simulations study.

The ability of arginine-rich peptides to cross the lipid bilayer and enter cytoplasm, unlike their lysine-based analogues, is intensively studied in the context of cell-penetrating peptides. Although the experiments have not yet reconstructed their internalization mechanism, the computational studies have shown that the type or charge of lipid polar groups is one of the crucial factors in their translocation. In order to gain more detailed insight into the interaction of guanidinium (Gdm+) and ammonium (NH4+) cations, as important building blocks in arginine and lysine amino acids, with lipid bilayers, we conducted the experimental and computational study that tackles this phenomenon. The adsorption of Gdm+ and NH4+ on lipid bilayers prepared from a zwitterionic (DPPC) and an anionic (DPPS) lipid was examined by thermoanalytic and spectroscopic techniques. Using temperature-dependent UV-Vis spectroscopy and DSC calorimetry we determined the impact of Gdm+ and NH4+ on the thermotropic properties of lipid bilayers. FTIR data, along with molecular dynamics simulations, unraveled the molecular-level details on the nature of their interactions, showing the proton transfer between NH4+ and DPPS, but not between Gdm+ and DPPS. The findings originated from this work imply that Gdm+ and NH4+ form qualitatively different interactions with lipids of different charge which is reflected in the physico-chemical interactions that arginine-and lysine-based peptides establish at a complex and chemically heterogeneous environment such as the biological membrane.

Effect of ultrasound on the structural characteristics and oxidative stability of walnut oil oleogel coated with soy protein isolate-phosphatidylserine.

In this study, the three-dimensional network system formed by rice bran wax (RBW) was used as the internal structure, and the external structure formed by soybean protein isolate (SPI) and phosphatidylserine (PS) was added on the basis of the internal structure to prepare walnut oil oleogel (SPI-PS-WOG). Ultrasonic treatment was applied to the mixed solution to make SPI-PS-WOG, on the basis, the effects of ultrasonic treatment on SPI-PS-WOG were investigated. The results showed that both ß and ß' crystalline forms were present in all SPI-PS-WOG samples. When the ultrasonic power was 450 W, the first weight loss peak in the thermogravimetric (TGA) curve appeared at 326 °C, which was shifted to the right compared to the peak that occurred when the ultrasonic power was 0 W, indicating that the thermal stability of the SPI-PS-WOG was improved by the ultrasonic treatment. Moreover, when the ultrasonic power was 450 W, the oil holding capacity (OHC) reached 95.3 %, which was the best compared with other groups. Both confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) showed that the ultrasonic treatment of appropriate power succeeded in making the SPI-PS-WOG samples more evenly dispersed in the internal structure and denser in the external structure. In terms of oxidative stability, it was found that the peroxide value of SPI-PS-WOG remained at 9.8 mmol/kg oil for 50 days under 450 W ultrasonic power treatment, which was significantly improved compared with liquid walnut oil (WO). These results provide a new idea for the preparation of oleogels, and also lay a theoretical foundation for the application of ultrasonic treatment in oleogels.

Negatively charged phospholipids accelerate the membrane fusion activity of the plant-specific insert domain of an aspartic protease.

Various plants use antimicrobial proteins/peptides to resist phytopathogens. In the potato, Solanum tuberosum, the plant-specific insert (PSI) domain of an aspartic protease performs this role by disrupting phytopathogen plasma membranes. However, the mechanism by which PSI selects target membranes has not been elucidated. Here, we studied PSI-induced membrane fusion, focusing on the effects of lipid composition on fusion efficiency. Membrane fusion by the PSI involves an intermediate state whereby adjacent liposomes share their bilayers. We found that increasing the concentration of negatively charged phosphatidylserine (PS) phospholipids substantially accelerated PSI-mediated membrane fusion. NMR data demonstrated that PS did not affect the binding between the PSI and liposomes but had seminal effects on the dynamics of PSI interaction with liposomes. In PS-free liposomes, the PSI underwent significant motion, which was suppressed on PS-contained liposomes. Molecular dynamics simulations showed that the PSI binds to PS-containing membranes with a dominant angle ranging from -31° to 30°, with respect to the bilayer, and is closer to the membrane surfaces. In contrast, PSI is mobile and exhibits multiple topological states on the surface of PS-free membranes. Taken together, our data suggested that PS lipids limit the motion of the anchored PSI, bringing it closer to the membrane surface and efficiently bridging different liposomes to accelerate fusion. As most phytopathogens have a higher content of negatively charged lipids as compared with host cells, these results indicate that the PSI selectively targets negatively charged lipids, which likely represents a way of distinguishing the pathogen from the host.

Secretion of Recombinant Human Annexin V in Fusion with the Super Folder GFP for Labelling Phosphatidylserine-Exposing Membranes.

Annexin V (ANXV), mostly characterized by its ability to interact with biological membranes in a calcium-dependent manner. ANXV interacts mainly with phosphatidylserine (PS), for that fluorescent ANXV widely produced and used as a sensitive and specific probe to mark apoptotic cells or any PS-containing bilayers membranes. Many reports described the prokaryotic expression of recombinant human ANXV. To overcome some of E. coli expression limitations, we aimed in this work to investigate unconventional alternative expression system in mammalian cells for producing secreted human ANXV in fusion with the super folder green fluorescent protein (sfGFP). HEK239T cells were transfected using polyethylenimine (PEI) and pcDNA-sfGFP-ANXV plasmid. Forty-eight hours post transfection, direct fluorescence measurement, immunoblotting and ELISA confirmed the presence of secreted sfGFP-ANXV in cells supernatant. The yield of secreted 6 × His-tagged sfGFP-ANXV after affinity purification was estimated to be around 2 µg per 1 ml of cells supernatant. The secretion system was proper to produce a fully functional sfGFP-ANXV fusion protein in quantities enough to recognize and bind PS-containing surfaces or liposomes. Besides, biological assays such as flow cytometry and fluorescent microscopy confirmed the capacity of the secreted sfGFP-ANXV to detect PS exposure on apoptotic cells. Taken together, we present mammalian expression as a quick, affordable and endotoxin-free system to produce sfGFP-ANXV fusion protein. The secreted sfGFP-ANXV in eukaryotic system is a promising biotechnological tool, it opens up new horizons for additional applications in the detection of PS bearing surfaces and apoptosis in vitro and in vivo assays.

Phosphatidylserine from Portunus trituberculatus Eggs Alleviates Insulin Resistance and Alters the Gut Microbiota in High-Fat-Diet-Fed Mice.

Portunus trituberculatus eggs contain phospholipids, whose components and bioactivity are unclear. Here, we investigated the fatty acid composition of phosphatidylserine from P. trituberculatus eggs (Pt-PS). Moreover, its effects on insulin resistance and gut microbiota were also evaluated in high-fat-diet-fed mice. Our results showed that Pt-PS accounted for 26.51% of phospholipids and contained abundant polyunsaturated fatty acids (more than 50% of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)). Animal experiments indicated that Pt-PS significantly decreased body weight and adipose weight gain, improved hyperglycemia and hyperinsulinemia, mitigated insulin resistance, and regulated circulatory cytokines. Pt-PS activated insulin receptor substrate 1 (IRS1) and increased the levels of IRS1-associated phosphatidylinositol 3-hydroxy kinase (PI3K), phosphorylated protein kinase B (Akt) protein, and plasma membrane glucose transporter 4 protein. Furthermore, Pt-PS modified the gut microbiota, inducing, especially, a dramatic decrease in the ratio of Firmicutes to Bacteroidetes at the phylum level, as well as a remarkable improvement in their subordinate categories. Pt-PS also reduced fecal lipopolysaccharide concentration and enhanced fecal acetate, propionate, and butyrate concentrations. Additionally, the effects of Pt-PS on alleviation of insulin resistance and regulation of intestinal bacteria were better than those of phosphatidylserine from soybean. These results suggest that Pt-PS mitigates insulin resistance by altering the gut microbiota. Therefore, Pt-PS may be developed as an effective food supplement for the inhibition of insulin resistance and the regulation of human gut health.

Recovery of brain DHA-containing phosphatidylserine and ethanolamine plasmalogen after dietary DHA-enriched phosphatidylcholine and phosphatidylserine in SAMP8 mice fed with high-fat diet.

BACKGROUND: Glycerophospholipids were the main components of cerebral cortex lipids, and there was a close association between lipid homeostasis and human health. It has been reported that dietary DHA-enriched phosphatidylcholine (DHA-PC) and phosphatidylserine (DHA-PS) could improve brain function. However, it was unclear that whether supplementation of DHA-PC and DHA-PS could change lipid profiles in the brain of dementia animals. METHODS: SAMP8 mice was fed with different diet patterns for 2 months, including high-fat diet and low-fat diet. After intervention with DHA-PC and DHA-PS for another 2 months, the lipid profile in cerebral cortex was determined by lipidomics in dementia mice. RESULTS: High-fat diet could significantly decrease the levels of DHA-containing PS/pPE, DPA-containing PS, and AA-containing PE, which might exhibit the potential of lipid biomarkers for the prevention and diagnosis of AD. Notably, DHA-PC and DHA-PS remarkably recovered the lipid homeostasis in dementia mice. These might provide a potential novel therapy strategy and direction of dietary intervention for patients with cognitive decline. CONCLUSIONS: DHA-PC and DHA-PS could recover the content of brain DHA-containing PS and pPE in SAMP8 mice fed with high-fat diet.

Peptide Disc Mediated Control of Membrane Protein Orientation in Supported Lipid Bilayers for Surface-Sensitive Investigations.

In vitro characterization of membrane proteins requires experimental approaches providing mimics of the microenvironment that proteins encounter in native membranes. In this context, supported lipid bilayers provide a suitable platform to investigate membrane proteins by a broad range of surface-sensitive techniques such as neutron reflectometry (NR), quartz crystal microbalance with dissipation monitoring (QCM-D), surface plasmon resonance (SPR), atomic force microscopy (AFM), and fluorescence microscopy. Nevertheless, the successful incorporation of membrane proteins in lipid bilayers with sufficiently high concentration and controlled orientation relative to the bilayer remains challenging. We propose the unconventional use of peptide discs made by phospholipids and amphipathic 18A peptides to mediate the formation of supported phospholipid bilayers with two different types of membrane proteins, CorA and tissue factor (TF). The membrane proteins are reconstituted in peptide discs, deposited on a solid surface, and the peptide molecules are then removed with extensive buffer washes. This leaves a lipid bilayer with a relatively high density of membrane proteins on the support surface. As a very important feature, the strategy allows membrane proteins with one large extramembrane domain to be oriented in the bilayer, thus mimicking the in vivo situation. The method is highly versatile, and we show its general applicability by characterizing with the above-mentioned surface-sensitive techniques two different membrane proteins, which were efficiently loaded in the supported bilayers with ∼0.6% mol/mol (protein/lipid) concentration corresponding to 35% v/v for CorA and 8% v/v for TF. Altogether, the peptide disc mediated formation of supported lipid bilayers with membrane proteins represents an attractive strategy for producing samples for structural and functional investigations of membrane proteins and for preparation of suitable platforms for drug testing or biosensor development.

Improved Intracellular Delivery of Polyarginine Peptides with Cargoes.

Complementary to endocytosis, cell-penetrating peptides (CPPs) at high concentrations can penetrate the cell membrane in a direct way, which further makes CPPs popular candidates for delivering therapeutic or diagnostic agents. Although featured as rapid uptake, the translocation efficiency and potential toxicity of the direct penetration are usually affected by cargoes, which is still unclear. Here, using coarse-grained molecular dynamics simulations, we show that the polyarginine (R8) peptides penetrate the membrane through a water pore in the membrane, and the transmembrane efficiency is improved by conjugating to small nanoparticles (NPs) with proper linkers. It can be attributed to both the extension of the lifetime of the water pore by the NPs and outward diffusion of negative lipids in the asymmetry membrane, which induces the surrounding R8-NP conjugates to the water pore before it is closed. The translocation efficiency is closely related to the length of the linkers, and it gets the maximum value when the length of the linkers is around half of the membrane thickness. Overlong linkers not only decrease the transmembrane efficiency because of the blockage of NPs in the water pore but may also cause cytotoxicity because of the unclosed water pore. The results provide insights into the internalization of CPPs and facilitate the design of CPP and drug conjugates with high efficiency and low toxicity.

Topotecan effect on the structure of normal and cancer plasma membrane lipid models: A multi-model approach.

Topotecan is a relatively large, planar, asymmetric and polar molecule with a lactone moiety. In neutral or basic aqueous solutions, this ring opens forming the carboxylate form of Topotecan that is biologically inactive and uncapable of passively cross membranes. Nevertheless, despite this inability to cross membranes at this form, Topotecan may still be able to interact with phospholipid bilayers, disturbing them. In this context, phospholipid models, mimicking normal (DMPC at pH 7.4) and cancer cell lipid membranes (DMPC:DMPS (5:1) at pH 6.5), were used to assess structural modifications upon interaction with Topotecan. Langmuir isotherms of monolayers coupled with Brewster angle microscopy, differential scanning calorimetry of liposomes and X-ray scattering of small and wide angle of stacked multilayers were used as complementary techniques. The overall results show that the interaction of Topotecan with lipid membranes is deeply conditioned by their composition and that Topotecan seems to have a preferential interaction with the glycerol backbone of phosphatidylcholine phospholipids.

Impact of Phospholipids and Tocopherols on the Oxidative Stability of Soybean Oil-in-Water Emulsions.

Phospholipids have been shown to act synergistically with tocopherols and delay lipid oxidation in bulk oil. The synergistic activity between phospholipids and tocopherols is due to the ability of amino-group-containing phospholipids (e.g., phosphatidylethanolamine (PE) and phosphatidylserine (PS)) to convert oxidized tocopherol back into tocopherols. This study shows the effect of PE and PS on the antioxidant activity of different tocopherol homologues in oil-in-water emulsions. Effect of emulsifier type on the interaction between tocopherols and phospholipids was also studied. δ-Tocopherol and PE exhibited greater antioxidant activity as compared to α-tocopherol and PE. PS displayed 1.5-3 times greater synergism than PE with Tween 20 as emulsifier whereas both PE and PS had a similar antioxidant activity in the presence of α-tocopherol when bovine serum albumin was used as the emulsifier. This study is the first to show that PE and PS can act synergistically with tocopherols to inhibit lipid oxidation in oil-in-water emulsions and can present a new clean label antioxidant strategy for food emulsions.

Fabrication of 3-O-sn-Phosphatidyl-L-serine Anchored PLGA Nanoparticle Bearing Amphotericin B for Macrophage Targeting.

PURPOSE: To fabricate, characterize and evaluate 3-O-sn-Phosphatidyl-L-serine (PhoS) anchored PLGA nanoparticles for macrophage targeted therapeutic intervention of VL. MATERIALS AND METHODS: PLGA-AmpB NPs were prepared by well-established nanoprecipitation method and decorated with Phos by thin film hydration method. Physico-chemical characterization of the formulation was done by Zetasizer nano ZS and atomic force microscopy. RESULTS: The optimized formulation (particle size, 157.3 ± 4.64 nm; zeta potential, - 42.51 ± 2.11 mV; encapsulation efficiency, ∼98%) showed initial rapid release up to 8 h followed by sustained release until 72 h. PhoS generated 'eat-me' signal driven augmented macrophage uptake, significant increase in in-vitro (with ∼82% parasite inhibition) and in-vivo antileishmanial activity with preferential accumulation in macrophage rich organs liver and spleen were found. Excellent hemo-compatibility justified safety profile of developed formulation in comparison to commercial formulations. CONCLUSION: The developed PhoS-PLGA-AmpB NPs have improved efficacy, and necessary stability which promisingly put itself as a better alternative to available commercial formulations for optimized treatment of VL.

Enhanced efficacy of curcumin with phosphatidylserine-decorated nanoparticles in the treatment of hepatic fibrosis.

Hepatic macrophages have been considered as a therapeutic target for liver fibrosis treatment, and phosphatidylserine (PS)-containing nanoparticles are commonly used to mimic apoptotic cells that can specifically regulate macrophage functions, resulting in anti-inflammatory effects. This study was designed to test the efficacy of PS-modified nanostructured lipid carriers (mNLCs) containing curcumin (Cur) (Cur-mNLCs) in the treatment of liver fibrosis in a rat model. Carbon tetrachloride-induced liver fibrosis in rats was used as an experimental model, and the severity of the disease was examined by both biochemical and histological methods. Here, we showed that mNLCs were spherical nanoparticles with decreased negative zeta potentials due to PS decoration, and significantly increased both mean residence time and area under the curve of Cur. In the rats with liver fibrosis, PS-modification of NLCs enhanced the nanoparticles targeting to the diseased liver, which was evidenced by their highest accumulation in the liver. As compared to all the controls, Cur-mNLCs were significantly more effective at reducing the liver damage and fibrosis, which were indicated by in Cur-mNLCs-treated rats the least increase in liver enzymes and pro-inflammatory cytokines in the circulation, along with the least increase in collagen fibers and alpha smooth muscle actin and the most increased hepatocyte growth factors (HGF) and matrix metalloprotease (MMP) two in the livers. In conclusion, PS-modified NLCs nanoparticles prolonged the retention time of Cur, and enhanced its bioavailability and delivery efficiency to the livers, resulting in reduced liver fibrosis and up-regulating hepatic expression of HGF and MMP-2.

Phosphatidylserine targeted single-walled carbon nanotubes for photothermal ablation of bladder cancer.

Bladder cancer has a 60%-70% recurrence rate most likely due to any residual tumour left behind after a transurethral resection (TUR). Failure to completely resect the cancer can lead to recurrence and progression into higher grade tumours with metastatic potential. We present here a novel therapy to treat superficial tumours with the potential to decrease recurrence. The therapy is a heat-based approach in which bladder tumour specific single-walled carbon nanotubes (SWCNTs) are delivered intravesically at a very low dose (0.1 mg SWCNT per kg body weight) followed 24 h later by a short 30 s treatment with a 360° near-infrared light that heats only the bound nanotubes. The energy density of the treatment was 50 J cm-2, and the power density that this treatment corresponds to is 1.7 W cm-2, which is relatively low. Nanotubes are specifically targeted to the tumour via the interaction of annexin V (AV) and phosphatidylserine, which is normally internalised on healthy tissue but externalised on tumours and the tumour vasculature. SWCNTs are conjugated to AV, which binds specifically to bladder cancer cells as confirmed in vitro and in vivo. Due to this specific localisation, NIR light can be used to heat the tumour while conserving the healthy bladder wall. In a short-term efficacy study in mice with orthotopic MB49 murine bladder tumours treated with the SWCNT-AV conjugate and NIR light, no tumours were visible on the bladder wall 24 h after NIR light treatment, and there was no damage to the bladder. In a separate survival study in mice with the same type of orthotopic tumours, there was a 50% cure rate at 116 days when the study was ended. At 116 days, no treatment toxicity was observed, and no nanotubes were detected in the clearance organs or bladder.

High-Yield and Sustainable Production of Phosphatidylserine in Purely Aqueous Solutions via Adsorption of Phosphatidylcholine on Triton-X-100-Modified Silica.

Triton X-100 was covalently bound to a surface of silica and acted as an anchor molecule to facilitate the adsorption of phosphatidylcholine (PC) in a purely aqueous solution. The silica-adsorbed PC obtained was successfully used for phospholipase D (PLD)-mediated transphosphatidylation in the production of phosphatidylserine (PS). Organic solvents were completely avoided in the whole production process. The PC loading and PS yield reached 98.9 and 99.0%, respectively. Two adsorption models were studied, and the relevant parameters were calculated to help us understand the adsorption and reaction processes deeply. In addition, the silica-adsorbed PC provides a promising way to continuously biosynthesize PS. A packed-bed reactor was employed to demonstrate the process flow of the continuous production of PS. The recyclability and stability of the Triton-X-100-modified silica were excellent, as demonstrated by its use 30 times during continuous operation without any loss of the productivity.

Functional reconstitution of cell-free synthesized purified Kv channels.

The study of ion channel activity and the screening of possible inhibitor molecules require reliable methods for production of active channel proteins, their insertion into artificial membranes and for the measurement of their activity. Here we report on cell-free expression of soluble and active Kv1.1 and Kv1.3 channels and their efficient insertion into liposomes. Two complementary methods for the determination of the electrical activity of the proteoliposome-embedded channels were compared using Kv1.1 as a model system: (1) single channel recordings in droplet interface bilayers (DIB) and (2) measurement of the membrane voltage potential generated by a potassium ion diffusion potential using the voltage-sensitive fluorescent dye oxonol VI. Single channel recordings in DIBs proved unreliable because of the non-reproducible fusion of proteoliposomes with an artificial membrane. Therefore, the use of the optical indicator oxonol VI was adapted for 96 well microtiter plates using the ionophore valinomycin as a positive control. The activity of Kv1.1 and Kv1.3 channels was then monitored in the absence and presence of different venom toxins, demonstrating that fluorescent dyes can be used very efficiently when screening small molecules for their channel blocking activity.

Biophysical study of the non-steroidal anti-inflammatory drugs (NSAID) ibuprofen, naproxen and diclofenac with phosphatidylserine bilayer membranes.

Non-steroidal anti-inflammatory drugs (NSAIDs) represent an effective pain treatment option and therefore one of the most sold therapeutic agents worldwide. The study of the molecular interactions responsible for their physiological activity, but also for their side effects, is therefore important. This report presents data on the interaction of the most consumed NSAIDs (ibuprofen, naproxen and diclofenac) with one main phospholipid in eukaryotic cells, dimyristoylphosphatidylserine (DMPS). The applied techniques are Fourier-transform infrared spectroscopy (FTIR), with which in transmission the gel to liquid crystalline phase transition of the acyl chains in the absence and presence of the NSAID are monitored, supplemented by differential scanning calorimetry (DSC) data on the phase transition. FTIR in reflection (ATR, attenuated total reflectance) is applied to record the dependence of the interactions of the NSAID with particular functional groups observed in the DMPS spectrum such as the ester carbonyl and phosphate vibrational bands. With Förster resonance energy transfer (FRET) a possible intercalation of the NSAID into the DMPS liposomes and with isothermal titration calorimetry (ITC) the thermodynamics of the interaction are monitored. The data show that the NSAID react in a particular way with this lipid, but in some parameters the three NSAID clearly differ, with which now a clear picture of the interaction processes is possible.

Membrane insertion of αA-crystallin is oligomer-size dependent.

Vertebrate lens is one of the tissues with the highest soluble protein concentration. The predominant soluble proteins in lens fiber cells are crystallins, and among them, α-crystallins belong to the small heat shock protein family with chaperone-like activity. Although α-crystallins are highly soluble in waters, α-crystallins have been detected in the membrane-bound fraction of lens, which will increase in the aged or cataractous lens. In this research, we found αA-crystallin exhibited a complex thermal transition with remarkable changes in secondary and quaternary structures. Treatment of αA-crystallin at high temperatures induced larger oliogomers with higher hydrophobic exposure. Both heat-treated and untreated αA-crystallin could insert into lipid monolayer directly as revealed by monolayer surface pressure experiments. Heat-treatment facilitated the membrane insertion of αA-crystallin and increased the membrane-bound fraction in the cells. The membrane-binding ability of αA-crystallin could be altered by cataract-causing mutations R116C, R116H and Y118D. Our results suggested that the irreversible changes in oligomer size induced by various stresses might promote the membrane association of αA-crystallin and therefore might play a role in aged cataract. Alternations in the membrane binding ability of α-crystallins might be important to the understanding of both aged and congenital cataracts.

Re-evaluation of the water exchange lifetime value across red blood cell membrane.

The water exchange lifetime (τ(i)) through red blood cell (RBC) membranes can be measured by analyzing the water protons bi-exponential T1 and T2 curves when RBCs are suspended in a medium supplemented with paramagnetic species. Since the seminal papers published in the early '70s of the previous century, paramagnetic Mn(2+) ions were used for doping the extracellular compartment in the RBCs suspension. The obtained τ(i) values fall in the range of 9.8-14 ms. Conversely, other physic-chemical measurements afforded longer τ(i) values. Herein, it is shown that the replacement of Mn(2+) with the highly stable, hydrophilic Gd(III) complexes used as paramagnetic magnetic resonance imaging (MRI) contrast agents led to measure τ(iI) values of 19.1 ± 0.65 ms at 25 ° C. The observed difference is ascribed to the occurrence of enhanced permeability of RBC membrane in the presence of Mn(2+) ions. This view finds support from the observation that an analogous behavior was shown in the presence of other divalent cations, such Ca(2+) and Zn(2+) ions. A possible role of scramblase has been hypothesized. Finally, τ(i) has been measured in presence of alcohols to show that the herein proposed method can detect minor changes in RBC membranes' stiffness upon the incorporation of aliphatic alcohols.

Sub-chronic (13-week) oral toxicity study, preceded by an in utero exposure phase and genotoxicity studies with fish source phosphatidylserine in rats.

The safety of fish phosphatidylserine (PS) conjugated to DHA (InCog™) was examined in a series of toxicology studies as first step to support future use in infants and general population using in vitro genotoxicity tests and in a sub-chronic toxicity study with an in-utero exposure phase. PS is a major lipid in the cell membrane, active in various membrane-mediated processes. PS-DHA, present in human milk, has been suggested to be important for early brain development. Rats were exposed to diets containing 1.5%, 3% or 4.5% InCog or two control diets. Parental (F0) animals were fed throughout mating, gestation and lactation. Subsequently, a subchronic, 13-week study was conducted on the F1 animals followed by 4 weeks of recovery. The genotoxicity tests showed no mutagenicity potential. No significant toxicological findings were found in the F0 rats or the F1 pups. In the 13-weeks study, an increase in the presence of renal minimal-mild multifocal corticomedullary mineralization was noted in nine females of the high-dose group. This change was not associated with any inflammatory or degenerative changes in the kidneys. The no-observed-adverse-effect level (NOAEL) in the present study was placed at 3% in the diet (mid-dose group), equivalent to an overall intake of at least 2.1 g InCog/kg bw/day in the F1 generation.