Properties Of Cell Membrane Research Articles

Stress-induced ethanol affects endocytic vesicle recycling and F-actin organisation in arabidopsis root apex cells

Ethanol (EtOH) is a short-chain alcohol that is abundant in nature. EtOH is endogenously produced by plants under hypoxic conditions, and exogenously applied EtOH improves plant stress tolerance at low concentrations (<1%). However, no direct observations have shown how EtOH affects cellular events in plants. In intact Arabidopsis roots, 0.1% EtOH promoted reactive oxygen species production in root apex cells. EtOH also accelerated exocytic vesicle recycling and altered F-actin organisation, both of which are closely related to cell membrane properties. In addition to exogenous EtOH application, hypoxic treatment resulted in EtOH production in roots and degradation of the cross-wall actin cytoskeleton in root epidermal cells. We conclude that hypoxia-induced EtOH production affects endocytic vesicle recycling and associated signalling pathways. • EtOH unbalances endocytic and exocytic vesicle recycling pathways in root apex transition zone cells • EtOH induces dispersion of F-actin bundles at root apex transition zone cross-walls • Hypoxia induces endogenous EtOH production and depletion of F-actin in cells of Arabidopsis root apex transition zone

Menthone Exerts its Antimicrobial Activity Against Methicillin Resistant Staphylococcus aureus by Affecting Cell Membrane Properties and Lipid Profile.

The characteristic constituents of essential oils from aromatic plants have been widely applied as antimicrobial agents in the last decades. However, their mechanisms of action remain obscure, especially from the metabolic perspective. The aim of the study was to explore the antimicrobial effect and mechanism of menthone, a main component of peppermint oil, against methicillin resistant Staphylococcus aureus (MRSA). An integrated approach including the microbiology and the high-coverage lipidomics was applied. The changes of membrane properties were studies by the fluorescence and electron microscopical observations. The lipid profile was analyzed by ultra-high performance liquid chromatography coupled with quadruple Exactive mass spectrometry (UHPLC-QE-MS). The lipid-related key targets which were associated with the inhibitory effect of menthone against MRSA, were studied by network analysis and molecular docking. Menthone exhibited antibacterial activities against MRSA, with minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of 3,540 and 7,080 μg/mL, respectively. The membrane potential and membrane integrity upon menthone treatment were observed to change strikingly. Further, lipids fingerprinting identified 136 significantly differential lipid species in MRSA cells exposed to menthone at subinhibitory level of 0.1× MIC. These metabolites span 30 important lipid classes belonging to glycerophospholipids, glycolipids, and sphingolipids. Lastly, the correlations of these altered lipids, as well as the potential metabolic pathways and targets associated with menthone treatment were deciphered preliminarily. Menthone had potent antibacterial effect on MRSA, and the mechanism of action involved the alteration of membrane structural components and corresponding properties. The interactions of identified key lipid species and their biological functions need to be further determined and verified, for the development of novel antimicrobial strategies against MRSA.

ANALYSIS OF OPINIONS OF RESPONDENTS ON THE KNOWLEDGE CONCERNING THE ROLE OF CHOLESTEROL IN PHYSIOLOGICAL PROCESSES

Interest in cholesterol has grown significantly, especially in recent years, as it has become clear that cholesterol is a substance necessary for the normal functioning of the body: it regulates the properties of cell membranes; participates in the synthesis of vitamin D, which maintains the proper level of calcium and phosphorus in the body and determines the development and maintenance of bones; stimulates the production of antibodies; participates in the synthesis of bile acids and steroid hormones. Purpose: The aim of this study was to analyze the opinions of women and men of all ages on the knowledge of the role of cholesterol in physiological processes. Methodology. The research was conducted in 2017-2019 among the inhabitants of Słupsk, Bytów, Miastko, Sławno, the commune of Potęgowo and Czarna Dąbrówka (Pomeranian and West-Pomeranian voivodeships). Overall, 330 respondents took part in the study, including 274 women (83%) and 56 men (17%). Scientific novelty. The respondents largely knew which group of chemical compounds cholesterol belongs to. Less than half of the respondents knew the cholesterol fractions. Most of the respondents correctly answered the question about the general functions of cholesterol. More than half of the respondents knew that the liver is the organ responsible for the production of cholesterol. Regarding the prevention of cardiovascular diseases, respondents knew that there are many factors influencing the prevention of cardiovascular diseases. The respondents did not know products that have a high cholesterol content. The respondents identify the elevated level of cholesterol mainly with problems with the cardiovascular system. Unfortunately, almost the entire group of respondents believes that elevated cholesterol should be reduced as soon as possible, regardless of the reason for its increase. Conclusions. The respondents associated cholesterol with something bad and suggested that its elevated level should be reduced in all situations. Cholesterol was also considered by the respondents as a factor that is very important in the development of cardiovascular diseases.

Identification and quantification of ionising radiation-induced oxysterol formation in membranes of lens fibre cells

Ionising radiation (IR) is a cause of lipid peroxidation, and epidemiological data have revealed a correlation between exposure to IR and the development of eye lens cataracts. Cataracts remain the leading cause of blindness around the world. The plasma membranes of lens fibre cells are one of the most cholesterolrich membranes in the human body, forming lipid rafts and contributing to the biophysical properties of lens fibre plasma membrane. Liquid chromatography followed by mass spectrometry was used to analyse bovine eye lens lipid membrane fractions after exposure to 5 and 50 Gy and eye lenses taken from wholebody 2 Gy-irradiated mice. Although cholesterol levels do not change significantly, IR dose-dependant formation of the oxysterols 7β-hydroxycholesterol, 7-ketocholesterol and 5, 6-epoxycholesterol in bovine lens nucleus membrane extracts was observed. Whole-body X-ray exposure (2 Gy) of 12-week old mice resulted in an increase in 7β-hydroxycholesterol and 7-ketocholesterol in their eye lenses. Their increase regressed over 24 h in the living lens cortex after IR exposure. This study also demonstrated that the IR-induced fold increase in oxysterols was greater in the mouse lens cortex than the nucleus. Further work is required to elucidate the mechanistic link(s) between oxysterols and IR-induced cataract, but these data evidence for the first time that IR exposure of mice results in oxysterol formation in their eye lenses.

Cell Membrane Features as Potential Breeding Targets to Improve Cold Germination Ability of Seeds.

Cold stress breeding that focuses on the improvement of chilling tolerance at the germination stage is constrained by the complexities of the trait which involves integrated cellular, biochemical, hormonal and molecular responses. Biological membrane serves as the first line of plant defense under stress. Membranes receive cold stress signals and transduce them into intracellular responses. Low temperature stress, in particular, primarily and effectively affects the structure, composition and properties of cell membranes, which ultimately disturbs cellular homeostasis. Under cold stress, maintenance of membrane integrity through the alteration of membrane lipid composition is of prime importance to cope with the stress. This review describes the critical role of cell membranes in cold stress responses as well as the physiological and biochemical manifestations of cold stress in plants. The potential of cell membrane properties as breeding targets in developing strategies to improve cold germination ability is discussed using cotton (Gossypium hirsutum L.) as a model.

Fatty Acid Composition of Comamonas testosteroni under Hexachlorobenzene Loading Conditions

Changes in the lipid composition in bacterial membranes are considered to be the most important adaptation mechanisms to adverse chemical factors. The aim of the study was to compare the hexachlorobenzene effects on the fatty acid composition of total lipids Comamonas testosteroni. Methods. The study was performed with C. testosteroni UCM B-400 and B-401, B-213 strains. Bacteria were grown in the Luria-Bertrani (LB) liquid medium containing 10 and 20 mg/L of hexachlorobenzene (HCB). After cultivation, the biomass was separated by centrifugation and the fatty acid composition of total lipids was determined through analyzing its methyl esters. To assess the cell membrane properties, such parameters as the lipid unsaturation index, the average carbon chain length of fatty acids, and the membrane viscosity index were determined. Results. In the fatty acids spectrum of C. testosteroni B-400 after cultivation in a medium containing 20 mg/L of HCB, the contents of unsaturated hexadecenoic (C16:1) and octadecenoic (C18:1) acids were lower by 10.6 and 5.5%, respectively, and that of saturated hexadecanoic (C16:0) acid was higher by 8.4%, compared to the control. The fatty acid composition of C. testosteroni B-401 was more stable compared to strain B-400. Collection strain C. testosteroni B-213 compared to strains isolated from soil with high HCB load, in the presence of 10 and 20 mg/L of HCB had the highest relative content of saturated hexadecanoic acid (C16:0) up to 38.33—40.7%. Unsaturated octadecenoic acid decreased at the doses 10 and 20 mg/L to 1.5—2% compared to the control. In all strains under the HCB impact, there was an increase in the relative content of C17-cyclopropanoic acid compared to control variants. Conclusions. C. testosteroni UCM B-400, B-401, and B-213 bacteria under cultivation conditions in HCB-containing medium, decreasing the degree of lipid unsaturation and increasing the relative content of C17-cyclopropanoic acid can be considered as the main mechanisms regulating the cytoplasmic membrane fluidity; the displaying of these protective reactions had a strain trait and did not depend on the adaptation in natural isolating places.

Benzoate group attachment to TEMPO provides enhanced discrimination of liposomes fabricated using human lung normal and carcinoma cells

It is widely accepted that the lipid compositions of the plasma membranes of healthy and cancer cells significantly differ from each other. During the cancer progression, cancer cells change the lipid constituent of the membranes resulting in the loss of lipid asymmetry between the membrane leaflets. Consequently, physicochemical properties of the cell membranes are also changed in response to altered lipid organization. Partitioning of the spin probe 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) into the membranes of the cells has broadly been applied to characterize membrane properties of various cells in health and disease conditions. In this work, we used liposomes fabricated using lipids extracted from normal and carcinoma cells. This system permits the determination of the properties of the healthy and cancer cell membranes provided exclusively by its lipid components. Application of TEMPO-benzoate, in which the benzoate group is attached to the TEMPO, indicates significantly enhanced discrimination of liposomes between cancer and normal cells. Partitioning experiments with TEMPO-benzoate revealed relatively enhanced incorporation efficiency for liposomes of cancer cells. On the contrary, TEMPO incorporation efficiency in the same liposomes of cancer cells was not much different compared to healthy cells. Data indicate that TEMPO-benzoate as a probe is more suitable than TEMPO to discriminate cancer cells from healthy cells. Free energy gain observed for TEMPO-benzoate resulted mainly from the hydrophobic effect of the benzoate group.

Microbial cell membrane properties and intracellular metabolism regulate individual level microbial responses to acid stress

Community- and individual-level soil microbial traits greatly depend on environmental conditions, especially soil pH. Given substantial spatio-temporal variations in soil pH, microorganisms exhibit acid tolerance to some extent, although the underlying mechanisms of microbial tolerance capacities remain understudied. In this study, ten microbial species with different functions (plant growth promoting, pathogen biocontrol, and nutrient cycling functions) were cultured under different acid treatments to investigate the acid tolerance capacity and the underlying physiological mechanisms for acid tolerance of these microbial species. Microbial species exhibited substantially different acid tolerance capacities, making them survival at pH from 3.5 to 5.0. Cellular physiological assays further indicated that three aspects could contribute to the acid tolerance capacity of soil microorganisms in the present study, i.e., shield effect by regulating the fluidity of cell membrane to prevent H+ from entering into cells, neutralizing effect by promoting the internal metabolic activities to produce metabolic chemicals such as amino acids and urea that could consume more intracellular H+, and pumping effect by regulating the H+-ATPase activity to extrude H+ out of cells, although it cannot be denied that other pathways may also function. However, these pathways did not make the same contribution to the acid tolerance capacity of different microbial species, suggesting that the regulators and underlying mechanisms depended greatly on microbial species. Further studies that combine omics technologies to discern tradeoff among microbial traits such as resource allocation, stress tolerance, etc., would be helpful to clarify the molecular mechanisms underlying microbial tolerance to the environmental stressors such as soil acidification.

Plasmalogens and platelet-activating factor roles in chronic inflammatory diseases.

Fatty acids and phospholipid molecules are essential for determining the structure and function of cell membranes, and they hence participate in many biological processes. Platelet activating factor (PAF) and its precursor plasmalogen, which represent two subclasses of ether phospholipids, have attracted increasing research attention recently due to their association with multiple chronic inflammatory, neurodegenerative, and metabolic disorders. These pathophysiological conditions commonly involve inflammatory processes linked to an excess presence of PAF and/or decreased levels of plasmalogens. However, the molecular mechanisms underlying the roles of plasmalogens in inflammation have remained largely elusive. While anti-inflammatory responses most likely involve the plasmalogen signal pathway; pro-inflammatory responses recruit arachidonic acid, a precursor of pro-inflammatory lipid mediators which is released from membrane phospholipids, notably derived from the hydrolysis of plasmalogens. Plasmalogens per se are vital membrane phospholipids in humans. Changes in their homeostatic levels my alter cell membrane properties, thus affecting key signaling pathways that mediate inflammatory cascades and immune responses. The plasmalogen analogs of PAF are also potentially important, considering that anti-PAF activity has strong anti-inflammatory effects. Plasmalogen replacement therapy was further identified as a promising anti-inflammatory strategy allowing for the relief of pathological hallmarks in patients affected by chronic diseases with an inflammatory component. The aim of this Short Review is to highlight the emerging roles and implications of plasmalogens in chronic inflammatory disorders, along with the promising outcomes of plasmalogen replacement therapy for the treatment of various PAF-related chronic inflammatory pathologies.

Sterylglucosides in Fungi.

Sterylglucosides (SGs) are sterol conjugates widely distributed in nature. Although their universal presence in all living organisms suggests the importance of this kind of glycolipids, they are yet poorly understood. The glycosylation of sterols confers a more hydrophilic character, modifying biophysical properties of cell membranes and altering immunogenicity of the cells. In fungi, SGs regulate different cell pathways to help overcome oxygen and pH challenges, as well as help to accomplish cell recycling and other membrane functions. At the same time, the level of these lipids is highly controlled, especially in wild-type fungi. In addition, modulating SGs metabolism is becoming a novel tool for vaccine and antifungal development. In the present review, we bring together multiple observations to emphasize the underestimated importance of SGs for fungal cell functions.

A SARS-CoV-2 impedimetric biosensor based on the immobilization of ACE-2 receptor-containing entire cell membranes as the biorecognition element

A SARS-CoV-2 biosensor based on the biorecognition of the spike protein to the angiotensin-converting enzyme 2 (ACE-2) transmembrane receptor was developed using entire cell membranes as the biorecognition layer. In this new SARS-CoV-2 detection platform, cellular membranes from VeroCCL81 (mVero) and Calu-3 (mCalu) cells (which overexpress the ACE-2 transmembrane receptors) were extracted and immobilized as vesicles on an indium tin oxide electrode (ITO). Electrochemical impedance spectroscopy was used to optimize the performance of the developed devices for SARS-CoV-2 detection. This novel biosensor comprises a low-cost system (less than one US$ dollar) that uses the unique properties of cell membranes combined with the catalytic properties of electrochemical platforms to allow spike proteins recognition. A linear response from 10 to 100 ng/mL was obtained from the optimized biosensors, a limit of detection of 10.0 pg/mL and 7.25 pg/mL and limit of quantification of 30.4 pg/mL and 21.9 pg/mL were achieved with satisfactory accuracy for ITO-APTES-mVero and ITO-APTES-mCalu, respectively. Selectivity studies revealed that this platform was able to differentiate the target spike proteins from NS1 proteins from dengue and Zika viruses. In addition, sensors comprising cell membranes devoid of the ACE-2 transmembrane receptor exhibited no biorecognition signal. The developed devices are suitable for SARS-CoV-2 detection based on spike protein recognition, and capable of providing a low-cost, accurate, and accessible tool for use in a pandemic and post-pandemic scenario.

Plasma membrane and brain dysfunction of the old: Do we age from our membranes?

One of the characteristics of aging is a gradual hypo-responsiveness of cells to extrinsic stimuli, mainly evident in the pathways that are under hormone control, both in the brain and in peripheral tissues. Age-related resistance, i.e., reduced response of receptors to their ligands, has been shown to Insulin and also to leptin, thyroid hormones and glucocorticoids. In addition, lower activity has been reported in aging for ß-adrenergic receptors, adenosine A2B receptor, and several other G-protein-coupled receptors. One of the mechanisms proposed to explain the loss of sensitivity to hormones and neurotransmitters with age is the loss of receptors, which has been observed in several tissues. Another mechanism that is finding more and more experimental support is related to the changes that occur with age in the lipid composition of the neuronal plasma membrane, which are responsible for changes in the receptors’ coupling efficiency to ligands, signal attenuation and pathway desensitization. In fact, recent works have shown that altered membrane composition—as occurs during neuronal aging—underlies reduced response to glutamate, to the neurotrophin BDNF, and to insulin, all these leading to cognition decay and epigenetic alterations in the old. In this review we present evidence that altered functions of membrane receptors due to altered plasma membrane properties may be a triggering factor in physiological decline, decreased brain function, and increased vulnerability to neuropathology in aging.

Charge of Phospholipids Determines the Rate of Lysozyme Aggregation but Not the Structure and Toxicity of Amyloid Aggregates.

Biophysical properties of plasma membranes are determined by a chemical structure of phospholipids, including saturation of fatty acids and charge of polar heads of these molecules. Phospholipids not only determine fluidity and plasticity of membranes but also play an important role in abrupt aggregation of misfolded proteins. In this study, we investigate the role of the charge of the most abundant phospholipids in the plasma membrane on the aggregation properties of the lysozyme. We found that the charge of phospholipids determines the aggregation rate of lysozyme and the morphology of the protein aggregates. However, the secondary structure and toxicity of these protein specimens are determined by the chemical nature rather than the charge of phospholipids. These findings show that the charge of phospholipids can be a key factor that determines the stability and aggregation mechanism of amyloidogenic proteins.

Erg6p is essential for antifungal drug resistance, plasma membrane properties and cell wall integrity in Candida glabrata.

ERG6 gene encodes C-24 methyltransferase, one of the specific enzymes that differ in mammalian and yeast sterol biosynthesis. To explore the function of CgErg6p in the yeast pathogen Candida glabrata, we have constructed the Cgerg6Δ deletion mutant. We found that C. glabrata cells lacking CgErg6p exhibit reduced susceptibility to both antifungal azoles and polyenes. The reduced content of ergosterol in the Cgerg6 deletion mutant was accompanied by increased expression of genes encoding the last steps of the ergosterol biosynthetic pathway. The absence of CgErg6p leads to plasma membrane hyperpolarization and decrease in its fluidity compared to the parental C. glabrata strain. The absence of sterols containing C-24 alkyls influenced the susceptibility of Cgerg6Δ mutant cells to alkali metal cations and several other metabolic inhibitors. Our results thus show that sterols lacking C-24 alkyls are not sufficient substitutes for maintaining yeast plasma membrane function. The absence of CgErg6p influences also the cell wall integrity and calcineurin signaling in C. glabrata.

Cell Membrane and V2C MXene-Based Electrochemical Immunosensor with Enhanced Antifouling Capability for Detection of CD44.

The inactive adsorption and interference of biomolecules in electrochemical biosensors is a topic of intense interest. Directly utilizing native cell membranes to endow electrochemical surfaces with antifouling and biocompatible features is a promising strategy, rather than attempting to synthetically replicate complex biological interface properties. In this study, we present a facial and sensitive sandwich-type antifouling immunoassay through platelet membrane/Au nanoparticle/delaminated V2C nanosheet (PM/AuNPs/d-V2C)-modified electrode as the substrate of sensing interface and methylene blue/aminated metal organic framework (MB@NH2-Fe-MOF-Zn) as an electrochemical signal probe. The biosensor perfectly integrates the high conductivity of AuNPs-loaded V2C MXene with the excellent loading property of NH2-Fe-MOF-Zn to improve the electrochemical sensing performance. In addition, the excellent antifouling properties of the homogeneous cell membrane can effectively prevent the non-specific adsorption of model proteins. The obtained antifouling biosensor possesses the capability of ultrasensitive detection of CD44 and CD44-positive cancer cell in complex liquids and exhibits good analytical performance for the analysis of CD44 with a linear range from 0.5 ng/mL to 500 ng/mL. This strategy of developing cell membrane-based biosensing systems with enhanced antifouling capability can be easily expanded to the construction of other complex biosensors, and the advanced biological probes and analytical methods provide a favorable means to accurately quantify biomarkers associated with tumor progression.

Biotransformation of sulfamethoxazole by a novel strain, Nitratireductor sp. GZWM139: Characterized performance, metabolic mechanism and application potential

A novel strain, Nitratireductor sp. GZWM139 capable of efficient removal of SMX was isolated from mariculture sewage, and Nitratireductor was reported to conduct the removal of antibiotics for the first time. Strain GZWM139 exhibited desirable adaptations to environmental factors with SMX removal efficiencies more than 90 % at temperatures of 28–38 °C, pH values of 4.5–8.5, salinities of 20–30 ‰, SMX levels of 1–5 mg/L and shaking speeds of 20–260 rpm. SMX removal was a cooperated process implemented by intracellular enzymes and extracellular enzymes, and was achieved through four proposed biotransformation pathways with the occurrences of demethylation, hydroxylation, nitration, formylation, oxidation, bond cleavage and ring opening. Strain GZWM139 responded to the SMX removal process by altering properties of cell membrane and motivating activities of xenobiotic-metabolizing enzymes and antioxidant system. Genomic analysis proved the existence of functional genes relevant to the SMX removal in strain GZWM139 and provided echoing genetic insights for revealing the SMX removal mechanism. Strain GZWM139 performed efficient detoxification of SMX and accomplished simultaneous removal of SMX and nitrogen in both mariculture sewage and domestic sewage. The findings are significant to the effective elimination of SMX pollution and comprehensive cognitions on metabolic mechanisms of SMX removal.

The effect of cholesterol on highly curved membranes measured by nanosecond Fluorescence Correlation Spectroscopy

Small lipid vesicles (with diameter ≤100 nm) with their highly curved membranes comprise a special class of biological lipid bilayers. The mechanical properties of such membranes are critical for their function, e.g. exocytosis. Cholesterol is a near-universal regulator of membrane properties in animal cells. Yet measurements of the effect of cholesterol on the mechanical properties of membranes have remained challenging, and the interpretation of such measurements has remained a matter of debate. Here we show that nanosecond fluorescence correlation spectroscopy (FCS) can directly measure the ns-microsecond rotational correlation time (τ r) of a lipid probe in high curvature vesicles with extraordinary sensitivity. Using a home-built 4-Pi fluorescence cross-correlation spectrometer containing polarization-modulating elements, we measure the rotational correlation time (τ r) of Nile Red in neurotransmitter vesicle mimics. As the cholesterol mole fraction increases from 0 to 50%, τ r increases from 17 ± 1 to 112 ± 12 ns, indicating a viscosity change of nearly a factor of 7. These measurements are corroborated by solid-state NMR results, which show that the order parameter of the lipid acyl chains increases by about 50% for the same change in cholesterol concentration. Additionally, we measured the spectral parameters of polarity-sensitive fluorescence dyes, which provide an indirect measure of viscosity. The green/red ratio of Nile Red and the generalized polarization of Laurdan show consistent increases of 1.3× and 2.6×, respectively. Our results demonstrate that rotational FCS can directly measure the viscosity of highly curved membranes with higher sensitivity and a wider dynamic range compared to other conventional techniques. Significantly, we observe that the viscosity of neurotransmitter vesicle mimics is remarkably sensitive to their cholesterol content.

Extraction of Cardiac Cell Membrane Fractional-Order Capacitance from Current Response to Voltage Step

The aim of the article is to examine the possibilities of modeling electrical properties of cardiac cell membrane using the Cole impedance model containing a fractional-order capacitor. A method is presented to determine the parameters of the model based on the current transient response to a voltage step applied to the membrane. The method is based on the numerical minimization of the least squares error between the recorded and calculated transient response of the model using the MATLAB function fminsearch. The use of a fractional-order capacitor in the membrane model is found to result in a reduction in the modeling error compared to that of the classic capacitor.

P2X7-mediated alteration of membrane fluidity is associated with the late stages of age-related macular degeneration.

We have shown deficits in monocyte phagocytosis from patients with age-related macular degeneration (AMD). Cell membrane fluidity is known to affect phagocytic capacity and leucocyte functionality more generally. Therefore, we examined membrane fluidity of peripheral blood leucocytes in human patients with AMD and in the P2X7 null mouse model of AMD using flow cytometry with a fluorescent probe for fluidity, TMA-DPH. The results showed that membrane fluidity was decreased in all leucocyte types of late AMD relative to healthy controls (HC) including monocytes, neutrophils and lymphocytes but this was not apparent in earlier stages of AMD. Further analysis of factors contributing to membrane fluidity indicated that pre-treatment of monocytes and lymphocytes with ATP greatly increased membrane fluidity in humans and mice. Evidence from P2X7 null mice and P2X7 antagonists confirmed that these ATP-driven increases in membrane fluidity were mediated by P2X7 but were not associated with the classic P2X7 functions of pore formation or phagocytosis. Analysis of P2X7 expression indicated that receptor levels were elevated in classic monocytes of late AMD patients, further suggesting the P2X7 may contribute to altered plasma membrane properties. Our findings identified a novel biological function of P2X7 in modulating membrane fluidity of leucocytes and demonstrated reduced membrane fluidity in cellular changes associated with the late stage of AMD.

Effects of subtoxic 3-methylmethcathinone concentrations on neuronal cell electrical properties

Synthetic cathinones are commonly abused novel psychoactive substances (NPS) that are consumed alone or in combination with other psychoactive drugs via the oral, intranasal, or intravenous routes. These substances induce physiological and subjective effects typical of psychostimulant drugs, although with a faster onset and shorter duration when compared to amphetamine derivatives. A common consequence of their short half-lives and duration of effects, is repeated administration or bingeing that results in an increase in the number of associated deaths. New synthetic cathinones, now in the fourth generation, have increasing potency and toxicity as compared to those from previous generations. NPS may interfere with the electrical activity of the catecholaminergic neuron, causing damages to neuronal function. The aim of this research is to investigate the effects of 3-methylmethcathinone (3-MMC) on excitability of neuronal cells to identify mechanisms underlying its neurotoxicity. Functional techniques detecting neuronal electrical properties including Fura-2-video imaging and current clamp electrophysiology were applied to single neurons exposed to subtoxic 3-MMC concentrations. Changes in intracellular calcium homeostasis were correlated to neuronal electrical signals during exposure. Neuronal viability was measured in cells exposed to a large range of 3-MMC concentrations by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. This assay measures metabolic cell activity by detecting the conversion of the water-soluble yellow MTT dye to an insoluble purple formazan by active mitochondrial reductases. Formazan is solubilized and its concentration determined by optical density at 570 nm. Under conditions of neuronal toxicity, a significant change in metabolic activity is identified enabling detection of cell stress from exposure to a toxic agent. 3-MMC did not affect neuronal viability at 10–300 nM concentrations. Time-lapse experiments measuring intracellular calcium concentrations ([Ca2+]i) after membrane depolarization showed that 3-MMC prevented [Ca2+]i increases elicited by high K+. At the same time current-clamp experiments revealed that 3-MMC shifts membrane potential to more depolarized values at the same range of concentrations. Collectively, before inducing neurotoxicity, 3-MMC may interfere with the electrical properties of neuronal plasma membranes, thus, dysregulating [Ca2+]i homeostasis. 3-MMC interfered with membrane depolarization at subtoxic concentrations, suggesting the ability of this synthetic cathinone to modulate an as yet unknown voltage-dependent channel(s) at the neuronal level. Due to the lack of knowledge about mechanisms underlying 3-MMC neurotoxicity, investigation of putative targets may be useful for the treatment and/or prevention of its toxic effects in humans.