Influence of zinc deficiency on cell-membrane fluidity in Jurkat, 3T3 and IMR-32 cells.

PubMed Central

Verstraeten, Sandra V; Zago, M Paola; MacKenzie, Gerardo G; Keen, Carl L; Oteiza, Patricia I

2004-01-01

We investigated whether zinc deficiency can affect plasma membrane rheology. Three cell lines, human leukaemia T-cells (Jurkat), rat fibroblasts (3T3) and human neuroblastoma cells (IMR-32), were cultured for 48 h in control medium, in zinc-deficient medium (1.5 microM zinc; 1.5 Zn), or in the zinc-deficient medium supplemented with 15 microM zinc (15 Zn). The number of viable cells was lower in the 1.5 Zn group than in the control and 15 Zn groups. The frequency of apoptosis was higher in the 1.5 Zn group than in the control and 15 Zn groups. Membrane fluidity was evaluated using the 6-(9-anthroyloxy)stearic acid and 16-(9-anthroyloxy)palmitic acid probes. Membrane fluidity was higher in 1.5 Zn cells than in the control cells; no differences were observed between control cells and 15 Zn cells. The effect of zinc deficiency on membrane fluidity at the water/lipid interface was associated with a higher phosphatidylserine externalization. The higher membrane fluidity in the hydrophobic region of the bilayer was correlated with a lower content of arachidonic acid. We suggest that the increased fluidity of the membrane secondary to zinc deficiency is in part due to a decrease in arachidonic acid content and the apoptosis-related changes in phosphatidylserine distribution. PMID:14629198

Rooster sperm plasma membrane protein and phospholipid organization and reorganization attributed to cooling and cryopreservation

USDA-ARS?s Scientific Manuscript database

Cholesterol to phospholipid ratio is used as a representation for membrane fluidity, and predictor of cryopreservation success but results are not consistent across species and ignore the impact of membrane proteins. Therefore, this research explored the modulation of membrane fluidity and protein ...

Vitamin E supplement improves erythrocyte membrane fluidity of thalassemia: an ESR spin labeling study.

PubMed

Sutipornpalangkul, Werasak; Morales, Noppawan Phumala; Unchern, Supeenun; Sanvarinda, Yupin; Chantharaksri, Udom; Fucharoen, Suthat

2012-01-01

Beta-thalassemia/Hemoglobin E (beta-thal/Hb E) is prevalent in Thailand. The imbalance of globin chains in red blood cells is the primary cause of this anemic disease. The excess alpha-globin in beta-thal/Hb E causes typical damage(s) to membrane of erythroblasts and erythrocytes. By using three paramagnetic labeled compounds (5-, 12-, and 16-spin labeled stearic acids, SLS), the changes of the molecular motion in the lipid bilayer of thalassemic RBCs that have structural modification can be detected. to investigate erythrocyte membrane fluidity and the effect of vitamin E treatment in beta-thalassemia/Hemoglobin E patients by using spin labeling techniques. The erythrocyte membrane fluidity was investigated in nine splenectomized and five non-splenectomized beta-thalassemia/hemoglobin E (beta-thal/Hb E) patients using EPR spin labeling techniques. To determine the effect of vitamin E on erythrocyte membrane fluidity, only the splenectomized patients were enrolled. Patients were divided into two groups. The first group received 350 mg vitamin E daily for a period of 1 month (n = 5) and the second group received placebo for an equal period (n = 4). Three paramagnetic fatty acid, 5-, 12-, and 16-doxyl stearic acids, (5-, 12- and 16-DS) were used to label phospholipids layer near both the surface (5-DS) and the deeper hydrophobic region of membrane (12-and 16-DS). Lipid peroxidation (TBARs) was measured using a colorimetric method. Vitamin E was measured with high performance liquid chromatography (HPLC). Significantly higher values of erythrocyte membrane fluidity were revealed with 12-, 16-DS in splenectomized patients, as compared with non-splenectomized patients and normal subjects. In 3-thal/Hb E patients, fluidity values, both outer hyperfine splitting (2T(//)) and order parameter (S) of 12-DS showed inverse correlation with serum TBARs. There was no significant difference between the fluidity values measured with 5-DS. After vitamin E supplementation, the erythrocyte membrane fluidity was decreased in almost all patients. In contrast to the vitamin E supplementation group, increased erythrocyte membrane fluidity was demonstrated in the placebo group. Vitamin E supplementation also had effect on other clinical parameters such as increased plasma vitamin E, decreased serum TBARs and no change in hemoglobin. The present results suggested the abnormal motion of lipid in the deeper phospholipids region of membrane. In addition, vitamin E supplementation may have a role in the prevention of erythrocyte membrane damage of these patients.

Leptin improves membrane fluidity of erythrocytes in humans via a nitric oxide-dependent mechanism--an electron paramagnetic resonance investigation.

PubMed

Tsuda, Kazushi; Kimura, Keizo; Nishio, Ichiro

2002-09-27

Abnormalities in physical properties of the cell membranes may underlie the defects that are strongly linked to hypertension, stroke, and other cardiovascular diseases. Recently, there has been an indication that leptin, the product of the human obesity gene, actively participates not only in the metabolic regulations but also in the control of cardiovascular functions. In the present study, to assess the role of leptin in the regulation of membrane properties, the effects of leptin on membrane fluidity of erythrocytes in humans are examined. The membrane fluidity of erythrocytes in healthy volunteers by means of an electron paramagnetic resonance (EPR) and spin-labeling method is determined. In an in vitro study, leptin decreased the order parameter (S) for 5-nitroxide stearate (5-NS) and the peak height ratio (ho/h-1) for 16-NS obtained from EPR spectra of erythrocyte membranes in a dose-dependent manner in healthy volunteers. The finding indicated that leptin increased the membrane fluidity and improved the microviscosity of erythrocytes. The effect of leptin on the membrane fluidity was significantly potentiated by the nitric oxide (NO) donors, L-arginine and S-nitroso-N-acetylpenicillamine (SNAP), and a cyclic guanosine monophosphate (cGMP) analog, 8-bromo-cGMP. In contrast, the change evoked by leptin was significantly attenuated in the presence of the NO synthase inhibitors, N(G)-nitro-L-arginine-methyl-ester (L-NAME) and asymmetric dimethyl-L-arginine (ADMA). The results of the present study showed that leptin increased the membrane fluidity and improved the rigidity of cell membranes to some extent via an NO- and cGMP-dependent mechanism. Furthermore, the data also suggest that leptin might have a crucial role in the regulation of rheological behavior of erythrocytes and microcirculation in humans.

Electron paramagnetic resonance investigation on modulatory effect of benidipine on membrane fluidity of erythrocytes in essential hypertension.

PubMed

Tsuda, Kazushi

2008-03-01

It has been shown that benidipine, a long-lasting calcium (Ca) channel blocker, may exert its protective effect against vascular disorders by increasing nitric oxide (NO) production. The purpose of the present study was to investigate whether orally administered benidipine might influence the membrane function in patients with essential hypertension. We measured the membrane fluidity of erythrocytes by using an electron paramagnetic resonance (EPR) and spin-labeling method. In the preliminary study using erythrocytes obtained from healthy volunteers, benidipine decreased the order parameter (S) for 5-nitroxide stearate (5-NS) and the peak height ratio (ho/h-1) for 16-NS in the EPR spectra in vitro. The finding indicated that benidipine increased the membrane fluidity and improved the microviscosity of erythrocytes. In addition, it was demonstrated that the effect of benidipine on membrane fluidity of erythrocytes was significantly potentiated by the NO-substrate, L-arginine. In the separate series of the study, we observed that orally administered benidipine for 4 weeks significantly increased the membrane fluidity of erythrocytes with a concomitant increase in plasma NO metabolite levels in hypertensive subjects. The results of the present study demonstrated that benidipine might increase the membrane fluidity and improve the microviscosity of erythrocytes both in vitro and in vivo, to some extent, by the NO-dependent mechanism. Furthermore, it is strongly suggested that orally administered benidipine might have a beneficial effect on the rheologic behavior of erythrocytes and the improvement of the microcirculation in hypertensive subjects.

A role for protein kinase C in the regulation of membrane fluidity and Ca²(+) flux at the endoplasmic reticulum and plasma membranes of HEK293 and Jurkat cells.

PubMed

Chen, Lihong; Meng, Qingli; Jing, Xian; Xu, Pingxiang; Luo, Dali

2011-02-01

Protein kinase C (PKC) plays a prominent role in the regulation of a variety of cellular functions, including Ca²(+) signalling. In HEK293 and Jurkat cells, the Ca²(+) release and Ca²(+) uptake stimulated by several different activators were attenuated by activation of PKC with phorbol myristate acetate (PMA) or 1-oleoyl-2-acetyl-sn-glycerol (OAG) and potentiated by PKC inhibition with Gö6983 or knockdown of PKCα or PKCβ using shRNA. Immunostaining and Western blotting analyses revealed that PKCα and PKCβII accumulated at the plasma membrane (PM) and that these isoforms, along with PKCβI, also translocated to the endoplasmic reticulum (ER) upon activation with PMA. Measurements of membrane fluidity showed that, like the cell membrane stabilizers bovine serum albumin (BSA) and ursodeoxycholate (UDCA), PMA and OAG significantly reduced the fluidity of both the PM and ER membranes; these effects were blocked in PKC-knockdown cells. Interestingly, both BSA and UDCA inhibited the Ca²(+) responses to agonists to the same extent as PMA, whereas Tween 20, which increases membrane fluidity, raised the internal Ca²(+) concentration. Thus, activation of PKC induces both translocation of PKC to the PM and ER membranes and downregulation of membrane fluidity, thereby negatively modulating Ca²(+) flux. Copyright © 2010 Elsevier Inc. All rights reserved.

Further investigation of relationships between membrane fluidity and ethanol tolerance in Saccharomyces cerevisiae.

PubMed

Ishmayana, Safri; Kennedy, Ursula J; Learmonth, Robert P

2017-11-27

Membrane lipid unsaturation index and membrane fluidity have been related to yeast ethanol stress tolerance in published studies, however findings have been inconsistent. In this study, viability reduction on exposure to 18% (v/v) ethanol was compared to membrane fluidity determined by laurdan generalized polarization. Furthermore, in the determination of viability reduction, we examined the effectiveness of two methods, namely total plate count and methylene violet staining. We found a strong negative correlation between ethanol tolerance and membrane fluidity, indicated by negative Pearson correlation coefficients of - 0.79, - 0.65 and - 0.69 for Saccharomyces cerevisiae strains A12, PDM and K7, respectively. We found that lower membrane fluidity leads to higher ethanol tolerance, as indicated by decreased viability reduction and higher laurdan generalized polarization in respiratory phase compared to respiro-fermentative phase cells. Total plate count better differentiated ethanol tolerance of yeast cells in different growth phases, while methylene violet staining was better to differentiate ethanol tolerance of the different yeast strains at a particular culture phase. Hence, both viability assessment methods have their own advantages and limitations, which should be considered when comparing stress tolerance in different situations.

Estriol improves membrane fluidity of erythrocytes by the nitric oxide-dependent mechanism: an electron paramagnetic resonance study.

PubMed

Tsuda, K; Shimamoto, Y; Kimura, K; Nishio, I; Masuyama, Y

2001-05-01

The present in vitro study was performed to investigate the effects of estriol (E3) on membrane fluidity of erythrocytes by means of an electron paramagnetic resonance (EPR) and spin-labeling method. E3 was shown to significantly decrease the order parameter (S) for 5-nitroxide stearate (5-NS) and the peak height ratio (ho/h-1) for 16-NS obtained from EPR spectra of erythrocyte membranes. This finding indicated that E3 might increase the membrane fluidity of erythrocytes. The effect of E3 was significantly potentiated by the nitric oxide (NO) donor, S-nitroso-N-acetylpenicillamine (SNAP), and a cyclic guanosine 3',5'-monophosphate (cGMP) analog, 8-bromo-cGMP. In contrast, the change in the membrane fluidity induced by E3 was antagonized by the NO synthase inhibitor, L-NG-nitroarginine-methyl-ester (L-NAME), and asymmetric dimethyl-L-arginine (ADMA). The results of the present study showed that E3 significantly increased the membrane fluidity and improved the microviscosity of erythrocyte membranes, partially mediated by an NO- and cGMP-dependent pathway. Furthermore, the data might be consistent with the hypothesis that E3 could have a beneficial effect on the rheological behavior of erythrocytes and may play a crucial role in the regulation of microcirculation.

Solanum Nigrum polysaccharide (SNL) extract effects in transplanted tumor-bearing mice--erythrocyte membrane fluidity and blocking of functions.

PubMed

Yuan, Hong-Liang; Liu, Xiao-Lei; Liu, Ying-Jie

2014-01-01

Solanum nigrum L. has been used in traditional Chinese medicine because of its diuretic and antipyretic effects. The present research concerned effects of crude polysaccharides isolated from Solanum nigrum L. on erythrocyte membranes of tumor-bearing S180 and H22 in mice. Fluorescence- labeled red blood cell membranes were used with DPH fluorescence spectrophotometry to examine erythrocyte membrane fluidity, and colorimetry to determine degree of erythrocyte surface membrane blocking. Extent of reaction by tumor-bearing mice with the enzyme erythrocyte membrane bubble shadow detection of red cell membrane variation in the degree of closure before and after administration. Solanum nigrum polysaccharide could significantly improve the S180 and H22 tumor-bearing mice erythrocyte membrane fluidity, compared with the control group, the difference was significant (p<0.01), SNL can significantly improve the red blood cell membrane and then S180 tumor-bearing mice sealing ability, compared with the negative control group, the difference was significant(p<0.05, p<0.01). H22 tumor-bearing mice can increase red cell membrane and then sealing ability, the difference was significant (p<0.05). Solanum nigrum polysaccharide degree of fluidity and blocking two transplanted tumors in mice restored the ability to raise the red cell membrane has a significant effect. Solanum nigrum L.-type mice transplanted tumor can affect the red blood cell membrane fluidity and re-closed, through the red cell membrane of red blood cells to enhance the immune function of the possibility of erythrocyte immunity against tumor formation garland provide experimental basis.

Subcellular membrane fluidity of Lactobacillus delbrueckii subsp. bulgaricus under cold and osmotic stress.

PubMed

Meneghel, Julie; Passot, Stéphanie; Cenard, Stéphanie; Réfrégiers, Matthieu; Jamme, Frédéric; Fonseca, Fernanda

2017-09-01

Cryopreservation of lactic acid bacteria may lead to undesirable cell death and functionality losses. The membrane is the first target for cell injury and plays a key role in bacterial cryotolerance. This work aimed at investigating at a subcellular resolution the membrane fluidity of two populations of Lactobacillus delbrueckii subsp. bulgaricus when subjected to cold and osmotic stresses associated to freezing. Cells were cultivated at 42 °C in mild whey medium, and they were exposed to sucrose solutions of different osmolarities (300 and 1800 mOsm L -1 ) after harvest. Synchrotron fluorescence microscopy was used to measure membrane fluidity of cells labeled with the cytoplasmic membrane probe 1-[4 (trimethylamino) phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH). Images were acquired at 25 and 0 °C, and more than a thousand cells were individually analyzed. Results revealed that a bacterial population characterized by high membrane fluidity and a homogeneous distribution of fluidity values appeared to be positively related to freeze-thaw resistance. Furthermore, rigid domains with different anisotropy values were observed and the occurrence of these domains was more important in the freeze-sensitive bacterial population. The freeze-sensitive cells exhibited a broadening of existing highly rigid lipid domains with osmotic stress. The enlargement of domains might be ascribed to the interaction of sucrose with membrane phospholipids, leading to membrane disorganization and cell degradation.

Fluidity of pea root plasma membranes under altered gravity

NASA Astrophysics Data System (ADS)

Klymchuk, D. O.; Baranenko, V. V.; Vorobyova, T. V.; Dubovoy, V. D.

This investigation aims to determine whether clinorotation 2 rev min of pea Pisum sativum L seedlings induces the alterations in the physical-chemical properties of cellular membranes including the plasma membrane fluidity The last is an important regulator of functional activity of membrane enzymes The plasma membranes were isolated by aqueous two-phase partitioning from roots of 6-day old pea seedlings The membrane fluidity was examined by fluorescence spectroscopy using pyrene probe The plasma membrane vesicles with known protein concentration were added to the incubation buffer to a final concentration of 50 mu g of protein per ml A small amount by 1 mu l of pyrene solution in 2-propanol was added to the incubation mixture to a final probe concentration 5 mu M at constant mixing Fluorescence spectra were measured using a Perkin-Elmer LS-50 spectrofluorometer Perkin-Elmer England Pyrene was excited at 337 nm and fluorescence intensity of monomers I M and excimers I E were measured at 393 and 470 nm respectively The I E I M ratios were 0 081 pm 0 003 and 0 072 pm 0 004 in preparations obtained from clinorotated and the control seedlings respectively This fact indicates that rotation on the clinostat increases the membrane fluidity Compared with controls clinorotated seedlings have also showed a reduced growth and a higher level of total unsaturated fatty acids determined by gas chromatography The factors that influence on the fluidity of membrane lipids in bilayer appear to be the

Nitric oxide improves membrane fluidity of erythrocytes in essential hypertension: An electron paramagnetic resonance investigation.

PubMed

Tsuda, K; Kimura, K; Nishio, I; Masuyama, Y

2000-09-07

It has been shown that rheological abnormality might be an etiological factor in hypertension. Recent studies have revealed that human erythrocytes possess a nitric oxide (NO) synthase and that this activation might be involved in the regulation of rheological properties of erythrocytes. The present study was undertaken to investigate the role of NO in the regulation of membrane functions of erythrocytes in patients with essential hypertension by means of an electron paramagnetic resonance (EPR) and spin-labeling method. The NO donor S-nitroso-N-acetylpenicillamine (SNAP) decreased the order parameter (S) for 5-nitroxide stearate (5-NS) and the peak height ratio (h(0)/h(-1)) for 16-NS obtained from EPR spectra of erythrocyte membranes in a dose-dependent manner. The finding indicated that the NO donor increased the membrane fluidity of erythrocytes. In addition, the effect of SNAP was significantly potentiated by 8-bromo-cyclic guanosine monophosphate. By contrast, the change of the fluidity induced by SNAP was reversed in the presence of L-N(G)-nitroarginine methyl ester and asymmetric dimethyl L-arginine. In patients with essential hypertension, the membrane fluidity of erythrocytes was significantly lower than in the normotensive subjects. The effect of SNAP was more pronounced in essential hypertension than in normotensive subjects. These results showed that NO increased the membrane fluidity and decreased the rigidity of cell membranes. Furthermore, the greater effect of NO on the fluidity in essential hypertension suggests that NO might actively participate in the regulation of rheological behavior of erythrocytes and have a crucial role in the improvement of microcirculation in hypertension. Copyright 2000 Academic Press.

Plasma membrane microorganization of LR73 multidrug-resistant cells revealed by FCS

NASA Astrophysics Data System (ADS)

Winckler, Pascale; Jaffiol, Rodolphe; Cailler, Aurélie; Morjani, Hamid; Jeannesson, Pierre; Deturche, Régis

2011-03-01

Tumoral cells could present a multidrug resistance (MDR) to chemotherapeutic treatments. This drug resistance would be associated to biomechanisms occurring at the plasma membrane level, involving modification of membrane fluidity, drug permeability, presence of microdomains (rafts, caveolae...), and membrane proteins overexpression such as Pglycoprotein. Fluorescence correlation spectroscopy (FCS) is the relevant method to investigate locally the fluidity of biological membranes through the lateral diffusion of a fluorescent membrane probe. Thus, we use FCS to monitor the plasma membrane local organization of LR73 carcinoma cells and three derived multidrug-resistant cancer cells lines. Measurements were conducted at the single cell level, which enabled us to get a detailed overview of the plasma membrane microviscosity distribution of each cell line studied. Moreover, we propose 2D diffusion simulation based on a Monte Carlo model to investigate the membrane organisation in terms of microdomains. This simulation allows us to relate the differences in the fluidity distributions with microorganization changes in plasma membrane of MDR cells.

Hydrostatic pressure decreases membrane fluidity and lipid desaturase expression in chondrocyte progenitor cells.

PubMed

Montagne, Kevin; Uchiyama, Hiroki; Furukawa, Katsuko S; Ushida, Takashi

2014-01-22

Membrane biomechanical properties are critical in modulating nutrient and metabolite exchange as well as signal transduction. Biological membranes are predominantly composed of lipids, cholesterol and proteins, and their fluidity is tightly regulated by cholesterol and lipid desaturases. To determine whether such membrane fluidity regulation occurred in mammalian cells under pressure, we investigated the effects of pressure on membrane lipid order of mouse chondrogenic ATDC5 cells and desaturase gene expression. Hydrostatic pressure linearly increased membrane lipid packing and simultaneously repressed lipid desaturase gene expression. We also showed that cholesterol mimicked and cholesterol depletion reversed those effects, suggesting that desaturase gene expression was controlled by the membrane physical state itself. This study demonstrates a new effect of hydrostatic pressure on mammalian cells and may help to identify the molecular mechanisms involved in hydrostatic pressure sensing in chondrocytes. © 2013 Elsevier Ltd. All rights reserved.

Influence of polyunsaturated fatty acid supplementation and membrane fluidity on ozone and nitrogen dioxide sensitivity of rat alveolar macrophages

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rietjens, I.M.; van Tilburg, C.A.; Coenen, T.M.

1987-01-01

The phospholipid polyunsaturated fatty acid (PUFA) content and the membrane fluidity of rat alveolar macrophages were modified dose-dependently and in different ways. This was done to study the importance of both membrane characteristics for the cellular sensitivity toward ozone and nitrogen dioxide. Cells preincubated with arachidonic acid (20:4) complexed to bovine serum albumin (BSA) demonstrated an increased in vitro sensitivity versus ozone and nitrogen dioxide. The phenomenon was only observed at the highest 20:4 concentrations tested, whereas the membrane fluidity of the 20:4-treated cells already showed a maximum increase at lower preincubation concentrations. Hence it could be concluded that themore » increased ozone and nitrogen dioxide sensitivity of PUFA-enriched cells is not caused by their increased membrane fluidity, resulting in an increased accessibility of sensitive cellular fatty acid moieties or amino acid residues. This conclusion receives further support from other observations. These results strongly support the involvement of lipid oxidation in the mechanism(s) of toxic action of both ozone and nitrogen dioxide in an intact cell system.« less

Roles of pyruvate dehydrogenase and branched-chain α-keto acid dehydrogenase in branched-chain membrane fatty acid levels and associated functions in Staphylococcus aureus.

PubMed

Singh, Vineet K; Sirobhushanam, Sirisha; Ring, Robert P; Singh, Saumya; Gatto, Craig; Wilkinson, Brian J

2018-04-01

Membrane fluidity to a large extent is governed by the presence of branched-chain fatty acids (BCFAs). Branched-chain α-keto acid dehydrogenase (BKD) is the key enzyme in BCFA synthesis. A Staphylococcus aureus BKD-deficient strain still produced substantial levels of BCFAs. Pyruvate dehydrogenase (PDH) with structural similarity to BKD has been speculated to contribute to BCFAs in S. aureus. This study was carried out using BKD-, PDH- and BKD : PDH-deficient derivatives of methicillin-resistant S. aureus strain JE2. Differences in growth kinetics were evaluated spectrophotometrically, membrane BCFAs using gas chromatography and membrane fluidity by fluorescence polarization. Carotenoid levels were estimated by measuring A465 of methanol extracts from 48 h cultures. MIC values were determined by broth microdilution.Results/Key findings. BCFAs made up 50 % of membrane fatty acids in wild-type but only 31 % in the BKD-deficient mutant. BCFA level was ~80 % in the PDH-deficient strain and 38 % in the BKD : PDH-deficient strain. BKD-deficient mutant showed decreased membrane fluidity, the PDH-deficient mutant showed increased membrane fluidity. The BKD- and PDH-deficient strains grew slower and the BKD : PDH-deficient strain grew slowest at 37 °C. However at 20 °C, the BKD- and BKD : PDH-deficient strains grew only a little followed by autolysis of these cells. The BKD-deficient strain produced higher levels of staphyloxanthin. The PDH-deficient and BKD : PDH-deficient strains produced very little staphyloxanthin. The BKD-deficient strain showed increased susceptibility to daptomycin. The BCFA composition of the cell membrane in S. aureus seems to significantly impact cell growth, membrane fluidity and resistance to daptomycin.

Inhibition of HIV-1 entry by the tricyclic coumarin GUT-70 through the modification of membrane fluidity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matsuda, Kouki; Hattori, Shinichiro; Kariya, Ryusho

Membrane fusion between host cells and HIV-1 is the initial step in HIV-1 infection, and plasma membrane fluidity strongly influences infectivity. In the present study, we demonstrated that GUT-70, a natural product derived from Calophyllum brasiliense, stabilized plasma membrane fluidity, inhibited HIV-1 entry, and down-regulated the expression of CD4, CCR5, and CXCR4. Since GUT-70 also had an inhibitory effect on viral replication through the inhibition of NF-κB, it is expected to be used as a dual functional and viral mutation resistant reagent. Thus, these unique properties of GUT-70 enable the development of novel therapeutic agents against HIV-1 infection.

Erythrocyte membrane fluidity and indices of plasmatic oxidative damage after acute physical exercise in humans.

PubMed

Berzosa, C; Gómez-Trullén, E M; Piedrafita, E; Cebrián, I; Martínez-Ballarín, E; Miana-Mena, F J; Fuentes-Broto, L; García, J J

2011-06-01

Optimal levels of membrane fluidity are essential for numerous cell functions including cell growth, solute transport and signal transduction. Since exercise enhances free radical production, our aim was to evaluate in healthy male subjects the effects of an acute bout of maximal and submaximal exercise on the erythrocyte membrane fluidity and its possible relation to the oxidative damage overproduction due to exercise. Subjects (n = 34) performed three cycloergometric tests: a continuous progressive exercise, a strenuous exercise until exhaustion and an acute bout of exercise at an intensity corresponding to 70% of maximal work capacity for 30 min. Venous blood samples were collected before and immediately after these exercises. Erythrocyte membrane fluidity was assessed by fluorescence spectroscopy. Plasma malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA) concentrations and carbonyl content of plasmatic proteins were used as an index of lipid and protein oxidation, respectively. Exercise produced a dramatic drop in the erythrocyte membrane fluidity as compared to resting time, but this was not accompanied by significant changes in the plasmatic MDA and 4-HDA concentrations. The highest erythrocyte membrane rigidity was detected immediately after strenuous exercise until exhaustion was performed. Protein carbonyl levels were higher after exhaustive exercises than at rest. Continuous progressive and strenuous exercises until exhaustion, but not submaximal workload, resulted in a significant enhanced accumulation of carbonylated proteins in the plasma. These findings are consistent with the idea that exercise exaggerates oxidative damage, which may contribute, at least partially, to explain the rigidity in the membrane of the erythrocytes due to acute exercise.

[The effects of electromagnetic pulse on fluidity and lipid peroxidation of mitochondrial membrane].

PubMed

Wang, Changzhen; Cong, Jianbo; Xian, Hong; Cao, Xiaozhe; Sun, Cunpu; Wu, Ke

2002-08-01

To study the effects of intense electromagnetic pulse(EMP) on the biological effects of mitochondrial membrane. Rat liver mitochondrial suspension was exposed to EMP at 60 kV/m level. The changes of membrane lipid fluidity and membrane protein mobility were detected by ESR and spin label technique. Malondialdehyde(MDA) was detected by spectrophotometer. The mobility of membrane protein decreased significantly(P < 0.05). Correlation time (tau c) of control group was (0.501 +/- 0.077) x 10(-9)s, and tau c of EMP group was (0.594 +/- 0.049) x 10(-9)s, indicating that the mobility of protein was restricted. The fluidity of mitochondrial membrane increased significantly(P < 0.05) at the same time. Order parameter(S) of mitochondrial membrane lipid in control group was 0.63 +/- 0.01, while S of EMP group was 0.61 +/- 0.01(P < 0.05). MDA decreased significantly. The mobility and lipid peroxidation of mitochondrial membrane may be disturbed after EMP exposure.

Smoking-induced alterations in platelet membrane fluidity and Na(+)/K(+)-ATPase activity in chronic cigarette smokers.

PubMed

Padmavathi, Pannuru; Reddy, Vaddi Damodara; Maturu, Paramahamsa; Varadacharyulu, Nallanchakravarthula

2010-06-30

Cigarette smoking is a recognized risk factor for cardiovascular diseases and has been implicated in the pathogenesis of atherosclerosis. Platelet adhesiveness and aggregation increases as a result of smoking. Cigarette smoking modifies haemostatic parameters via thrombosis with a consequently higher rate of cardiovascular events, but smoking-induced alterations of platelet membrane fluidity and other changes have not been studied. Thirty experimental and control subjects (mean age 35+/-8) were selected for the study. Experimental subjects had smoked 10+/-2 cigarettes per day for 7-10 years. The plasma lipid profile, platelet carbonyls, sulfhydryl groups, Na(+)/k(+)-ATPase activity, fluidity using a fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH), total cholesterol and phospholipids as well individual phospholipids were determined. Increases in the platelet membrane cholesterol phospholipid (C/P) ratio, phosphotidylethanolamine, phosphotidylserine with decreased phosphotidylcholine, Na(+)/k(+)-ATPase activity, fluidity and no significant change in phosphotidylinositol and sphingomylein, as well as increases in plasma total cholesterol, LDL-cholesterol, protein carbonyls with decreased HDL-cholesterol and sulfhydryl groups were observed in cigarette smokers. Platelet membrane total phospholipids were positively correlated with plasma LDL-cholesterol (r=0.568) and VLDL-cholesterol (r=0.614) in cigarette smokers. Increased plasma LDL-cholesterol, VLDL-cholesterol and total cholesterol might have resulted in the increased C/P ratio and decreased platelet membrane fluidity of cigarette smokers.

Membrane fluidity profiles as deduced by saturation-recovery EPR measurements of spin-lattice relaxation times of spin labels

NASA Astrophysics Data System (ADS)

Mainali, Laxman; Feix, Jimmy B.; Hyde, James S.; Subczynski, Witold K.

2011-10-01

There are no easily obtainable EPR spectral parameters for lipid spin labels that describe profiles of membrane fluidity. The order parameter, which is most often used as a measure of membrane fluidity, describes the amplitude of wobbling motion of alkyl chains relative to the membrane normal and does not contain explicitly time or velocity. Thus, this parameter can be considered as nondynamic. The spin-lattice relaxation rate ( T1-1) obtained from saturation-recovery EPR measurements of lipid spin labels in deoxygenated samples depends primarily on the rotational correlation time of the nitroxide moiety within the lipid bilayer. Thus, T1-1 can be used as a convenient quantitative measure of membrane fluidity that reflects local membrane dynamics. T1-1 profiles obtained for 1-palmitoyl-2-( n-doxylstearoyl)phosphatidylcholine ( n-PC) spin labels in dimyristoylphosphatidylcholine (DMPC) membranes with and without 50 mol% cholesterol are presented in parallel with profiles of the rotational diffusion coefficient, R⊥, obtained from simulation of EPR spectra using Freed's model. These profiles are compared with profiles of the order parameter obtained directly from EPR spectra and with profiles of the order parameter obtained from simulation of EPR spectra. It is shown that T1-1 and R⊥ profiles reveal changes in membrane fluidity that depend on the motional properties of the lipid alkyl chain. We find that cholesterol has a rigidifying effect only to the depth occupied by the rigid steroid ring structure and a fluidizing effect at deeper locations. These effects cannot be differentiated by profiles of the order parameter. All profiles in this study were obtained at X-band (9.5 GHz).

A selective estrogen receptor modulator, tamoxifen, and membrane fluidity of erythrocytes in normotensive and hypertensive postmenopausal women: an electron paramagnetic resonance investigation.

PubMed

Tsuda, Kazushi; Nishio, Ichiro

2005-08-01

Recent studies have shown that tamoxifen, which belongs to a group called selective estrogen receptor modulators (SERM), may exert protective effects against cardiovascular diseases and stroke in postmenopausal women. On the other hand, abnormalities in physical properties of the cell membranes may underlie the defects that are strongly linked to hypertension, stroke, and other cardiovascular diseases. The present study was performed to investigate the effects of tamoxifen on cell membrane fluidity (a reciprocal value of membrane microviscosity) in normotensive and hypertensive postmenopausal women. We used an electron paramagnetic resonance (EPR) and spin-labeling method. Tamoxifen significantly decreased the order parameter (S) for 5-nitroxide stearate (5-NS) and the peak height ratio (h(o)/h(-1)) for 16-NS obtained from EPR spectra of erythrocyte membranes in normotensive postmenopausal women (mean +/- SEM, order parameter value; control 0.719 +/- 0.002, n = 41; tamoxifen 1 x 10(-7) mol/L 0.704 +/- 0.002, n = 41, P < .0001; tamoxifen 1 x 10(-6) mol/L 0.696 +/- 0.002, n = 41, P < .0001; tamoxifen 1 x 10(-5) mol/L 0.692 +/- 0.002, n = 41, P < .0001). The finding indicated that tamoxifen increased the membrane fluidity and improved the membrane microviscosity of erythrocytes. The membrane action of tamoxifen was antagonized by the estrogen receptor antagonist ICI 182,780. The effect of tamoxifen was significantly potentiated by the nitric oxide (NO) donors, l-arginine and S-nitroso-N-acetylpenicillamine, and a cGMP analog 8-bromo-cGMP. In contrast, the change evoked by tamoxifen was counteracted by the NO synthase inhibitors N(G)-nitro-l-arginine-methyl-ester and asymmetric dimethyl-l-arginine. In hypertensive postmenopausal women, the membrane fluidity of erythrocytes was significantly lower than in normotensive postmenopausal women. The effect of tamoxifen on the membrane fluidity was more pronounced in hypertensive postmenopausal women than in normotensive postmenopausal women. These results showed that tamoxifen increased the membrane fluidity of erythrocytes and improved the rigidity of cell membranes in postmenopausal women, to some extent, through the NO- and cGMP-dependent mechanisms. Furthermore, the greater effect of tamoxifen in hypertensive postmenopausal women suggests that tamoxifen could have a beneficial effect in regulating the blood rheologic behavior and in the improvement of the microcirculation in hypertension.

Influence of MLS laser radiation on erythrocyte membrane fluidity and secondary structure of human serum albumin.

PubMed

Pasternak, Kamila; Nowacka, Olga; Wróbel, Dominika; Pieszyński, Ireneusz; Bryszewska, Maria; Kujawa, Jolanta

2014-03-01

The biostimulating activity of low level laser radiation of various wavelengths and energy doses is widely documented in the literature, but the mechanisms of the intracellular reactions involved are not precisely known. The aim of this paper is to evaluate the influence of low level laser radiation from an multiwave locked system (MLS) of two wavelengths (wavelength = 808 nm in continuous emission and 905 nm in pulsed emission) on the human erythrocyte membrane and on the secondary structure of human serum albumin (HSA). Human erythrocytes membranes and HSA were irradiated with laser light of low intensity with surface energy density ranging from 0.46 to 4.9 J cm(-2) and surface energy power density 195 mW cm(-2) (1,000 Hz) and 230 mW cm(-2) (2,000 Hz). Structural and functional changes in the erythrocyte membrane were characterized by its fluidity, while changes in the protein were monitored by its secondary structure. Dose-dependent changes in erythrocyte membrane fluidity were induced by near-infrared laser radiation. Slight changes in the secondary structure of HSA were also noted. MLS laser radiation influences the structure and function of the human erythrocyte membrane resulting in a change in fluidity.

Effect of superparamagnetic iron oxide nanoparticles on fluidity and phase transition of phosphatidylcholine liposomal membranes

PubMed Central

Santhosh, Poornima Budime; Drašler, Barbara; Drobne, Damjana; Kreft, Mateja Erdani; Kralj, Slavko; Makovec, Darko; Ulrih, Nataša Poklar

2015-01-01

Superparamagnetic iron oxide nanoparticles (SPIONs) with multifunctional properties have shown great promise in theranostics. The aim of our work was to compare the effects of SPIONs on the fluidity and phase transition of the liposomal membranes prepared with zwitterionic phosphatidylcholine lipids. In order to study if the surface modification of SPIONs has any influence on these membrane properties, we have used four types of differently functionalized SPIONs, such as: plain SPIONs (primary size was shown to bê11 nm), silica-coated SPIONs, SPIONs coated with silica and functionalized with positively charged amino groups or negatively charged carboxyl groups (the primary size of all the surface-modified SPIONs was ~20 nm). Small unilamellar vesicles prepared with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine lipids and multilamellar vesicles prepared with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine lipids were encapsulated or incubated with the plain and surface-modified SPIONs to determine the fluidity and phase transition temperature of the bilayer lipids, respectively. Fluorescent anisotropy and differential scanning calorimetric measurements of the liposomes that were either encapsulated or incubated with the suspension of SPIONs did not show a significant difference in the lipid ordering and fluidity; though the encapsulated SPIONs showed a slightly increased effect on the fluidity of the model membranes in comparison with the incubated SPIONs. This indicates the low potential of the SPIONs to interact with the nontargeted cell membranes, which is a desirable factor for in vivo applications. PMID:26491286

Eicosapentaenoic acid reduces membrane fluidity, inhibits cholesterol domain formation, and normalizes bilayer width in atherosclerotic-like model membranes.

PubMed

Mason, R Preston; Jacob, Robert F; Shrivastava, Sandeep; Sherratt, Samuel C R; Chattopadhyay, Amitabha

2016-12-01

Cholesterol crystalline domains characterize atherosclerotic membranes, altering vascular signaling and function. Omega-3 fatty acids reduce membrane lipid peroxidation and subsequent cholesterol domain formation. We evaluated non-peroxidation-mediated effects of eicosapentaenoic acid (EPA), other TG-lowering agents, docosahexaenoic acid (DHA), and other long-chain fatty acids on membrane fluidity, bilayer width, and cholesterol domain formation in model membranes. In membranes prepared at 1.5:1 cholesterol-to-phospholipid (C/P) mole ratio (creating pre-existing domains), EPA, glycyrrhizin, arachidonic acid, and alpha linolenic acid promoted the greatest reductions in cholesterol domains (by 65.5%, 54.9%, 46.8%, and 45.2%, respectively) compared to controls; other treatments had modest effects. EPA effects on cholesterol domain formation were dose-dependent. In membranes with 1:1 C/P (predisposing domain formation), DHA, but not EPA, dose-dependently increased membrane fluidity. DHA also induced cholesterol domain formation without affecting temperature-induced changes in-bilayer unit cell periodicity relative to controls (d-space; 57Å-55Å over 15-30°C). Together, these data suggest simultaneous formation of distinct cholesterol-rich ordered domains and cholesterol-poor disordered domains in the presence of DHA. By contrast, EPA had no effect on cholesterol domain formation and produced larger d-space values relative to controls (60Å-57Å; p<0.05) over the same temperature range, suggesting a more uniform maintenance of lipid dynamics despite the presence of cholesterol. These data indicate that EPA and DHA had different effects on membrane bilayer width, membrane fluidity, and cholesterol crystalline domain formation; suggesting omega-3 fatty acids with differing chain length or unsaturation may differentially influence membrane lipid dynamics and structural organization as a result of distinct phospholipid/sterol interactions. Copyright © 2016. Published by Elsevier B.V.

Cholesterol modulates open probability and desensitization of NMDA receptors

PubMed Central

Korinek, Miloslav; Vyklicky, Vojtech; Borovska, Jirina; Lichnerova, Katarina; Kaniakova, Martina; Krausova, Barbora; Krusek, Jan; Balik, Ales; Smejkalova, Tereza; Horak, Martin; Vyklicky, Ladislav

2015-01-01

NMDA receptors (NMDARs) are glutamate-gated ion channels that mediate excitatory neurotransmission in the CNS. Although these receptors are in direct contact with plasma membrane, lipid–NMDAR interactions are little understood. In the present study, we aimed at characterizing the effect of cholesterol on the ionotropic glutamate receptors. Whole-cell current responses induced by fast application of NMDA in cultured rat cerebellar granule cells (CGCs) were almost abolished (reduced to 3%) and the relative degree of receptor desensitization was increased (by seven-fold) after acute cholesterol depletion by methyl-β-cyclodextrin. Both of these effects were fully reversible by cholesterol repletion. By contrast, the responses mediated by AMPA/kainate receptors were not affected by cholesterol depletion. Similar results were obtained in CGCs after chronic inhibition of cholesterol biosynthesis by simvastatin and acute enzymatic cholesterol degradation to 4-cholesten-3-one by cholesterol oxidase. Fluorescence anisotropy measurements showed that membrane fluidity increased after methyl-β-cyclodextrin pretreatment. However, no change in fluidity was observed after cholesterol enzymatic degradation, suggesting that the effect of cholesterol on NMDARs is not mediated by changes in membrane fluidity. Our data show that diminution of NMDAR responses by cholesterol depletion is the result of a reduction of the open probability, whereas the increase in receptor desensitization is the result of an increase in the rate constant of entry into the desensitized state. Surface NMDAR population, agonist affinity, single-channel conductance and open time were not altered in cholesterol-depleted CGCs. The results of our experiments show that cholesterol is a strong endogenous modulator of NMDARs. Key points NMDA receptors (NMDARs) are tetrameric cation channels permeable to calcium; they mediate excitatory synaptic transmission in the CNS and their excessive activation can lead to neurodegeneration. Although these receptors are in direct contact with plasma membrane, lipid–NMDAR interactions are little understood. Using cultured rat cerebellar granule cells, we show that acute and chronic pretreatments resulting in cell cholesterol depletion profoundly diminish NMDAR responses and increase NMDAR desensitization, and also that cholesterol enrichment potentiates NMDAR responses; however, cholesterol manipulation has no effect on the amplitude of AMPA/kainate receptor responses. Diminution of NMDAR responses by cholesterol depletion is the result of a reduction of the ion channel open probability, whereas the increase in receptor desensitization is the result of an increase in the rate constant of entry into the desensitized state. These results demonstrate the physiological role of membrane lipids in the modulation of NMDAR activity. PMID:25651798

Hyperforin modifies neuronal membrane properties in vivo.

PubMed

Eckert, Gunter P; Keller, Jan-Henning; Jourdan, Claudia; Karas, Michael; Volmer, Dietrich A; Schubert-Zsilavecz, Manfred; Müller, Walter E

2004-09-02

Hyperforin, the major active constituent of St. John Wort (SJW) extract, affects several neurotransmitter systems in the brain putatively by modulation of the physical state of neuronal membranes. Accordingly, we tested the effects of SJW extract and of hyperforin on the properties of murine brain membrane fluidity. Oral administration of SJW extract and of hyperforin sodium salt results in significant hyperforin brain levels. Treatment of mice with hyperforin leads to decreased annular- and bulk fluidity and increased acyl-chain flexibility of brain membranes. All hyperforin related changes of membrane properties were significantly correlated with the corresponding hyperforin brain levels. Our data emphasises a membrane interaction of hyperforin that possibly contributes to its pharmacological effects.

Plasmonic Techniques for Viral Membrane Characterization

NASA Astrophysics Data System (ADS)

Feizpour, Amin

The lipid bilayer membrane of enveloped viruses, such as human immunodeficiency virus type 1 (HIV-1), plays an important role in key steps of the infection, including cell binding and uptake. Phosphatidylserine (PS) and monosialotetrahexosylganglioside (GM1) are examples of two host-derived lipids in the membrane of enveloped virus particles that are known to contribute to virus attachment, uptake, and ultimately dissemination. A quantitative characterization of their contribution to the functionality of the virus requires information about their relative concentrations in the viral membrane. In this dissertation, a gold nanoparticle (NP) binding assay for probing relative PS and GM1 lipid concentrations in the outer leaflet of different virus-like particles (VLPs) using small sample sizes is introduced. The assay evaluates both scattering intensity and resonance wavelength and determines relative NP densities through plasmon coupling as a measure for the target lipid concentrations in the NP-labeled VLP membrane. The performed studies reveal significant differences in the membrane of HIV-1 and Ebola VLPs that assemble at different intracellular sites and pave the way to an optical quantification of lipid concentration in virus particles at physiological titers. In addition, this technique was used in another application to improve the understanding of the relationship between the membrane PS lipid and the infectivity of HIV-2 and murine leukemia virus (MLV). The composition of the membrane, in particular the cholesterol (chol) content, determines its fluidity. As differences in the membrane composition of individual virus particles can lead to different intracellular fates, biophysical tools capable of probing the membrane fluidity on the single-virus level are required. In this dissertation, we demonstrate that fluctuations in the polarization of light scattered off gold or silver nanoparticle (NP)-labeled virus-like-particles (VLPs) encode information about the membrane fluidity of individual VLPs. We developed a plasmonic polarization fluctuation tracking microscopy (PFTM) which facilitated, for the first time, the investigation of the effect of chol content on the membrane fluidity and its dependence on temperature on the single-VLP level. Chol extraction studies with different methyl-beta-cyclodextrin (MbetaCD) concentrations yielded a gradual decrease in polarization fluctuations as function of time. The PFTM revealed chol content and fluidity heterogeneities of an HIV-1 VLP population.

A calcium channel blocker, benidipine, improves cell membrane fluidity in human subjects via a nitric oxide-dependent mechanism. An electron paramagnetic resonance investigation.

PubMed

Tsuda, Kazushi; Nishio, Ichiro

2004-12-01

Recent studies have revealed that benidipine, a long-acting dihydropyridine-type of calcium (Ca) channel blocker, may exert its protective effect against vascular disorders by increasing nitric oxide (NO) production. The purpose of the present study was to investigate the effects of benidipine and NO on the membrane function in human subjects. We measured the membrane fluidity of erythrocytes by using an electron paramagnetic resonance (EPR) and spin-labeling method. Benidipine decreased the order parameter (S) for 5-nitroxide stearate (5-NS) and the peak height ratio (h(o)/h(-1)) for 16-NS obtained from EPR spectra of erythrocyte membranes in a dose-dependent manner in normotensive volunteers. The finding indicated that benidipine increased the membrane fluidity and improved the microviscosity of erythrocytes. The effect of benidipine was significantly potentiated by the NO donor, S-nitroso-n-acetylpenicillamine, and by the cyclic guanosine 3', 5'-monophosphate (cGMP) analog, 8-bromo-cGMP. In contrast, the change evoked by benidipine was counteracted by the NO synthase inhibitors, N(G)-nitro-L-arginine-methyl-ester and asymmetric dimethyl-L-arginine. These results demonstrated that benidipine increased the membrane fluidity of erythrocytes, at least in part, via the NO- and cGMP-dependent mechanism. Furthermore, the data strongly suggest that benidipine might have a beneficial effect on the rheologic behavior of erythrocytes and the improvement of the microcirculation in humans.

Effect of growth temperature, surface type and incubation time on the resistance of Staphylococcus aureus biofilms to disinfectants.

PubMed

Abdallah, Marwan; Chataigne, Gabrielle; Ferreira-Theret, Pauline; Benoliel, Corinne; Drider, Djamel; Dhulster, Pascal; Chihib, Nour-Eddine

2014-03-01

The goal of this study was to investigate the effect of the environmental conditions such as the temperature change, incubation time and surface type on the resistance of Staphylococcus aureus biofilms to disinfectants. The antibiofilm assays were performed against biofilms grown at 20 °C, 30 °C and 37 °C, on the stainless steel and polycarbonate, during 24 and 48 h. The involvement of the biofilm matrix and the bacterial membrane fluidity in the resistance of sessile cells were investigated. Our results show that the efficiency of disinfectants was dependent on the growth temperature, the surface type and the disinfectant product. The increase of growth temperature from 20 °C to 37 °C, with an incubation time of 24 h, increased the resistance of biofilms to cationic antimicrobials. This change of growth temperature did not affect the major content of the biofilm matrix, but it decreased the membrane fluidity of sessile cells through the increase of the anteiso-C19 relative amount. The increase of the biofilm resistance to disinfectants, with the rise of the incubation time, was dependent on both growth temperature and disinfectant product. The increase of the biofilm age also promoted increases in the matrix production and the membrane fluidity of sessile cells. The resistance of S. aureus biofilm seems to depend on the environment of the biofilm formation and involves both extracellular matrix and membrane fluidity of sessile cells. Our study represents the first report describing the impact of environmental conditions on the matrix production, sessile cells membrane fluidity and resistance of S. aureus biofilms to disinfectants.

Influence of membrane fatty acid composition and fluidity on airborne survival of Escherichia coli.

PubMed

Ng, Tsz Wai; Chan, Wing Lam; Lai, Ka Man

2018-04-01

Finding ways to predict and control the survival of bacterial aerosols can contribute to the development of ways to alleviate a number of crucial microbiological problems. Significant damage in the membrane integrity of Escherichia coli during aerosolization and airborne suspension has been revealed which has prompted the question of how the membrane fatty acid composition and fluidity influence the survival of airborne bacteria. Two approaches of using isogenic mutants and different growth temperatures were selected to manipulate the membrane fatty acid composition of E. coli before challenging the bacteria with different relative humidity (RH) levels in an aerosol chamber. Among the mutants (fabR - , cfa. fadA - ), fabR - had the lowest membrane fluidity index (FI) and generally showed a higher survival than the parental strain. Surprisingly, its resistance to airborne stress was so strong that its viability was fully maintained even after airborne suspension at 40% RH, a harsh RH level to bacterial survival. Moreover, E. coli cultured at 20 °C with a higher FI than that at 30 and 37 °C generally had a lower survival after aerosolization and airborne suspension. Unlike FI, individual fatty acid and cyclopropane fatty acid composition did not relate to the bacterial survival. Lipid peroxidation of the membrane was undetected in all the bacteria. Membrane fluidity plays a stronger role in determining the bacteria survival during airborne suspension than during aerosolization. Certain relationships between FI and bacteria survival were identified, which could help predict the transmission of bacteria under different conditions.

A biophysical approach to daunorubicin interaction with model membranes: relevance for the drug's biological activity.

PubMed

Alves, Ana Catarina; Ribeiro, Daniela; Horta, Miguel; Lima, José L F C; Nunes, Cláudia; Reis, Salette

2017-08-01

Daunorubicin is extensively used in chemotherapy for diverse types of cancer. Over the years, evidence has suggested that the mechanisms by which daunorubicin causes cytotoxic effects are also associated with interactions at the membrane level. The aim of the present work was to study the interplay between daunorubicin and mimetic membrane models composed of different ratios of 1,2-dimyristoyl- sn -glycero- 3 -phosphocholine (DMPC), sphingomyelin (SM) and cholesterol (Chol). Several biophysical parameters were assessed using liposomes as mimetic model membranes. Thereby, the ability of daunorubicin to partition into lipid bilayers, its apparent location within the membrane and its effect on membrane fluidity were investigated. The results showed that daunorubicin has higher affinity for lipid bilayers composed of DMPC, followed by DMPC : SM, DMPC : Chol and lastly by DMPC : SM : Chol. The addition of SM or Chol into DMPC membranes not only increases the complexity of the model membrane but also decreases its fluidity, which, in turn, reduces the amount of anticancer drug that can partition into these mimetic models. Fluorescence quenching studies suggest a broad distribution of the drug across the bilayer thickness, with a preferential location in the phospholipid tails. The gathered data support that daunorubicin permeates all types of membranes to different degrees, interacts with phospholipids through electrostatic and hydrophobic bonds and causes alterations in the biophysical properties of the bilayers, namely in membrane fluidity. In fact, a decrease in membrane fluidity can be observed in the acyl region of the phospholipids. Ultimately, such outcomes can be correlated with daunorubicin's biological action, where membrane structure and lipid composition have an important role. In fact, the results indicate that the intercalation of daunorubicin between the phospholipids can also take place in rigid domains, such as rafts that are known to be involved in different receptor processes, which are important for cellular function. © 2017 The Author(s).

In vivo influence of extract from Aronia melanocarpa on the erythrocyte membranes in patients with hypercholesterolemia.

PubMed

Duchnowicz, Piotr; Nowicka, Agmieszka; Koter-Michalak, Maria; Broncel, Marlena

2012-09-01

Hypercholesterolemia increases cholesterol concentration in erythrocyte membranes, which results in decrease of membrane fluidity and decreases the deformability of red blood cells. The fruits of Arona melanocarpa contains many of polyphenols and other compounds that have beneficial health effects. The aim of the study was to estimate the influence of 2-month supplementation of extract from Aronia melanocarpa (100 mg Aronox, three times per day) on cholesterol concentration, lipid peroxidation, membrane fluidity, level of thiol groups and activity of ATPase in erythrocytes from patients with hypercholesterolemia. The study involved 25 patients with hypercholesterolemia without pharmacological treatment and 20 healthy individuals as a control group. Blood samples were collected before, and after 1 and 2 months of Aronia administration. The 2-month Aronia supplementation resulted in a decrease of cholesterol concentration (by 22%) and a decrease of lipid peroxidation (by 40%), and an increase of membrane fluidity. No statistically significant increase of the concentration of thiol groups and of ATPase activity were observed. Our study shows that supplementation of extract from Aronia melanocarpa has a beneficial effect on rheological properties of erythrocytes.

Microscopic mechanism analyses on influence of metabolism of erythrocyte membrane-lipid etc. by LLLIB

NASA Astrophysics Data System (ADS)

Xu, Lin; Zhang, Canbang; Wen, Yuanbin; Liu, Shuxiao; Zhou, Lingyun

2009-08-01

Some cases with cerebral infarction were treated by He-Ne laser irradiation on blood. In the treatment before and after, membrane-cholesterol(C)/membrane-phosphatide(P), membrane fluidity(F) and deformability of erythrocyte were determined. The results showed that low level laser irradiation on blood (LLLIB) can sure reduce the ratio of (C)/(P), can heighten fluidity and improve deformability of erythrocyte .Thus the metabolism ability of erythrocyte membrane-lipid ,the blood circulation and the properties of hemorheology can be improved. In this paper, the microscopic mechanism of those aforesaid action effects by low level laser irradiation on blood were analyzed by means of Quantum theory and some corresponding models.

Vitamin E supplementation protects erythrocyte membranes from oxidative stress in healthy Chinese middle-aged and elderly people.

PubMed

Sun, Yongye; Ma, Aiguo; Li, Yong; Han, Xiuxia; Wang, Qiuzhen; Liang, Hui

2012-05-01

Elderly people are subject to higher levels of oxidative stress than are young people. Vitamin E, as a powerful antioxidant residing mainly in biomembranes, may provide effective protection against oxidative membrane damage and resultant age-related deterioration, especially in the elderly. We hypothesized that appropriate levels of vitamin E supplementation would protect erythrocyte membranes from oxidative stress and thus improve membrane fluidity in healthy middle-aged and elderly people. To test this, we conducted a 4-month double-blind, randomized trial in which 180 healthy subjects (55-70 years old) were randomly divided into 4 groups: group C (control), and 3 treatment groups in which daily doses of 100 mg (VE1), 200 mg (VE2), and 300 mg (VE3) dl-α-tocopheryl acetate were administered. We measured plasma α-tocopherol concentration, malondialdehyde, and superoxide dismutase levels, erythrocyte hemolysis, and erythrocyte membrane fluidity at the beginning and end of the trial. After 4 months supplementation, plasma α-tocopherol concentrations in the 3 treatment groups had increased by 71%, 78%, and 95%, respectively (all P < .01), and significant decreases in plasma malondialdehyde concentrations were observed in these groups (all P < .05). Erythrocyte hemolysis was decreased by 20% to 38% after vitamin E supplementation (all P < .05), and in addition, groups VE2 and VE3 showed dramatic improvements in erythrocyte membrane fluidity (P < .01). Surprisingly, superoxide dismutase activity also decreased significantly in the treatment groups (all P < .05). In summary, vitamin E supplementation apparently alleviates oxidative stress in healthy middle-aged to elderly people, at least in part by improving erythrocyte membrane fluidity and reducing erythrocyte hemolysis. Copyright © 2012 Elsevier Inc. All rights reserved.

Cholesterol-loaded-cyclodextrins improve the post-thaw quality of stallion sperm.

PubMed

Murphy, C; English, A M; Holden, S A; Fair, S

2014-03-01

An unacceptable proportion of stallion sperm do not survive the freeze-thaw process. The hypothesis of this study was that adding cholesterol to a stallion semen extender would stabilise the sperm membrane, resulting in an improved post-thaw semen quality in terms of increased sperm viability, membrane integrity and fluidity, and reduced oxidative stress. Semen was collected from three stallions and diluted in four extenders: TALP; TALP+0.75mg methyl-β-cyclodextrin-cholesterol (MβCD)/mL (MβCD0.75); TALP+1.5mg MβCD-cholesterol/mL (MβCD1.5); and Equipro. Following 15min incubation, samples were centrifuged and diluted to 100×10(6)sperm/mL, frozen in 0.5mL straws and stored in liquid nitrogen. Sperm from each treatment was assessed for progressive linear motility (PLM) and acceptable membrane integrity under hypotonic conditions on a phase contrast microscope at 1000× while viability, membrane fluidity and superoxide generation were assessed by flow cytometry. The MβCD1.5 and MβCD0.75 treatments had a greater proportion of viable sperm than the TALP treatment (P<0.01). There was no effect of treatment on PLM or membrane integrity. The MβCD1.5 treatment had a greater proportion of viable sperm positive for membrane fluidity than the TALP treatment (P<0.05). The MβCD1.5 and MβCD0.75 treatments had a lesser proportion of viable sperm positive for superoxide generation than the TALP treatment (P<0.001). This study has demonstrated that adding cholesterol to stallion sperm prior to cryopreservation increases post-thaw viability, with these viable sperm being of better quality in terms of increased membrane fluidity and reduced superoxide generation. Copyright © 2014 Elsevier B.V. All rights reserved.

The actin homologue MreB organizes the bacterial cell membrane

PubMed Central

Strahl, Henrik; Bürmann, Frank; Hamoen, Leendert W.

2014-01-01

The eukaryotic cortical actin cytoskeleton creates specific lipid domains, including lipid rafts, which determine the distribution of many membrane proteins. Here we show that the bacterial actin homologue MreB displays a comparable activity. MreB forms membrane-associated filaments that coordinate bacterial cell wall synthesis. We noticed that the MreB cytoskeleton influences fluorescent staining of the cytoplasmic membrane. Detailed analyses combining an array of mutants, using specific lipid staining techniques and spectroscopic methods, revealed that MreB filaments create specific membrane regions with increased fluidity (RIFs). Interference with these fluid lipid domains (RIFs) perturbs overall lipid homeostasis and affects membrane protein localization. The influence of MreB on membrane organization and fluidity may explain why the active movement of MreB stimulates membrane protein diffusion. These novel MreB activities add additional complexity to bacterial cell membrane organization and have implications for many membrane-associated processes. PMID:24603761

The actin homologue MreB organizes the bacterial cell membrane.

PubMed

Strahl, Henrik; Bürmann, Frank; Hamoen, Leendert W

2014-03-07

The eukaryotic cortical actin cytoskeleton creates specific lipid domains, including lipid rafts, which determine the distribution of many membrane proteins. Here we show that the bacterial actin homologue MreB displays a comparable activity. MreB forms membrane-associated filaments that coordinate bacterial cell wall synthesis. We noticed that the MreB cytoskeleton influences fluorescent staining of the cytoplasmic membrane. Detailed analyses combining an array of mutants, using specific lipid staining techniques and spectroscopic methods, revealed that MreB filaments create specific membrane regions with increased fluidity (RIFs). Interference with these fluid lipid domains (RIFs) perturbs overall lipid homeostasis and affects membrane protein localization. The influence of MreB on membrane organization and fluidity may explain why the active movement of MreB stimulates membrane protein diffusion. These novel MreB activities add additional complexity to bacterial cell membrane organization and have implications for many membrane-associated processes.

The Effect of Combined Ezetimibe/Atorvastatin Therapy vs. Atorvastatin Monotherapy on the Erythrocyte Membrane Structure in Patients with Coronary Artery Disease: A Pilot Study.

PubMed

Jackowska, Paulina; Pytel, Edyta; Koter-Michalak, Maria; Olszewska-Banaszczyk, Małgorzata; Legęza, Aleksandra; Broncel, Marlena

2016-01-01

Erythrocytes play an important role in atherogenesis. An excessive accumulation of cholesterol in erythrocyte membranes leads to disruption of the erythrocytes. The aim of the study was to compare the effect of two different hypolipidemic therapies on the structure of erythrocyte membranes. The study included 18 patients with angiographic confirmed coronary artery disease who, despite at least 6 months of hypolipidemic treatment, had not achieved LDL-C < 70 mg/dL and 18 healthy individuals as the control group. The following parameters were studied: total cholesterol level and erythrocyte membrane fluidity, lipid peroxidation, SH groups in membrane protein and plasma lipids. We observed a decrease in TC (20%), LDL-C (35%), level of lipid peroxidation (25%) and total cholesterol in erythrocytes (23%), and an increase in HDL-C (8%) and erythrocyte membrane fluidity of subsurface layers (14%) after 6 months of 10 mg atorvastatin + 10 mg ezetimibe therapy, in comparison with healthy controls. In the group treated with 40 mg atorvastatin for 6 months, decreased LDL-C (23%), lipid peroxidation (37%) and membrane cholesterol concentration (18%) was noted, as well as an increase in erythrocyte membrane fluidity in the subsurface layers (12%). Both the combination therapy and the monotherapy lead to an improvement of erythrocyte membrane structure, whose parameters reached values close to those in the control healthy group.

Effect of Pulsed Electric Field on Membrane Lipids and Oxidative Injury of Salmonella typhimurium

PubMed Central

Yun, Ou; Zeng, Xin-An; Brennan, Charles S.; Han, Zhong

2016-01-01

Salmonella typhimurium cells were subjected to pulsed electric field (PEF) treatment at 25 kV/cm for 0–4 ms to investigate the effect of PEF on the cytoplasmic membrane lipids and oxidative injury of cells. Results indicated that PEF treatment induced a decrease of membrane fluidity of Salmonella typhimurium (S. typhimuriumi), possibly due to the alterations of fatty acid biosynthesis-associated gene expressions (down-regulation of cfa and fabA gene expressions and the up-regulation of fabD gene expression), which, in turn, modified the composition of membrane lipid (decrease in the content ratio of unsaturated fatty acids to saturated fatty acids). In addition, oxidative injury induced by PEF treatment was associated with an increase in the content of malondialdehyde. The up-regulation of cytochrome bo oxidase gene expressions (cyoA, cyoB, and cyoC) indicated that membrane damage was induced by PEF treatment, which was related to the repairing mechanism of alleviating the oxidative injury caused by PEF treatment. Based on these results, we achieved better understanding of microbial injury induced by PEF, suggesting that micro-organisms tend to decrease membrane fluidity in response to PEF treatment and, thus, a greater membrane fluidity might improve the efficiency of PEF treatment to inactivate micro-organisms. PMID:27556460

Not changes in membrane fluidity but proteotoxic stress triggers heat shock protein expression in Chlamydomonas reinhardtii.

PubMed

Rütgers, Mark; Muranaka, Ligia Segatto; Schulz-Raffelt, Miriam; Thoms, Sylvia; Schurig, Juliane; Willmund, Felix; Schroda, Michael

2017-12-01

A conserved reaction of all organisms exposed to heat stress is an increased expression of heat shock proteins (HSPs). Several studies have proposed that HSP expression in heat-stressed plant cells is triggered by an increased fluidity of the plasma membrane. Among the main lines of evidence in support of this model are as follows: (a) the degree of membrane lipid saturation was higher in cells grown at elevated temperatures and correlated with a lower amplitude of HSP expression upon a temperature upshift, (b) membrane fluidizers induce HSP expression at physiological temperatures, and (c) membrane rigidifier dimethylsulfoxide dampens heat-induced HSP expression. Here, we tested whether this holds also for Chlamydomonas reinhardtii. We show that heat-induced HSP expression in cells grown at elevated temperatures was reduced because they already contained elevated levels of cytosolic HSP70A/90A that apparently act as negative regulators of heat shock factor 1. We find that membrane rigidifier dimethylsulfoxide impaired translation under heat stress conditions and that membrane fluidizer benzyl alcohol not only induced HSP expression but also caused protein aggregation. These findings support the classical model for the cytosolic unfolded protein response, according to which HSP expression is induced by the accumulation of unfolded proteins. Hence, the membrane fluidity model should be reconsidered. © 2017 John Wiley & Sons Ltd.

Characterization of Phospholipids in Insulin Secretory Granules and Mitochondria in Pancreatic Beta Cells and Their Changes with Glucose Stimulation*

PubMed Central

MacDonald, Michael J.; Ade, Lacmbouh; Ntambi, James M.; Ansari, Israr-Ul H.; Stoker, Scott W.

2015-01-01

The lipid composition of insulin secretory granules (ISG) has never previously been thoroughly characterized. We characterized the phospholipid composition of ISG and mitochondria in pancreatic beta cells without and with glucose stimulation. The phospholipid/protein ratios of most phospholipids containing unsaturated fatty acids were higher in ISG than in whole cells and in mitochondria. The concentrations of negatively charged phospholipids, phosphatidylserine, and phosphatidylinositol in ISG were 5-fold higher than in the whole cell. In ISG phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin, fatty acids 12:0 and 14:0 were high, as were phosphatidylserine and phosphatidylinositol containing 18-carbon unsaturated FA. With glucose stimulation, the concentration of many ISG phosphatidylserines and phosphatidylinositols increased; unsaturated fatty acids in phosphatidylserine increased; and most phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophosphatidylcholines were unchanged. Unsaturation and shorter fatty acid length in phospholipids facilitate curvature and fluidity of membranes, which favors fusion of membranes. Recent evidence suggests that negatively charged phospholipids, such as phosphatidylserine, act as coupling factors enhancing the interaction of positively charged regions in SNARE proteins in synaptic or secretory vesicle membrane lipid bilayers with positively charged regions in SNARE proteins in the plasma membrane lipid bilayer to facilitate docking of vesicles to the plasma membrane during exocytosis. The results indicate that ISG phospholipids are in a dynamic state and are consistent with the idea that changes in ISG phospholipids facilitate fusion of ISG with the plasma membrane-enhancing glucose-stimulated insulin exocytosis. PMID:25762724

In vivo influence of extract from Aronia melanocarpa on the erythrocyte membranes in patients with hypercholesterolemia

PubMed Central

Duchnowicz, Piotr; Nowicka, Agnieszka; Koter-Michalak, Maria; Broncel, Marlena

2012-01-01

Summary Background Hypercholesterolemia increases cholesterol concentration in erythrocyte membranes, which results in decrease of membrane fluidity and decreases the deformability of red blood cells. The fruits of Arona melanocarpa contains many of polyphenols and other compounds that have beneficial health effects. Material/Methods The aim of the study was to estimate the influence of 2-month supplementation of extract from Aronia melanocarpa (100 mg Aronox, three times per day) on cholesterol concentration, lipid peroxidation, membrane fluidity, level of thiol groups and activity of ATPase in erythrocytes from patients with hypercholesterolemia. The study involved 25 patients with hypercholesterolemia without pharmacological treatment and 20 healthy individuals as a control group. Blood samples were collected before, and after 1 and 2 months of Aronia administration. Results The 2-month Aronia supplementation resulted in a decrease of cholesterol concentration (by 22%) and a decrease of lipid peroxidation (by 40%), and an increase of membrane fluidity. No statistically significant increase of the concentration of thiol groups and of ATPase activity were observed. Conclusions Our study shows that supplementation of extract from Aronia melanocarpa has a beneficial effect on rheological properties of erythrocytes. PMID:22936193

Interaction of cationic carbosilane dendrimers and their complexes with siRNA with erythrocytes and red blood cell ghosts.

PubMed

Wrobel, Dominika; Kolanowska, Katarzyna; Gajek, Arkadiusz; Gomez-Ramirez, Rafael; de la Mata, Javier; Pedziwiatr-Werbicka, Elżbieta; Klajnert, Barbara; Waczulikova, Iveta; Bryszewska, Maria

2014-03-01

We have investigated the interactions between cationic NN16 and BDBR0011 carbosilane dendrimers with red blood cells or their cell membranes. The carbosilane dendrimers used possess 16 cationic functional groups. Both the dendrimers are made of water-stable carbon-silicon bonds, but NN16 possesses some oxygen-silicon bonds that are unstable in water. The nucleic acid used in the experiments was targeted against GAG-1 gene from the human immunodeficiency virus, HIV-1. By binding to the outer leaflet of the membrane, carbosilane dendrimers decreased the fluidity of the hydrophilic part of the membrane but increased the fluidity of the hydrophobic interior. They induced hemolysis, but did not change the morphology of the cells. Increasing concentrations of dendrimers induced erythrocyte aggregation. Binding of short interfering ribonucleic acid (siRNA) to a dendrimer molecule decreased the availability of cationic groups and diminished their cytotoxicity. siRNA-dendrimer complexes changed neither the fluidity of biological membranes nor caused cell hemolysis. Addition of dendriplexes to red blood cell suspension induced echinocyte formation. Copyright © 2013 Elsevier B.V. All rights reserved.

Correlation of polyunsaturated fatty acids with the cold adaptation of Rhodotorula glutinis.

PubMed

He, Jing; Yang, Zhaojie; Hu, Binbin; Ji, Xiuling; Wei, Yunlin; Lin, Lianbing; Zhang, Qi

2015-11-01

This study aimed to investigate the correlation between the cold adaptation of Rhodotorula glutinis YM25079 and the membrane fluidity, content of polyunsaturated fatty acids and mRNA expression level of the Δ(12)-desaturase gene. The optimum temperature for YM25079 growth was analysed first, then the composition changes of membrane lipid in YM25079 were detected by GC-MS and membrane fluidity was evaluated by 1-anilinonaphthalene-8-sulphonate (ANS) fluorescence. Meanwhile, the encoding sequence of Δ(12)-fatty acid desaturase in YM25079 was cloned and further transformed into Saccharomyces cerevisiae INVScl for functional analysis. The mRNA expression levels of Δ(12)-fatty acid desaturase at 15°C and 25°C were analysed by real-time PCR. YM25079 could grow at 5-30°C, with the optimum temperature of 15°C. The membrane fluidity of YM25079 was not significantly reduced when the culture temperature decreased from 25°C to 15°C, but the content of polyunsaturated fatty acids (PUFAs), including linoleic acid and α-Linolenic acid increased significantly from 29.4% to 55.39%. Furthermore, a novel Δ(12)-fatty acid desaturase gene YM25079RGD12 from YM25079 was successfully identified and characterized, and the mRNA transcription level of the Δ(12)-desaturase gene was about five-fold higher in YM25079 cells grown at 15°C than that at 25°C. These results suggests that the cold adaptation of Rhodotorula glutinis YM25079 might result from higher expression of genes, especially the Δ(12)-fatty acid desaturase gene, during polyunsaturated fatty acids biosynthesis, which increased the content of PUFAs in the cell membrane and maintained the membrane fluidity at low temperature. Copyright © 2015 John Wiley & Sons, Ltd.

Higher membrane fluidity mediates the increased subcutaneous fatty acid content in pigs fed reduced protein diets.

PubMed

Lopes, P A; Martins, A P; Martins, S V; Madeira, M S; Santos, N C; Moura, T F; Prates, J A M; Soveral, G

2017-04-01

The production of pork with moderate amounts of intramuscular fat (IMF) without an increase in subcutaneous fat is highly desirable for the meat industry. Several studies indicate that dietary protein reduction during the growing-finishing period of pigs enhances IMF content, but its consequence on carcass fat deposition is still contradictory. In this study, we hypothesized that the effects of reduced protein diets (RPD), corrected or not with the limiting amino acid lysine, on subcutaneous fat deposition from pigs with distinct genotypes are mediated by adipose membranes biophysical properties. In total, 36 crossbred (Large White×Landrace×Pietrain - a lean genotype) and purebred (Alentejana breed - a fatty genotype) male pigs were randomly assigned to the control group, the RPD group or the reduced protein diet equilibrated for lysine (RPDL) group, allowing a 2×3 factorial arrangement (n=6). Backfat thickness and total fatty acid content were higher in Alentejana relative to crossbred pigs. Although dietary treatments did not change backfat thickness, RPD and RPDL increased total fatty acids content of subcutaneous fat. In order to understand this effect, adipose tissue membranes isolated from pig's subcutaneous fat were assayed for glycerol permeability and fluidity, using 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-(trimethylamino)-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) probes. The glycerol transport across adipose membranes was not mediated by aquaglyceroporins and remained unchanged across dietary groups. Regardless of lysine correction, RPD increased membrane fluidity at the hydrocarbon region (lower DPH fluorescence anisotropy) in both genotypes of pigs. This result was associated with a lower ratio between oleic acid and linoleic acid on membrane's fatty acid composition. Adipose membrane's cholesterol content was independent from genotype and diet. Taken together, the present study shows that dietary protein reduction is successful in maintaining backfat thickness, although a negative side effect was observed on total fatty acids in subcutaneous fat, which may be due to changes in the fluidity of adipose membranes.

Effects of thallium(I) and thallium(III) on liposome membrane physical properties.

PubMed

Villaverde, Marcela S; Verstraeten, Sandra V

2003-09-15

The hypothesis that thallium (Tl) interaction with membrane phospholipids could result in the alteration of membrane physical properties was investigated. Working with liposomes composed of brain phosphatidylcholine and phosphatidylserine, we found that Tl(+), Tl(3+), and Tl(OH)(3) (0.5-25 microM): (a) increased membrane surface potential, (b) decreased the fluidity of the anionic regions of the membrane, in association with an increased fluidity in the cationic regions, and (c) promoted the rearrangement of lipids through lateral phase separation. The magnitude of these effects followed the order Tl(3+), Tl(OH)(3)>Tl(+). In addition, Tl(3+) also decreased the hydration of phospholipid polar headgroups and induced membrane permeabilization. The present results show that Tl interacts with membranes inducing major alterations in the rheology of the bilayer, which could be partially responsible for the neurotoxic effects of this metal.

Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction.

PubMed

Heuser, J E; Reese, T S

1973-05-01

When the nerves of isolated frog sartorius muscles were stimulated at 10 Hz, synaptic vesicles in the motor nerve terminals became transiently depleted. This depletion apparently resulted from a redistribution rather than disappearance of synaptic vesicle membrane, since the total amount of membrane comprising these nerve terminals remained constant during stimulation. At 1 min of stimulation, the 30% depletion in synaptic vesicle membrane was nearly balanced by an increase in plasma membrane, suggesting that vesicle membrane rapidly moved to the surface as it might if vesicles released their content of transmitter by exocytosis. After 15 min of stimulation, the 60% depletion of synaptic vesicle membrane was largely balanced by the appearance of numerous irregular membrane-walled cisternae inside the terminals, suggesting that vesicle membrane was retrieved from the surface as cisternae. When muscles were rested after 15 min of stimulation, cisternae disappeared and synaptic vesicles reappeared, suggesting that cisternae divided to form new synaptic vesicles so that the original vesicle membrane was now recycled into new synaptic vesicles. When muscles were soaked in horseradish peroxidase (HRP), this tracerfirst entered the cisternae which formed during stimulation and then entered a large proportion of the synaptic vesicles which reappeared during rest, strengthening the idea that synaptic vesicle membrane added to the surface was retrieved as cisternae which subsequently divided to form new vesicles. When muscles containing HRP in synaptic vesicles were washed to remove extracellular HRP and restimulated, HRP disappeared from vesicles without appearing in the new cisternae formed during the second stimulation, confirming that a one-way recycling of synaptic membrane, from the surface through cisternae to new vesicles, was occurring. Coated vesicles apparently represented the actual mechanism for retrieval of synaptic vesicle membrane from the plasma membrane, because during nerve stimulation they proliferated at regions of the nerve terminals covered by Schwann processes, took up peroxidase, and appeared in various stages of coalescence with cisternae. In contrast, synaptic vesicles did not appear to return directly from the surface to form cisternae, and cisternae themselves never appeared directly connected to the surface. Thus, during stimulation the intracellular compartments of this synapse change shape and take up extracellular protein in a manner which indicates that synaptic vesicle membrane added to the surface during exocytosis is retrieved by coated vesicles and recycled into new synaptic vesicles by way of intermediate cisternae.

EVIDENCE FOR RECYCLING OF SYNAPTIC VESICLE MEMBRANE DURING TRANSMITTER RELEASE AT THE FROG NEUROMUSCULAR JUNCTION

PubMed Central

Heuser, J. E.; Reese, T. S.

1973-01-01

When the nerves of isolated frog sartorius muscles were stimulated at 10 Hz, synaptic vesicles in the motor nerve terminals became transiently depleted. This depletion apparently resulted from a redistribution rather than disappearance of synaptic vesicle membrane, since the total amount of membrane comprising these nerve terminals remained constant during stimulation. At 1 min of stimulation, the 30% depletion in synaptic vesicle membrane was nearly balanced by an increase in plasma membrane, suggesting that vesicle membrane rapidly moved to the surface as it might if vesicles released their content of transmitter by exocytosis. After 15 min of stimulation, the 60% depletion of synaptic vesicle membrane was largely balanced by the appearance of numerous irregular membrane-walled cisternae inside the terminals, suggesting that vesicle membrane was retrieved from the surface as cisternae. When muscles were rested after 15 min of stimulation, cisternae disappeared and synaptic vesicles reappeared, suggesting that cisternae divided to form new synaptic vesicles so that the original vesicle membrane was now recycled into new synaptic vesicles. When muscles were soaked in horseradish peroxidase (HRP), this tracerfirst entered the cisternae which formed during stimulation and then entered a large proportion of the synaptic vesicles which reappeared during rest, strengthening the idea that synaptic vesicle membrane added to the surface was retrieved as cisternae which subsequently divided to form new vesicles. When muscles containing HRP in synaptic vesicles were washed to remove extracellular HRP and restimulated, HRP disappeared from vesicles without appearing in the new cisternae formed during the second stimulation, confirming that a one-way recycling of synaptic membrane, from the surface through cisternae to new vesicles, was occurring. Coated vesicles apparently represented the actual mechanism for retrieval of synaptic vesicle membrane from the plasma membrane, because during nerve stimulation they proliferated at regions of the nerve terminals covered by Schwann processes, took up peroxidase, and appeared in various stages of coalescence with cisternae. In contrast, synaptic vesicles did not appear to return directly from the surface to form cisternae, and cisternae themselves never appeared directly connected to the surface. Thus, during stimulation the intracellular compartments of this synapse change shape and take up extracellular protein in a manner which indicates that synaptic vesicle membrane added to the surface during exocytosis is retrieved by coated vesicles and recycled into new synaptic vesicles by way of intermediate cisternae. PMID:4348786

Biochemical characterization and membrane fluidity of membranous vesicles isolated from boar seminal plasma.

PubMed

Piehl, Lidia L; Cisale, Humberto; Torres, Natalia; Capani, Francisco; Sterin-Speziale, Norma; Hager, Alfredo

2006-05-01

Mammalian seminal plasma contains membranous vesicles (MV), which differ in composition and origin. Among these particles, human prostasomes and equine prostasome-like MV have been the most studied. The aim of the present work is to characterize the biochemical composition and membrane fluidity of MV isolated from boar seminal plasma. The MV from boar seminal plasma were isolated by ultracentrifugation and further purification by gel filtration on Sephadex G-200. The MV were examined by electron microscopy (EM), amount of cholesterol, total phospholipid, protein content, and phospholipid composition were analyzed. Membrane fluidity of MV and spermatozoa were estimated from the electron spin resonance (ESR) spectra of the 5-doxilstearic acid incorporated into the vesicle membranes by the order parameter (S). The S parameter gives a measure of degree of structural order in the membrane and is defined as the ratio of the spectral anisotropy in the membranes to the maximum anisotropy obtained in a rigidly oriented system. The S parameter takes into consideration that S = 1 for a rapid spin-label motion of about only one axis and S = 0 for a rapid isotropic motion. Intermediate S values between S = 0 and S = 1 represents the consequence of decreased membrane fluidity. The EM revealed the presence of bilaminar and multilaminar electron-dense vesicles. Cholesterol to phospholipid molar ratio from the isolated MV was 1.8. Phospholipid composition showed a predominance of sphingomyelin. The S parameter for porcine MV and for boar spermatozoa was 0.73 +/- 0.02 and 0.644 +/- 0.008, respectively, with the S for MV being greater (p < 0.001) than the S for spermatozoa. The high order for S found for boar MV was in agreement with the greater cholesterol/phospholipids ratio and the lesser ratio for phosphatidylcholine/sphingomyelin. Results obtained in the present work indicate that MV isolated from boar semen share many biochemical and morphological characteristics with equine prostasome-like MV and human prostasomes. The characteristics of the porcine MV of the seminal plasma, however, differed from those of boar sperm plasma membranes.

The protective effect of a 17°C holding time on boar sperm plasma membrane fluidity after exposure to 5°C.

PubMed

Casas, I; Althouse, G C

2013-02-01

The holding time (HT) is the period during which an ejaculate, either in a raw or diluted state, is held at 17°C before further processing for cold-storage. In boars, the HT positively influences select sperm quality parameters of semen cooled from 15 to 5°C, a range in temperature during which plasma membrane remodeling occurs. Objective insight into the effect of HT on plasma membrane organization remains unknown. Therefore, the present work sought to elucidate if HT contributes to minimizing alterations in boar sperm plasma membrane fluidity at the initial step of the cooling process in a cryopreservation practice (holding at 5°C) and in relation with select sperm quality parameters. Nineteen ejaculates from five boars were collected and processed according to different treatments: T1) Fresh diluted semen, 0h at 17°C; T2) Fresh diluted semen, 24h at 17°C (HT); T3) Sperm from T1 in a lactose-egg yolk (LEY) extender, 3h at 5°C; T4) Sperm from T2 in LEY, 3h at 5°C; T5) Sperm from T1 in LEY, 24h at 5°C; T6) Sperm from T2 in LEY, 24h at 5°C. Sperm motility was assessed using CASA, and sperm plasma membrane integrity and fluidity were evaluated by flow cytometry with dual labeling (M540/YO-PRO®-1). Results demonstrated that the lack of exposure to a HT (T5) results in reduced sample motility compared to those having a HT (T6), with sperm exposed to HT exhibiting less plasma membrane fluidity. Collectively, these results provide empirical evidence that incorporation of a HT in semen processing protects boar sperm against cold injury through maintenance of lipid architecture of the plasma membrane. Copyright © 2012 Elsevier Inc. All rights reserved.

Electron paramagnetic resonance studies of the membrane fluidity of the foodborne pathogenic psychrotroph Listeria monocytogenes.

PubMed

Edgcomb, M R; Sirimanne, S; Wilkinson, B J; Drouin, P; Morse, R D

2000-01-15

Listeria monocytogenes is a foodborne psychrotrophic pathogen that grows at refrigeration temperatures. Previous studies of fatty acid profiles of wild-type and cold-sensitive, branched-chain fatty acid deficient mutants of L. monocytogenes suggest that the fatty acid 12-methyltetradecanoic (anteiso-C(15:0)) plays a critical role in low-temperature growth of L. monocytogenes, presumably by maintaining membrane fluidity. The fluidity of isolated cytoplasmic membranes of wild-type (SLCC53 and 10403S), and a cold-sensitive mutant (cld-1) of L. monocytogenes, grown with and without the supplementation of 2-methylbutyric acid, has been studied using a panel of hydrocarbon-based nitroxides (2N10, 3N10, 4N10, and 5N10) and spectral deconvolution and simulation methods to obtain directly the Lorentzian line widths and hence rotational correlation times (tau(c)) and motional anisotropies of the nitroxides in the fast motional region. tau(c) values over the temperature range of -7 degrees C to 50 degrees C were similar for the membranes of strains SLCC53 and 10403S grown at 10 degrees C and 30 degrees C, and for strain cld-1 grown with 2-methylbutyric acid supplementation (which restores branched-chain fatty acids) at 30 degrees C. However, strain cld-1 exhibited a threefold higher tau(c) when grown without 2-methylbutyric acid supplementation (deficient in branched-chain fatty acids) compared to strains SLCC53, 10403S, and supplemented cld-1. No evidence was seen for a clear lipid phase transition in any sample. We conclude that the fatty acid anteiso-C(15:0) imparts an essential fluidity to the L. monocytogenes membrane that permits growth at refrigeration temperatures.

Interrelationships between Fatty Acid Composition, Staphyloxanthin Content, Fluidity, and Carbon Flow in the Staphylococcus aureus Membrane.

PubMed

Tiwari, Kiran B; Gatto, Craig; Wilkinson, Brian J

2018-05-17

Fatty acids play a major role in determining membrane biophysical properties. Staphylococcus aureus produces branched-chain fatty acids (BCFAs) and straight-chain saturated fatty acids (SCSFAs), and can directly incorporate exogenous SCSFAs and straight-chain unsaturated fatty acids (SCUFAs). Many S. aureus strains produce the triterpenoid pigment staphyloxanthin, and the balance of BCFAs, SCSFAs and staphyloxanthin determines membrane fluidity. Here, we investigated the relationship of fatty acid and carotenoid production in S. aureus using a pigmented strain (Pig1), its carotenoid-deficient mutant (Pig1Δ crtM ) and the naturally non-pigmented Staphylococcus argenteus that lacks carotenoid biosynthesis genes and is closely related to S. aureus . Fatty acid compositions in all strains were similar under a given culture condition indicating that staphyloxanthin does not influence fatty acid composition. Strain Pig1 had decreased membrane fluidity as measured by fluorescence anisotropy compared to the other strains under all conditions indicating that staphyloxanthin helps maintain membrane rigidity. We could find no evidence for correlation of expression of crtM and fatty acid biosynthesis genes. Supplementation of medium with glucose increased SCSFA production and decreased BCFA and staphyloxanthin production, whereas acetate-supplementation also decreased BCFAs but increased staphyloxanthin production. We believe that staphyloxanthin levels are influenced more through metabolic regulation than responding to fatty acids incorporated into the membrane.

Lipid engineering reveals regulatory roles for membrane fluidity in yeast flocculation and oxygen-limited growth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Degreif, Daniel; de Rond, Tristan; Bertl, Adam

Cells modulate lipid metabolism in order to maintain membrane homeostasis. In this paper, we use a metabolic engineering approach to manipulate the stoichiometry of fatty acid unsaturation, a regulator of cell membrane fluidity, in Saccharomyces cerevisiae. Unexpectedly, reduced lipid unsaturation triggered cell-cell adhesion (flocculation), a phenomenon characteristic of industrial yeast but uncommon in laboratory strains. We find that ER lipid saturation sensors induce expression of FLO1 – encoding a cell wall polysaccharide binding protein – independently of its canonical regulator. In wild-type cells, Flo1p-dependent flocculation occurs under oxygen-limited growth, which reduces unsaturated lipid synthesis and thus serves as the environmentalmore » trigger for flocculation. Transcriptional analysis shows that FLO1 is one of the most highly induced genes in response to changes in lipid unsaturation, and that the set of membrane fluidity-sensitive genes is globally activated as part of the cell's long-term response to hypoxia during fermentation. Finally, our results show how the lipid homeostasis machinery of budding yeast is adapted to carry out a broad response to an environmental stimulus important in biotechnology.« less

Lipid engineering reveals regulatory roles for membrane fluidity in yeast flocculation and oxygen-limited growth.

PubMed

Degreif, Daniel; de Rond, Tristan; Bertl, Adam; Keasling, Jay D; Budin, Itay

2017-05-01

Cells modulate lipid metabolism in order to maintain membrane homeostasis. Here we use a metabolic engineering approach to manipulate the stoichiometry of fatty acid unsaturation, a regulator of cell membrane fluidity, in Saccharomyces cerevisiae. Unexpectedly, reduced lipid unsaturation triggered cell-cell adhesion (flocculation), a phenomenon characteristic of industrial yeast but uncommon in laboratory strains. We find that ER lipid saturation sensors induce expression of FLO1 - encoding a cell wall polysaccharide binding protein - independently of its canonical regulator. In wild-type cells, Flo1p-dependent flocculation occurs under oxygen-limited growth, which reduces unsaturated lipid synthesis and thus serves as the environmental trigger for flocculation. Transcriptional analysis shows that FLO1 is one of the most highly induced genes in response to changes in lipid unsaturation, and that the set of membrane fluidity-sensitive genes is globally activated as part of the cell's long-term response to hypoxia during fermentation. Our results show how the lipid homeostasis machinery of budding yeast is adapted to carry out a broad response to an environmental stimulus important in biotechnology. Copyright © 2017 International Metabolic Engineering Society. All rights reserved.

The effect of MLS laser radiation on cell lipid membrane.

PubMed

Pasternak, Kamila; Wróbel, Dominika; Nowacka, Olga; Pieszyński, Ireneusz; Bryszewska, Maria; Kujawa, Jolanta

2018-03-14

Authors of numerous publications have proved the therapeutic effect of laser irradiation on biological material, but the mechanisms at cellular and subcellular level are not yet well understood. The aim of this study was to assess the effect of laser radiation emitted by the MLS M1 system (Multiwave Locked System) at two wavelengths (808 nm continuous and 905 nm pulsed) on the stability and fluidity of liposomes with a lipid composition similar to that of human erythrocyte membrane or made of phosphatidylocholine. Liposomes were exposed to low-energy laser radiation at surface densities 195 mW/cm² (frequency 1,000 Hz) and 230 mW/cm² (frequency 2,000 Hz). Different doses of radiation energy in the range 0-15 J were applied. The surface energy density was within the range 0.46 - 4.9 J/cm ². The fluidity and stability of liposomes subjected to such irradiation changed depending on the parameters of radiation used. Since MLS M1 laser radiation, depending on the parameters used, affects fluidity and stability of liposomes with the lipid content similar to erythrocyte membrane, it may also cause structural and functional changes in cell membranes.

Changing rooster sperm membranes to facilitate cryopreservation

USDA-ARS?s Scientific Manuscript database

Cryopreservation damages rooster sperm membranes. Part of this damage is due to membrane transitioning from the fluid to the gel state as temperature is reduced. This damage may be prevented by increasing membrane fluidity at low temperatures by incorporating cholesterol or unsaturated lipids into t...

Preparation of synaptic plasma membrane and postsynaptic density proteins using a discontinuous sucrose gradient.

PubMed

Bermejo, Marie Kristel; Milenkovic, Marija; Salahpour, Ali; Ramsey, Amy J

2014-09-03

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in the late 1960's, proteins associated with the synaptic plasma membrane can be isolated by ultracentrifugation on a sucrose density gradient. Once synaptic membranes are isolated, the macromolecular complex known as the post-synaptic density can be subsequently isolated due to its detergent insolubility. The techniques used to isolate synaptic plasma membranes and post-synaptic density proteins remain essentially the same after 40 years, and are widely used in current neuroscience research. This article details the fractionation of proteins associated with the synaptic plasma membrane and post-synaptic density using a discontinuous sucrose gradient. Resulting protein preparations are suitable for western blotting or 2D DIGE analysis.

Characterization of phospholipids in insulin secretory granules and mitochondria in pancreatic beta cells and their changes with glucose stimulation.

PubMed

MacDonald, Michael J; Ade, Lacmbouh; Ntambi, James M; Ansari, Israr-Ul H; Stoker, Scott W

2015-04-24

The lipid composition of insulin secretory granules (ISG) has never previously been thoroughly characterized. We characterized the phospholipid composition of ISG and mitochondria in pancreatic beta cells without and with glucose stimulation. The phospholipid/protein ratios of most phospholipids containing unsaturated fatty acids were higher in ISG than in whole cells and in mitochondria. The concentrations of negatively charged phospholipids, phosphatidylserine, and phosphatidylinositol in ISG were 5-fold higher than in the whole cell. In ISG phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin, fatty acids 12:0 and 14:0 were high, as were phosphatidylserine and phosphatidylinositol containing 18-carbon unsaturated FA. With glucose stimulation, the concentration of many ISG phosphatidylserines and phosphatidylinositols increased; unsaturated fatty acids in phosphatidylserine increased; and most phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophosphatidylcholines were unchanged. Unsaturation and shorter fatty acid length in phospholipids facilitate curvature and fluidity of membranes, which favors fusion of membranes. Recent evidence suggests that negatively charged phospholipids, such as phosphatidylserine, act as coupling factors enhancing the interaction of positively charged regions in SNARE proteins in synaptic or secretory vesicle membrane lipid bilayers with positively charged regions in SNARE proteins in the plasma membrane lipid bilayer to facilitate docking of vesicles to the plasma membrane during exocytosis. The results indicate that ISG phospholipids are in a dynamic state and are consistent with the idea that changes in ISG phospholipids facilitate fusion of ISG with the plasma membrane-enhancing glucose-stimulated insulin exocytosis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Increased Isoprenoid Quinone Concentration Modulates Membrane Fluidity in Listeria monocytogenes at Low Growth Temperatures.

PubMed

Seel, Waldemar; Flegler, Alexander; Zunabovic-Pichler, Marija; Lipski, André

2018-07-01

Listeria monocytogenes is a food pathogen capable of growing at a broad temperature range from 50°C to refrigerator temperatures. A key requirement for bacterial activity and growth at low temperatures is the ability to adjust the membrane lipid composition to maintain cytoplasmic membrane fluidity. In this study, we confirmed earlier findings that the extents of fatty acid profile adaptation differed between L. monocytogenes strains. We were able to demonstrate for isolates from food that growth rates at low temperatures and resistance to freeze-thaw stress were not impaired by a lower adaptive response of the fatty acid composition. This indicated the presence of a second adaptation mechanism besides temperature-regulated fatty acid synthesis. For strains that showed weaker adaptive responses in their fatty acid profiles to low growth temperature, we could demonstrate a significantly higher concentration of isoprenoid quinones. Three strains even showed a higher quinone concentration after growth at 6°C than at 37°C, which is contradictory to the reduced respiratory activity at lower growth temperatures. Analyses of the membrane fluidity in vivo by measuring generalized polarization and anisotropy revealed modulation of the transition phase. Strains with increased quinone concentrations showed an expanded membrane transition phase in contrast to strains with pronounced adaptations of fatty acid profiles. The correlation between quinone concentration and membrane transition phase expansion was confirmed by suppression of quinone synthesis. A reduced quinone concentration resulted in a narrower transition phase. Expansion of the phase transition zone by increasing the concentration of non-fatty acid membrane lipids is discussed as an additional mechanism improving adaptation to temperature shifts for L. monocytogenes strains. IMPORTANCE Listeria monocytogenes is a foodborne pathogen with an outstanding temperature range for growth. The ability for growth at temperatures close to the freezing point constitutes a serious contamination potential for cold stored food. The only known mechanism of the species for adaptation of membrane fluidity is modification of the membrane fatty acid composition. We were able to demonstrate that, at least for some strains, this adaptation mechanism is supported by regulation of the menaquinone concentration. The increase of this neutral membrane lipid is correlated with fluidization of the membrane under low-temperature conditions and therefore represents a fatty acid-independent mechanism for adaptation to low temperatures. Copyright © 2018 American Society for Microbiology.

Membrane Fluidity Changes, A Basic Mechanism of Interaction of Gravity with Cells?

NASA Astrophysics Data System (ADS)

Kohn, Florian; Hauslage, Jens; Hanke, Wolfgang

2017-10-01

All life on earth has been established under conditions of stable gravity of 1g. Nevertheless, in numerous experiments the direct gravity dependence of biological processes has been shown on all levels of organization, from single molecules to humans. According to the underlying mechanisms a variety of questions, especially about gravity sensation of single cells without specialized organelles or structures for gravity sensing is being still open. Biological cell membranes are complex structures containing mainly lipids and proteins. Functional aspects of such membranes are usually attributed to membrane integral proteins. This is also correct for the gravity dependence of cells and organisms which is well accepted since long for a wide range of biological systems. However, it is as well established that parameters of the lipid matrix are directly modifying the function of proteins. Thus, the question must be asked, whether, and how far plain lipid membranes are affected by gravity directly. In principle it can be said that up to recently no real basic mechanism for gravity perception in single cells has been presented or verified. However, it now has been shown that as a basic membrane parameter, membrane fluidity, is significantly dependent on gravity. This finding might deliver a real basic mechanism for gravity perception of living organisms on all scales. In this review we summarize older and more recent results to demonstrate that the finding of membrane fluidity being gravity dependent is consistent with a variety of published laboratory experiments. We additionally point out to the consequences of these recent results for research in the field life science under space condition.

Photopolymerization of Dienoyl Lipids Creates Planar Supported Poly(lipid) Membranes with Retained Fluidity.

PubMed

Orosz, Kristina S; Jones, Ian W; Keogh, John P; Smith, Christopher M; Griffin, Kaitlyn R; Xu, Juhua; Comi, Troy J; Hall, H K; Saavedra, S Scott

2016-02-16

Polymerization of substrate-supported bilayers composed of dienoylphosphatidylcholine (PC) lipids is known to greatly enhance their chemical and mechanical stability; however, the effects of polymerization on membrane fluidity have not been investigated. Here planar supported lipid bilayers (PSLBs) composed of dienoyl PCs on glass substrates were examined to assess the degree to which UV-initiated polymerization affects lateral lipid mobility. Fluorescence recovery after photobleaching (FRAP) was used to measure the diffusion coefficients (D) and mobile fractions of rhodamine-DOPE in unpolymerized and polymerized PSLBs composed of bis-sorbyl phosphatidylcholine (bis-SorbPC), mono-sorbyl-phosphatidylcholine (mono-SorbPC), bis-dienoyl-phosphatidylcholine (bis-DenPC), and mono-dienoyl phosphatidylcholine (mono-DenPC). Polymerization was performed in both the Lα and Lβ phase for each lipid. In all cases, polymerization reduced membrane fluidity; however, measurable lateral diffusion was retained which is attributed to a low degree of polymerization. The D values for sorbyl lipids were less than those of the denoyl lipids; this may be a consequence of the distal location of polymerizable group in the sorbyl lipids which may facilitate interleaflet bonding. The D values measured after polymerization were 0.1-0.8 of those measured before polymerization, a range that corresponds to fluidity intermediate between that of a Lα phase and a Lβ phase. This D range is comparable to ratios of D values reported for liquid-disordered (Ld) and liquid-ordered (Lo) lipid phases and indicates that the effect of UV polymerization on lateral diffusion in a dienoyl PSLB is similar to the transition from a Ld phase to a Lo phase. The partial retention of fluidity in UV-polymerized PSLBs, their enhanced stability, and the activity of incorporated transmembrane proteins and peptides is discussed.

Photopolymerization of dienoyl lipids creates planar supported poly(lipid) membranes with retained fluidity

PubMed Central

Orosz, Kristina S.; Jones, Ian W.; Keogh, John P.; Smith, Christopher M.; Griffin, Kaitlyn R.; Xu, Juhua; Comi, Troy J.; Hall, H. K.

2016-01-01

Polymerization of substrate-supported bilayers composed of dienoyl phosphatidylcholine (PC) lipids is known to greatly enhance their chemical and mechanical stability, however the effects of polymerization on membrane fluidity have not been investigated. Here planar supported lipid bilayers (PSLBs) composed of dienoyl PCs on glass substrates were examined to assess the degree to which UV-initiated polymerization affects lateral lipid mobility. Fluorescence recovery after photobleaching (FRAP) was used to measure the diffusion coefficients (D) and mobile fractions of rhodamine-DOPE in unpolymerized and polymerized PSLBs composed of bis-sorbyl phosphatidylcholine (bis-SorbPC), mono-sorbyl phosphatidylcholine (mono-SorbPC), bis-dienoyl phosphatidylcholine (bis-DenPC) and mono-dienoyl phosphatidylcholine (mono-DenPC). Polymerization was performed in both the Lα and Lβ phase for each lipid. In all cases, polymerization reduced membrane fluidity, however measurable lateral diffusion was retained which is attributed to a low degree of polymerization. The D values for sorbyl lipids were less than those of the denoyl lipids; this may be a consequence of the distal location of polymerizable group in the sorbyl lipids which may facilitate inter-leaflet bonding. The D values measured after polymerization were 0.1 to 0.8 of those measured before polymerization, a range that corresponds to fluidity intermediate between that of a Lα phase and a Lβ phase. This D range is comparable to ratios of D values reported for liquid-disordered (Ld) and liquid-ordered (Lo) lipid phases, and indicates that the effect of UV polymerization on lateral diffusion in a dienoyl PSLB is similar to the transition from a Ld phase to a Lo phase. The partial retention of fluidity in UV polymerized PSLBs, their enhanced stability, and the activity of incorporated transmembrane proteins and peptides is discussed. PMID:26794208

Dynamics of sperm subpopulations based on motility and plasma membrane status in thawed ram spermatozoa incubated under conditions that support in vitro capacitation and fertilisation.

PubMed

García-Álvarez, Olga; Maroto-Morales, Alejandro; Ramón, Manuel; del Olmo, Enrique; Jiménez-Rabadán, Pilar; Fernández-Santos, M Rocio; Anel-López, Luis; Garde, J Julián; Soler, Ana J

2014-06-01

The present study evaluated modifications occurring in thawed ram spermatozoa during incubation in different media that supported in vitro capacitation and fertilisation, and examines how these changes relate to IVF. Thawed sperm samples were incubated under capacitating (Cap) and non-capacitating (non-Cap) conditions for 0, 1 and 2h and used in an IVF test. During incubation, changes related to membrane status and the motility pattern of spermatozoa were assessed, the latter being used to characterise sperm subpopulations. A significantly greater increase (P≤0.05) in the percentage of spermatozoa with higher membrane fluidity was observed in samples incubated with Cap medium from the beginning of incubation. In addition, changes over time in the distribution of the motile subpopulation were particularly evident when spermatozoa were incubated with Cap medium, with a noted increase in spermatozoa classified as 'hyperactivated like', with major changes occurring after 1h incubation. Both characteristics (i.e. membrane fluidity and the percentage of the hyperactivated-like subpopulation) were significantly related with in vitro fertility, and only sperm samples incubated with the Cap medium were capable of fertilising oocytes. These results support the idea that changes in sperm membrane fluidity and motility pattern (i.e. an increase in hyperactivated spermatozoa) are needed for fertilisation to take place.

Pancreatic β-Cell Membrane Fluidity and Toxicity Induced by Human Islet Amyloid Polypeptide Species

NASA Astrophysics Data System (ADS)

Pilkington, Emily H.; Gurzov, Esteban N.; Kakinen, Aleksandr; Litwak, Sara A.; Stanley, William J.; Davis, Thomas P.; Ke, Pu Chun

2016-02-01

Aggregation of human islet amyloid polypeptide (hIAPP) into fibrils and plaques is associated with pancreatic β-cell loss in type 2 diabetes (T2D). However, due to the rapidness of hIAPP conversion in aqueous phase, exactly which hIAPP species is responsible for the observed toxicity and through what mechanisms remains ambiguous. In light of the importance of understanding hIAPP toxicity for T2D here we show a biophysical scheme based on the use of a lipophilic Laurdan dye for examining MIN6 cell membranes upon exposure to fresh and oligomeric hIAPP as well as mature amyloid. It has been found that all three hIAPP species, especially fresh hIAPP, enhanced membrane fluidity and caused losses in cell viability. The cell generation of reactive oxygen species (ROS), however, was the most pronounced with mature amyloid hIAPP. The correlation between changes in membrane fluidity and cell viability and their lack of correlation with ROS production suggest hIAPP toxicity is elicited through both physical and biochemical means. This study offers a new insight into β-cell toxicity induced by controlled hIAPP species, as well as new biophysical methodologies that may prove beneficial for the studies of T2D as well as neurological disorders.

Slow Freezing Coupled Static Magnetic Field Exposure Enhances Cryopreservative Efficiency—A Study on Human Erythrocytes

PubMed Central

Lin, Chun-Yen; Wei, Po-Li; Chang, Wei-Jen; Huang, Yung-Kai; Feng, Sheng-Wei; Lin, Che-Tong; Lee, Sheng-Yang; Huang, Haw-Ming

2013-01-01

The aim of this study was to assess the cryoprotective effect of static magnetic fields (SMFs) on human erythrocytes during the slow cooling procedure. Human erythrocytes suspended in 20% glycerol were slowly frozen with a 0.4-T or 0.8-T SMF and then moved to a −80°C freezer for 24 hr. The changes in survival rate, morphology, and metabolites of the thawed erythrocytes were examined. To understand possible cryoprotective mechanisms of SMF, membrane fluidity and dehydration stability of SMF-exposed erythrocytes were tested. For each test, sham-exposed erythrocytes were used as controls. Our results showed that freezing coupled with 0.4-T or 0.8-T SMFs significantly increased the relative survival ratios of the frozen-thawed erythrocytes by 10% and 20% (p<0.001), respectively. The SMFs had no effect on erythrocyte morphology and metabolite levels. However, membrane fluidity of the samples exposed to 0.8-T SMF decreased significantly (p<0.05) in the hydrophobic regions. For the dehydration stability experiments, the samples exposed to 0.8-T SMF exhibited significantly lower (p<0.05) hemolysis. These results demonstrate that a 0.8-T SMF decreases membrane fluidity and enhances erythrocyte membrane stability to resist dehydration damage caused by slow cooling procedures. PMID:23520546

Using spin-label W-band EPR to study membrane fluidity profiles in samples of small volume

NASA Astrophysics Data System (ADS)

Mainali, Laxman; Hyde, James S.; Subczynski, Witold K.

2013-01-01

Conventional and saturation-recovery (SR) EPR at W-band (94 GHz) using phosphatidylcholine spin labels (labeled at the alkyl chain [n-PC] and headgroup [T-PC]) to obtain profiles of membrane fluidity has been demonstrated. Dimyristoylphosphatidylcholine (DMPC) membranes with and without 50 mol% cholesterol have been studied, and the results have been compared with similar studies at X-band (9.4 GHz) (L. Mainali, J.B. Feix, J.S. Hyde, W.K. Subczynski, J. Magn. Reson. 212 (2011) 418-425). Profiles of the spin-lattice relaxation rate (T1-1) obtained from SR EPR measurements for n-PCs and T-PC were used as a convenient quantitative measure of membrane fluidity. Additionally, spectral analysis using Freed's MOMD (microscopic-order macroscopic-disorder) model (E. Meirovitch, J.H. Freed J. Phys. Chem. 88 (1984) 4995-5004) provided rotational diffusion coefficients (R⊥ and R||) and order parameters (S0). Spectral analysis at X-band provided one rotational diffusion coefficient, R⊥. T1-1, R⊥, and R|| profiles reflect local membrane dynamics of the lipid alkyl chain, while the order parameter shows only the amplitude of the wobbling motion of the lipid alkyl chain. Using these dynamic parameters, namely T1-1, R⊥, and R||, one can discriminate the different effects of cholesterol at different depths, showing that cholesterol has a rigidifying effect on alkyl chains to the depth occupied by the rigid steroid ring structure and a fluidizing effect at deeper locations. The nondynamic parameter, S0, shows that cholesterol has an ordering effect on alkyl chains at all depths. Conventional and SR EPR measurements with T-PC indicate that cholesterol has a fluidizing effect on phospholipid headgroups. EPR at W-band provides more detailed information about the depth-dependent dynamic organization of the membrane compared with information obtained at X-band. EPR at W-band has the potential to be a powerful tool for studying membrane fluidity in samples of small volume, ˜30 nL, compared with a representative sample volume of ˜3 μL at X-band.

Influence of selenium and fluoride on blood antioxidant capacity of rats.

PubMed

Feng, Pei; Wei, Jun-ren; Zhang, Zi-gui

2012-09-01

This study is to explore the effect of selenium and fluoride on blood antioxidant capacity of rats, and try to find out the optimal level of selenium in drinking water against fluorosis. Animals were divided into control group, sodium fluoride treated group (NaF, 50 mg/L) and selenium+NaF treated group (sodium selenite 0.375, 0.75, 1.5 mg/L) in water were respectively administered to male rats, which were decapitated after 6 months. Their blood was collected for GSH-Px activity, plasma SOD activity, T-AOC assay, uric acid assay, sialic acid (SA) content and MDA content, and the fluidity of erythrocyte membrane by electron spin resonance (ESR) was analyzed. The results showed that, compared with the control group, the blood antioxidant capacity of the rats exposed to fluoride was down-regulated significantly (P<0.05, P<0.01), MDA content increased significantly (P<0.05), the fluidity of erythrocyte membrane decreased (P<0.05, P<0.01). Meanwhile, the treatments of selenium along with NaF compared with fluorosis group, SOD activity, GSH-Px activity and T-AOC assay increased respectively, MDA content decreased significantly (P<0.05) in NaF+Se (Se 0.75, 1.5 mg/L) treated groups, uric acid level was up-regulated, but had no statistical significant difference (P>0.05). The fluidity of erythrocyte membrane showed significant increase (P<0.05), the content of SA was lower. Fluorosis could induce the decline of blood antioxidant capacity and the fluidity of erythrocyte membrane, as evident in this study, and Se at different levels possess some antagonistic effects on blood induced by fluoride. However, high dose of selenium (1.5 mg/L) is the optimum concentration. Copyright © 2010 Elsevier GmbH. All rights reserved.

Structural Degradation and Swelling of Lipid Bilayer under the Action of Benzene.

PubMed

Odinokov, Alexey; Ostroumov, Denis

2015-12-03

Benzene and other nonpolar organic solvents can accumulate in the lipid bilayer of cellular membranes. Their effect on the membrane structure and fluidity determines their toxic properties and antibiotic action of the organic solvents on the bacteria. We performed molecular dynamics simulations of the interaction of benzene with the dimyristoylphosphatidylcholine (DMPC) bilayer. An increase in the membrane surface area and fluidity was clearly detected. Changes in the acyl chain ordering, tilt angle, and overall bilayer thickness were, however, much less marked. The dependence of all computed quantities on the benzene content showed two regimes separated by the solubility limit of benzene in water. When the amount of benzene exceeded this point, a layer of almost pure benzene started to grow between the membrane leaflets. This process corresponds to the nucleation of a new phase and provides a molecular mechanism for the mechanical rupture of the bilayer under the action of nonpolar compounds.

The effects of low level microwaves on the fluidity of photoreceptor cell membrane.

PubMed

Pologea-Moraru, Roxana; Kovacs, Eugenia; Iliescu, Karina Roxana; Calota, Violeta; Sajin, Gheorghe

2002-05-15

Due to the extensive use of electromagnetic fields in everyday life, more information is required for the detection of mechanisms of interaction and the possible side effects of electromagnetic radiation on the structure and function of the organism. In this paper, we study the effects of low-power microwaves (2.45 GHz) on the membrane fluidity of rod photoreceptor cells. The retina is expected to be very sensitive to microwave irradiation due to the polar character of the photoreceptor cells [Biochim. Biophys. Acta 1273 (1995) 217] as well as to its high water content [Stud. Biophys. 81 (1981) 39].

Plasma membrane order and fluidity are diversely triggered by elicitors of plant defence

PubMed Central

Sandor, Roman; Der, Christophe; Grosjean, Kevin; Anca, Iulia; Noirot, Elodie; Leborgne-Castel, Nathalie; Lochman, Jan; Simon-Plas, Françoise; Gerbeau-Pissot, Patricia

2016-01-01

Although plants are exposed to a great number of pathogens, they usually defend themselves by triggering mechanisms able to limit disease development. Alongside signalling events common to most such incompatible interactions, modifications of plasma membrane (PM) physical properties could be new players in the cell transduction cascade. Different pairs of elicitors (cryptogein, oligogalacturonides, and flagellin) and plant cells (tobacco and Arabidopsis) were used to address the issue of possible modifications of plant PM biophysical properties induced by elicitors and their links to other events of the defence signalling cascade. We observed an increase of PM order whatever the elicitor/plant cell pair used, provided that a signalling cascade was induced. Such membrane modification is dependent on the NADPH oxidase-mediated reactive oxygen species production. Moreover, cryptogein, which is the sole elicitor able to trap sterols, is also the only one able to trigger an increase in PM fluidity. The use of cryptogein variants with altered sterol-binding properties confirms the strong correlation between sterol removal from the PM and PM fluidity enhancement. These results propose PM dynamics as a player in early signalling processes triggered by elicitors of plant defence. PMID:27604805

You Sank My Lipid Rafts!

ERIC Educational Resources Information Center

Campbell, Tessa N.

2009-01-01

The plasma membrane is the membrane that serves as a boundary between the interior of a cell and its extracellular environment. Lipid rafts are microdomains within a cellular membrane that possess decreased fluidity due to the presence of cholesterol, glycolipids, and phospholipids containing longer fatty acids. These domains are involved in many…

Fluidization of Membrane Lipids Enhances the Tolerance of Saccharomyces cerevisiae to Freezing and Salt Stress▿

PubMed Central

Rodríguez-Vargas, Sonia; Sánchez-García, Alicia; Martínez-Rivas, Jose Manuel; Prieto, Jose Antonio; Randez-Gil, Francisca

2007-01-01

Unsaturated fatty acids play an essential role in the biophysical characteristics of cell membranes and determine the proper function of membrane-attached proteins. Thus, the ability of cells to alter the degree of unsaturation in their membranes is an important factor in cellular acclimatization to environmental conditions. Many eukaryotic organisms can synthesize dienoic fatty acids, but Saccharomyces cerevisiae can introduce only a single double bond at the Δ9 position. We expressed two sunflower (Helianthus annuus) oleate Δ12 desaturases encoded by FAD2-1 and FAD2-3 in yeast cells of the wild-type W303-1A strain (trp1) and analyzed their effects on growth and stress tolerance. Production of the heterologous desaturases increased the content of dienoic fatty acids, especially 18:2Δ9,12, the unsaturation index, and the fluidity of the yeast membrane. The total fatty acid content remained constant, and the level of monounsaturated fatty acids decreased. Growth at 15°C was reduced in the FAD2 strains, probably due to tryptophan auxotrophy, since the trp1 (TRP1) transformants that produced the sunflower desaturases grew as well as the control strain did. Our results suggest that changes in the fluidity of the lipid bilayer affect tryptophan uptake and/or the correct targeting of tryptophan transporters. The expression of the sunflower desaturases, in either Trp+ or Trp− strains, increased NaCl tolerance. Production of dienoic fatty acids increased the tolerance to freezing of wild-type cells preincubated at 30°C or 15°C. Thus, membrane fluidity is an essential determinant of stress resistance in S. cerevisiae, and engineering of membrane lipids has the potential to be a useful tool of increasing the tolerance to freezing in industrial strains. PMID:17071783

Higher lipophilic index indicates higher risk of coronary heart disease in postmenopausal women

USDA-ARS?s Scientific Manuscript database

Fatty acids are essential components of cell membranes and play an integral role in membrane fluidity. The lipophilic index (LI, defined as the sum of the products between fatty acid levels and melting points (degrees Celsius), divided by the total amount of fatty acids is thought to reflect membran...

Stochastic lattice model of synaptic membrane protein domains.

PubMed

Li, Yiwei; Kahraman, Osman; Haselwandter, Christoph A

2017-05-01

Neurotransmitter receptor molecules, concentrated in synaptic membrane domains along with scaffolds and other kinds of proteins, are crucial for signal transmission across chemical synapses. In common with other membrane protein domains, synaptic domains are characterized by low protein copy numbers and protein crowding, with rapid stochastic turnover of individual molecules. We study here in detail a stochastic lattice model of the receptor-scaffold reaction-diffusion dynamics at synaptic domains that was found previously to capture, at the mean-field level, the self-assembly, stability, and characteristic size of synaptic domains observed in experiments. We show that our stochastic lattice model yields quantitative agreement with mean-field models of nonlinear diffusion in crowded membranes. Through a combination of analytic and numerical solutions of the master equation governing the reaction dynamics at synaptic domains, together with kinetic Monte Carlo simulations, we find substantial discrepancies between mean-field and stochastic models for the reaction dynamics at synaptic domains. Based on the reaction and diffusion properties of synaptic receptors and scaffolds suggested by previous experiments and mean-field calculations, we show that the stochastic reaction-diffusion dynamics of synaptic receptors and scaffolds provide a simple physical mechanism for collective fluctuations in synaptic domains, the molecular turnover observed at synaptic domains, key features of the observed single-molecule trajectories, and spatial heterogeneity in the effective rates at which receptors and scaffolds are recycled at the cell membrane. Our work sheds light on the physical mechanisms and principles linking the collective properties of membrane protein domains to the stochastic dynamics that rule their molecular components.

Short branched-chain C6 carboxylic acids result in increased growth, novel 'unnatural' fatty acids and increased membrane fluidity in a Listeria monocytogenes branched-chain fatty acid-deficient mutant.

PubMed

Sen, Suranjana; Sirobhushanam, Sirisha; Hantak, Michael P; Lawrence, Peter; Brenna, J Thomas; Gatto, Craig; Wilkinson, Brian J

2015-10-01

Listeria monocytogenes is a psychrotolerant food borne pathogen, responsible for the high fatality disease listeriosis, and expensive food product recalls. Branched-chain fatty acids (BCFAs) of the membrane play a critical role in providing appropriate membrane fluidity and optimum membrane biophysics. The fatty acid composition of a BCFA-deficient mutant is characterized by high amounts of straight-chain fatty acids and even-numbered iso fatty acids, in contrast to the parent strain where odd-numbered anteiso fatty acids predominate. The presence of 2-methylbutyrate (C5) stimulated growth of the mutant at 37°C and restored growth at 10°C along with the content of odd-numbered anteiso fatty acids. The C6 branched-chain carboxylic acids 2-ethylbutyrate and 2-methylpentanoate also stimulated growth to a similar extent as 2-methylbutyrate. However, 3-methylpentanoate was ineffective in rescuing growth. 2-Ethylbutyrate and 2-methylpentanoate led to novel major fatty acids in the lipid profile of the membrane that were identified as 12-ethyltetradecanoic acid and 12-methylpentadecanoic acid respectively. Membrane anisotropy studies indicated that growth of strain MOR401 in the presence of these precursors increased its membrane fluidity to levels of the wild type. Cells supplemented with 2-methylpentanoate or 2-ethylbutyrate at 10°C shortened the chain length of novel fatty acids, thus showing homeoviscous adaptation. These experiments use the mutant as a tool to modulate the membrane fatty acid compositions through synthetic precursor supplementation, and show how existing enzymes in L. monocytogenes adapt to exhibit non-native activity yielding unique 'unnatural' fatty acid molecules, which nevertheless possess the correct biophysical properties for proper membrane function in the BCFA-deficient mutant. Copyright © 2015 Elsevier B.V. All rights reserved.

Properties of Plasma Membrane from Pea Root Seedlings under Altered Gravity

NASA Astrophysics Data System (ADS)

Klymchuk, D.; Baranenko, V.; Vorobyova, T. V.; Kurylenko, I.; Chyzhykova, O.; Dubovoy, V.

In this study, the properties of pea (Pisum sativum L.) plasma membrane were examined to determine how the membrane structure and functions are regulated in response to clinorotation (2 rev/min) conditions. Membrane preparations enriched by plasma membrane vesicles were obtained by aqueous two-phase partitioning from 6-day seedling roots. The specific characteristics of H^+-ATPase, lípid composition and peroxidation intensity as well as fluidity of lipid bilayer were analysed. ATP hydrolytic activity was inhibited by ortovanadate and was insensitive to aside and nitrate in sealed plasma membrane vesicles isolated from both clinorotated and control seedlings. Plasma membrane vesicles from clinorotated seedlings in comparison to controls were characterised by increase in the total lipid/protein ratio, ATP hydrolytic activity and intensifying of lipid peroxidation. Sitosterol and campesterol were the predominant free sterol species. Clinorotated seedlings contained a slightly higher level of unsaturated fatty acid than controls. Plasma membrane vesicles were labelled with pyrene and fluorescence originating from monomeric (I_M) molecules and excimeric (I_E) aggregates were measured. The calculated I_E/I_M values were higher in clinorotated seedlings compared with controls reflecting the reduction in membrane microviscosity. The involvement of the changes in plasma membrane lipid content and composition, fluidity and H^+-ATPase activity in response of pea seedlings to altered gravity is discussed.

Plasma membrane order and fluidity are diversely triggered by elicitors of plant defence.

PubMed

Sandor, Roman; Der, Christophe; Grosjean, Kevin; Anca, Iulia; Noirot, Elodie; Leborgne-Castel, Nathalie; Lochman, Jan; Simon-Plas, Françoise; Gerbeau-Pissot, Patricia

2016-09-01

Although plants are exposed to a great number of pathogens, they usually defend themselves by triggering mechanisms able to limit disease development. Alongside signalling events common to most such incompatible interactions, modifications of plasma membrane (PM) physical properties could be new players in the cell transduction cascade. Different pairs of elicitors (cryptogein, oligogalacturonides, and flagellin) and plant cells (tobacco and Arabidopsis) were used to address the issue of possible modifications of plant PM biophysical properties induced by elicitors and their links to other events of the defence signalling cascade. We observed an increase of PM order whatever the elicitor/plant cell pair used, provided that a signalling cascade was induced. Such membrane modification is dependent on the NADPH oxidase-mediated reactive oxygen species production. Moreover, cryptogein, which is the sole elicitor able to trap sterols, is also the only one able to trigger an increase in PM fluidity. The use of cryptogein variants with altered sterol-binding properties confirms the strong correlation between sterol removal from the PM and PM fluidity enhancement. These results propose PM dynamics as a player in early signalling processes triggered by elicitors of plant defence. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yi, Jae-Sung; Choo, Hyo-Jung; Cho, Bong-Rae

Lipid rafts are plasma membrane platforms mediating signal transduction pathways for cellular proliferation, differentiation and apoptosis. Here, we show that membrane fluidity was increased in HeLa cells following treatment with ginsenoside Rh2 (Rh2), as determined by cell staining with carboxy-laurdan (C-laurdan), a two-photon dye designed for measuring membrane hydrophobicity. In the presence of Rh2, caveolin-1 appeared in non-raft fractions after sucrose gradient ultracentrifugation. In addition, caveolin-1 and GM1, lipid raft landmarkers, were internalized within cells after exposure to Rh2, indicating that Rh2 might disrupt lipid rafts. Since cholesterol overloading, which fortifies lipid rafts, prevented an increase in Rh2-induced membrane fluidity,more » caveolin-1 internalization and apoptosis, lipid rafts appear to be essential for Rh2-induced apoptosis. Moreover, Rh2-induced Fas oligomerization was abolished following cholesterol overloading, and Rh2-induced apoptosis was inhibited following treatment with siRNA for Fas. This result suggests that Rh2 is a novel lipid raft disruptor leading to Fas oligomerization and apoptosis.« less

Growth-Environment Dependent Modulation of Staphylococcus aureus Branched-Chain to Straight-Chain Fatty Acid Ratio and Incorporation of Unsaturated Fatty Acids.

PubMed

Sen, Suranjana; Sirobhushanam, Sirisha; Johnson, Seth R; Song, Yang; Tefft, Ryan; Gatto, Craig; Wilkinson, Brian J

2016-01-01

The fatty acid composition of membrane glycerolipids is a major determinant of Staphylococcus aureus membrane biophysical properties that impacts key factors in cell physiology including susceptibility to membrane active antimicrobials, pathogenesis, and response to environmental stress. The fatty acids of S. aureus are considered to be a mixture of branched-chain fatty acids (BCFAs), which increase membrane fluidity, and straight-chain fatty acids (SCFAs) that decrease it. The balance of BCFAs and SCFAs in USA300 strain JE2 and strain SH1000 was affected considerably by differences in the conventional laboratory medium in which the strains were grown with media such as Mueller-Hinton broth and Luria broth resulting in high BCFAs and low SCFAs, whereas growth in Tryptic Soy Broth and Brain-Heart Infusion broth led to reduction in BCFAs and an increase in SCFAs. Straight-chain unsaturated fatty acids (SCUFAs) were not detected. However, when S. aureus was grown ex vivo in serum, the fatty acid composition was radically different with SCUFAs, which increase membrane fluidity, making up a substantial proportion of the total (<25%) with SCFAs (>37%) and BCFAs (>36%) making up the rest. Staphyloxanthin, an additional major membrane lipid component unique to S. aureus, tended to be greater in content in cells with high BCFAs or SCUFAs. Cells with high staphyloxanthin content had a lower membrane fluidity that was attributed to increased production of staphyloxanthin. S. aureus saves energy and carbon by utilizing host fatty acids for part of its total fatty acids when growing in serum, which may impact biophysical properties and pathogenesis given the role of SCUFAs in virulence. The nutritional environment in which S. aureus is grown in vitro or in vivo in an infection is likely to be a major determinant of membrane fatty acid composition.

ATP-induced lipid membrane reordering in the myelinated nerve fiber identified using Raman spectroscopy

NASA Astrophysics Data System (ADS)

Kutuzov, N. P.; Brazhe, A. R.; Yusipovich, A. I.; Maksimov, G. V.; Dracheva, O. E.; Lyaskovskiy, V. L.; Bulygin, F. V.; Rubin, A. B.

2013-07-01

We demonstrate a successful application of Raman spectroscopy to the problem of lipid ordering with microscopic resolution in different regions of the myelinated nerve fiber. Simultaneous collection of Raman spectra of lipids and carotenoids has enabled us to characterize membrane fluidity and the degree of lipid ordering based on intensity ratios for the 1527/1160 and 2940/2885 cm-1 bands. We show that the intensity profiles of the major Raman bands vary significantly between the three major regions of myelinated nerve fiber: internode, paranode and the node of Ranvier. Mapping Raman peak intensities over these areas suggested that the carotenoid molecules are localized in the myelin membranes of nerve cells. Paranodal membranes were sensitive to extracellular ATP. ATP solutions (7 mM) influenced the 1527/1160 and 2940/2885 cm-1 intensity ratios. Changes in both carotenoid and lipid Raman spectra were in accord and indicated an increase in lipid ordering degree and decrease in membrane fluidity under ATP administration. The collected data provide evidence for the existence of a regulatory purinergic signaling pathway in the peripheral nervous system.

Influence of lecithin-lipid composition on physico-chemical properties of nanoliposomes loaded with a hydrophobic molecule.

PubMed

Bouarab, Lynda; Maherani, Behnoush; Kheirolomoom, Azadeh; Hasan, Mahmoud; Aliakbarian, Bahar; Linder, Michel; Arab-Tehrany, Elmira

2014-03-01

In this work, we studied the effect of nanoliposome composition based on phospholipids of docosahexaenoic acid (PL-DHA), salmon and soya lecithin, on physico-chemical characterization of vector. Cinnamic acid was encapsulated as a hydrophobic molecule in nanoliposomes made of three different lipid sources. The aim was to evaluate the influence of membrane lipid structure and composition on entrapment efficiency and membrane permeability of cinnamic acid. These properties are important for active molecule delivery. In addition, size, electrophoretic mobility, phase transition temperature, elasticity and membrane fluidity were measured before and after encapsulation. The results showed a correlation between the size of the nanoliposome and the entrapment. The entrapment efficiency of cinnamic acid was found to be the highest in liposomes prepared from salmon lecithin. The nanoliposomes composed of salmon lecithin presented higher capabilities as a carrier for cinnamic acid encapsulation. These vesicles also showed a high stability which in turn increases the membrane rigidity of nanoliposome as evaluated by their elastic properties, membrane fluidity and phase transition temperature. Copyright © 2013 Elsevier B.V. All rights reserved.

Plasma membrane ordering agent pluronic F-68 (PF-68) reduces neurotransmitter uptake and release and produces learning and memory deficits in rats

NASA Technical Reports Server (NTRS)

Clarke, M. S.; Prendergast, M. A.; Terry, A. V. Jr

1999-01-01

A substantial body of evidence indicates that aged-related changes in the fluidity and lipid composition of the plasma membrane contribute to cellular dysfunction in humans and other mammalian species. In the CNS, reductions in neuronal plasma membrane order (PMO) (i.e., increased plasma membrane fluidity) have been attributed to age as well as the presence of the beta-amyloid peptide-25-35, known to play an important role in the neuropathology of Alzheimer's disease (AD). These PMO increases may influence neurotransmitter synthesis, receptor binding, and second messenger systems as well as signal transduction pathways. The effects of neuronal PMO on learning and memory processes have not been adequately investigated, however. Based on the hypothesis that an increase in PMO may alter a number of aspects of synaptic transmission, we investigated several neurochemical and behavioral effects of the membrane ordering agent, PF-68. In cell culture, PF-68 (nmoles/mg SDS extractable protein) reduced [3H]norepinephrine (NE) uptake into differentiated PC-12 cells as well as reduced nicotine stimulated [3H]NE release. The compound (800-2400 microg/kg, i.p., resulting in nmoles/mg SDS extractable protein in the brain) decreased step-through latencies and increased the frequencies of crossing into the unsafe side of the chamber in inhibitory avoidance training. In the Morris water maze, PF-68 increased the latencies and swim distances required to locate a hidden platform and reduced the time spent and distance swam in the previous target quadrant during transfer (probe) trials. PF-68 did not impair performance of a well-learned working memory task, the rat delayed stimulus discrimination task (DSDT), however. Studies with 14C-labeled PF-68 indicated that significant (pmoles/mg wet tissue) levels of the compound entered the brain from peripheral (i.p.) injection. No PF-68 related changes were observed in swim speeds or in visual acuity tests in water maze experiments, rotorod performance, or in tests of general locomotor activity. Furthermore, latencies to select a lever in the DSDT were not affected. These results suggest that PF-68 induced deficits in learning and memory without confounding peripheral motor, sensory, or motivational effects at the tested doses. Furthermore, none of the doses induced a conditioned taste aversion to a novel 0.1% saccharin solution indicating a lack of nausea or gastrointestinal malaise induced by the compound. The data indicate that increases in neuronal plasma membrane order may have significant effects on neurotransmitter function as well as learning and memory processes. Furthermore, compounds such as PF-68 may also offer novel tools for studying the role of neuronal PMO in mnemonic processes and changes in PMO resulting from age-related disorders such as AD.

Growth, fatty acid profile in major lipid classes and lipid fluidity of Aurantiochytrium mangrovei SK-02 As a function of growth temperature.

PubMed

Chodchoey, Kanokwan; Verduyn, Cornelis

2012-01-01

Aurantiochytrium mangrovei Sk-02 was grown in a medium containing glucose (40 g/l), yeast extract (10 g/L) and sea salts (15 g/L) at temperatures ranging from 12 to 35°C. The fastest growth (µmax= 0.15 h(-1)) and highest fatty acid content of 415 mg/g-dry cell weight were found in the cells grown at 30°C. However, the cells grown at 12°C showed the highest percentage of polyunsaturated fatty acid (PUFA) (48.6% of total fatty acid). The percentage of docosahexaenoic acid (DHA) and pentadecanoic acid (C15:0) decreased with an increase in the growth temperature, whereas, palmitic acid (C16:0), stearic acid (C18:0) and DPA (C22:5n6) increased with an increase in the growth temperature. The composition of the major lipid class (%w/w) was slightly affected by the growth temperature. The fluidity of the organelle membrane or intracellular lipid (by DPH measurement) decreased with an increase in the growth temperatures, while the plasma membrane fluidity (by TMA-DPH measurement) could still maintain its fluidity in a wide range of temperatures (15 - 37°C). Furthermore, the distribution of DHA was found to be higher (36 - 54%) in phospholipid (PL) as compared to neutral lipid (NL) (20 - 41%).

Bivariate and multivariate analyses of the correlations between stability of the erythrocyte membrane, serum lipids and hematological variables.

PubMed

Bernardino Neto, M; de Avelar, E B; Arantes, T S; Jordão, I A; da Costa Huss, J C; de Souza, T M T; de Souza Penha, V A; da Silva, S C; de Souza, P C A; Tavares, M; Penha-Silva, N

2013-01-01

The observation that the fluidity must remain within a critical interval, outside which the stability and functionality of the cell tends to decrease, shows that stability, fluidity and function are related and that the measure of erythrocyte stability allows inferences about the fluidity or functionality of these cells. This study determined the biochemical and hematological variables that are directly or indirectly related to erythrocyte stability in a population of 71 volunteers. Data were evaluated by bivariate and multivariate analysis. The erythrocyte stability showed a greater association with hematological variables than the biochemical variables. The RDW stands out for its strong correlation with the stability of erythrocyte membrane, without being heavily influenced by other factors. Regarding the biochemical variables, the erythrocyte stability was more sensitive to LDL-C. Erythrocyte stability was significantly associated with RDW and LDL-C. Thus, the level of LDL-C is a consistent link between stability and functionality, suggesting that a measure of stability could be more one indirect parameter for assessing the risk of degenerative processes associated with high levels of LDL-C.

Deoxycholic acid modulates cell death signaling through changes in mitochondrial membrane properties[S

PubMed Central

Sousa, Tânia; Castro, Rui E.; Pinto, Sandra N.; Coutinho, Ana; Lucas, Susana D.; Moreira, Rui; Rodrigues, Cecília M. P.; Prieto, Manuel; Fernandes, Fábio

2015-01-01

Cytotoxic bile acids, such as deoxycholic acid (DCA), are responsible for hepatocyte cell death during intrahepatic cholestasis. The mechanisms responsible for this effect are unclear, and recent studies conflict, pointing to either a modulation of plasma membrane structure or mitochondrial-mediated toxicity through perturbation of mitochondrial outer membrane (MOM) properties. We conducted a comprehensive comparative study of the impact of cytotoxic and cytoprotective bile acids on the membrane structure of different cellular compartments. We show that DCA increases the plasma membrane fluidity of hepatocytes to a minor extent, and that this effect is not correlated with the incidence of apoptosis. Additionally, plasma membrane fluidity recovers to normal values over time suggesting the presence of cellular compensatory mechanisms for this perturbation. Colocalization experiments in living cells confirmed the presence of bile acids within mitochondrial membranes. Experiments with active isolated mitochondria revealed that physiologically active concentrations of DCA change MOM order in a concentration- and time-dependent manner, and that these changes preceded the mitochondrial permeability transition. Importantly, these effects are not observed on liposomes mimicking MOM lipid composition, suggesting that DCA apoptotic activity depends on features of mitochondrial membranes that are absent in protein-free mimetic liposomes, such as the double-membrane structure, lipid asymmetry, or mitochondrial protein environment. In contrast, the mechanism of action of cytoprotective bile acids is likely not associated with changes in cellular membrane structure. PMID:26351365

Comparative study of hypocholesterolemic and hypolipidemic effects of conjugated linolenic acid isomers against induced biochemical perturbations and aberration in erythrocyte membrane fluidity.

PubMed

Saha, Siddhartha S; Chakraborty, Anirban; Ghosh, Santinath; Ghosh, Mahua

2012-06-01

The purpose of the study was to evaluate hypolipidemic and hypocholesterolemic activities of conjugated linolenic acid (CLnA) isomers, present in bitter gourd and snake gourd seed, in terms of amelioration of plasma lipid profile, lipoprotein oxidation and erythrocyte membrane fluidity after oral administration. Male albino rats were divided into six groups. Group 1 was control, and others were induced with oxidative stress by oral gavage of sodium arsenite (Sa). Group 2 was kept as treated control, and groups 3-6 were further treated with different oral doses of seed oils to maintaining definite concentration of CLnA isomers (0.5 and 1.0% of total lipid for each CLnA isomer). CLnA isomers normalized cholesterol, LDL-cholesterol, HDL-cholesterol and triglyceride contents in plasma and body weight of experimental rats and decreased cholesterol synthesis by reducing hepatic HMG-CoA reductase activity. Administration of Sa caused alteration in erythrocyte membrane fluidity due to increase in cholesterol and decrease in phospholipid content. Tissue cholesterol and lipid contents were also increased by Sa administration. These altered parameters were reversed by experimental oil administration. Protective effect of CLnA isomers on erythrocyte morphology was observed by atomic force microscopy (AFM). Fatty acid composition of erythrocyte membrane showed decrease in polyunsaturated fatty acid (PUFA) and increase in arachidonic acid content after Sa administration, which was normalized with the treatment of these oils. Supplementation of CLnA isomers restored erythrocyte membrane (EM) lipid peroxidation and lipoprotein oxidation. CLnA isomers, present in vegetable oils, showed potent hypolipidemic and hypocholesterolemic activities against biochemical perturbations.

Fluorescent Probe Study of AOT Vesicle Membranes and Their Alteration upon Addition of Aniline or the Aniline Dimer p-Aminodiphenylamine (PADPA).

PubMed

Iwasaki, Fumihiko; Luginbühl, Sandra; Suga, Keishi; Walde, Peter; Umakoshi, Hiroshi

2017-02-28

Artificial vesicles formed from sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in aqueous solution are used successfully as additives for enzymatic oligomerizations or polymerizations of aniline or the aniline dimer p-aminodiphenylamine (PADPA) under slightly acidic conditions (e.g., pH 4.3 with horseradish peroxidase and hydrogen peroxide as oxidants). In these systems, the reactions occur membrane surface-confined. Therefore, (i) the physicochemical properties of the vesicle membrane and (ii) the interaction of aniline or PADPA with the AOT membrane play crucial roles in the progress and final outcome of the reactions. For this reason, the properties of AOT vesicles with and without added aniline or PADPA were investigated by using two fluorescent membrane probes: 1,6-diphenyl-1,3,5-hexatriene (DPH) and 6-lauroyl-2-dimethylaminonaphthalene (Laurdan). DPH and Laurdan were used as "sensors" of the membrane fluidity, surface polarity, and membrane phase state. Moreover, the effect of hexanol, alone or in combination with aniline or PADPA, as a possible modifier of the AOT membrane, was also studied with the aim of evaluating whether the membrane fluidity and surface polarity is altered significantly by hexanol, which, in turn, may have an influence on the mentioned types of reactions. The data obtained indicate that the AOT vesicle membrane at room temperature and pH 4.3 (0.1 M NaH 2 PO 4 ) is more fluid and has a more polar surface than in the case of fluid phospholipid vesicle membranes formed from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Furthermore, the fluorescence measurements indicate that mixed AOT-hexanol membranes are less fluid than pure AOT membranes and that they have a lower surface polarity than pure AOT membranes. PADPA strongly binds to AOT and to mixed AOT/hexanol membranes and leads to drastic changes in the membrane properties (decrease in fluidity and surface polarity), resulting in Laurdan fluorescence spectra, which are characteristic for intramembrane phase separations (coexistence of ordered and disordered domains). This means that highly fluid AOT membranes transform upon the addition of PADPA into membranes that have ordered domains. Although the relevance of this finding for the enzymatic oligomerization of PADPA is not yet clear, it is also of interest if one likes to use heterogeneous vesicle membranes as additives for carrying out membrane surface-confined reactions that do not necessarily involve PADPA as a reactant.

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons.

PubMed

Villarreal, Seth; Lee, Sung Hoon; Wu, Ling-Gang

2017-09-04

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Cryopreservation of canine semen - new challenges.

PubMed

Farstad, W

2009-07-01

Egg yolk (EY) protects cell membranes against cold shock, and it prevents or restores the loss of phospholipids from the membrane. EY has been widely used in semen extenders. It has been added to Tris-Glucose buffer and has been widely used for cooling and cryopreservation of canine semen. EY is not a defined entity, but a complex biological compound containing proteins, vitamins, phospholipids, glucose and antioxidants which are all potentially useful for cell membrane integrity. Unfortunately, it also is a biologically hazardous compound. Hence, whole EY needs to be replaced by other chemically defined components for semen processing in dogs. Freezing poor semen does not improve its quality, so attention must be focused on how to cope with dogs whose semen does not freeze well, and on designing individual freezing extenders for semen from such males. Furthermore, differences have been found among canid species in the ability of their spermatozoa to withstand freezing. There are differences in sperm membrane fatty acid composition among species, which may explain part of these differences. If the presence of long-chained polyunsaturated fatty acids contributes to increased membrane fluidity, this relationship may be biphasic, i.e. either too much membrane fluidity, or too little, could compromise successful sperm cryopreservation. An increase in fluidity of the outer leaflet of the plasma membrane has been shown in frozen thawed dog spermatozoa. The protective effect of exogenous lipids may lie in close association with the membrane rather than in modification or rearrangement of the membrane. This also points at lipids as an important, if not entirely new group of substances, which may substitute standard EY-based diluents in preserving sperm survival during freezing. EY-derived phospholipids or lecithin could be used to replace whole EY. Vegetable lecithin is currently investigated to avoid using substances of animal origin. EY also contains antioxidants which prevent cells from oxidative damage due to the generation of reactive oxygen species. An increasing number of publications now recognize the significance of protecting sperm from this damage during processing by using dietary or diluent supplemented antioxidants. This paper aims at looking at some of the new challenges in freezing of dog semen.

On the Teneurin track: a new synaptic organization molecule emerges

PubMed Central

Mosca, Timothy J.

2015-01-01

To achieve proper synaptic development and function, coordinated signals must pass between the pre- and postsynaptic membranes. Such transsynaptic signals can be comprised of receptors and secreted ligands, membrane associated receptors, and also pairs of synaptic cell adhesion molecules. A critical open question bridging neuroscience, developmental biology, and cell biology involves identifying those signals and elucidating how they function. Recent work in Drosophila and vertebrate systems has implicated a family of proteins, the Teneurins, as a new transsynaptic signal in both the peripheral and central nervous systems. The Teneurins have established roles in neuronal wiring, but studies now show their involvement in regulating synaptic connections between neurons and bridging the synaptic membrane and the cytoskeleton. This review will examine the Teneurins as synaptic cell adhesion molecules, explore how they regulate synaptic organization, and consider how some consequences of human Teneurin mutations may have synaptopathic origins. PMID:26074772

Detergent-dependent separation of postsynaptic density, membrane rafts and other subsynaptic structures from the synaptic plasma membrane of rat forebrain.

PubMed

Zhao, LiYing; Sakagami, Hiroyuki; Suzuki, Tatsuo

2014-10-01

We systematically investigated the purification process of post-synaptic density (PSD) and post-synaptic membrane rafts (PSRs) from the rat forebrain synaptic plasma membranes by examining the components and the structures of the materials obtained after the treatment of synaptic plasma membranes with TX-100, n-octyl β-d-glucoside (OG) or 3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate (CHAPSO). These three detergents exhibited distinct separation profiles for the synaptic subdomains. Type I and type II PSD proteins displayed mutually exclusive distribution. After TX-100 treatment, type I PSD was recovered in two fractions: a pellet and an insoluble fraction 8, which contained partially broken PSD-PSR complexes. Conventional PSD was suggested to be a mixture of these two PSD pools and did not contain type II PSD. An association of type I PSD with PSRs was identified in the TX-100 treatment, and those with type II PSD in the OG and CHAPSO treatments. An association of GABA receptors with gephyrin was easily dissociated. OG at a high concentration solubilized the type I PSD proteins. CHAPSO treatment resulted in a variety of distinct fractions, which contained certain novel structures. Two different pools of GluA, either PSD or possibly raft-associated, were identified in the OG and CHAPSO treatments. These results are useful in advancing our understanding of the structural organization of synapses at the molecular level. We systematically investigated the purification process of post-synaptic density (PSD) and synaptic membrane rafts by examining the structures obtained after treatment of the SPMs with TX-100, n-octyl β-d-glucoside or CHAPSO. Differential distribution of type I and type II PSD, synaptic membrane rafts, and other novel subdomains in the SPM give clues to understand the structural organization of synapses at the molecular level. © 2014 International Society for Neurochemistry.

Flexible Proton-Gated Oxide Synaptic Transistors on Si Membrane.

PubMed

Zhu, Li Qiang; Wan, Chang Jin; Gao, Ping Qi; Liu, Yang Hui; Xiao, Hui; Ye, Ji Chun; Wan, Qing

2016-08-24

Ion-conducting materials have received considerable attention for their applications in fuel cells, electrochemical devices, and sensors. Here, flexible indium zinc oxide (InZnO) synaptic transistors with multiple presynaptic inputs gated by proton-conducting phosphorosilicate glass-based electrolyte films are fabricated on ultrathin Si membranes. Transient characteristics of the proton gated InZnO synaptic transistors are investigated, indicating stable proton-gating behaviors. Short-term synaptic plasticities are mimicked on the proposed proton-gated synaptic transistors. Furthermore, synaptic integration regulations are mimicked on the proposed synaptic transistor networks. Spiking logic modulations are realized based on the transition between superlinear and sublinear synaptic integration. The multigates coupled flexible proton-gated oxide synaptic transistors may be interesting for neuroinspired platforms with sophisticated spatiotemporal information processing.

Role of DHA in aging-related changes in mouse brain synaptic plasma membrane proteome.

PubMed

Sidhu, Vishaldeep K; Huang, Bill X; Desai, Abhishek; Kevala, Karl; Kim, Hee-Yong

2016-05-01

Aging has been related to diminished cognitive function, which could be a result of ineffective synaptic function. We have previously shown that synaptic plasma membrane proteins supporting synaptic integrity and neurotransmission were downregulated in docosahexaenoic acid (DHA)-deprived brains, suggesting an important role of DHA in synaptic function. In this study, we demonstrate aging-induced synaptic proteome changes and DHA-dependent mitigation of such changes using mass spectrometry-based protein quantitation combined with western blot or messenger RNA analysis. We found significant reduction of 15 synaptic plasma membrane proteins in aging brains including fodrin-α, synaptopodin, postsynaptic density protein 95, synaptic vesicle glycoprotein 2B, synaptosomal-associated protein 25, synaptosomal-associated protein-α, N-methyl-D-aspartate receptor subunit epsilon-2 precursor, AMPA2, AP2, VGluT1, munc18-1, dynamin-1, vesicle-associated membrane protein 2, rab3A, and EAAT1, most of which are involved in synaptic transmission. Notably, the first 9 proteins were further reduced when brain DHA was depleted by diet, indicating that DHA plays an important role in sustaining these synaptic proteins downregulated during aging. Reduction of 2 of these proteins was reversed by raising the brain DHA level by supplementing aged animals with an omega-3 fatty acid sufficient diet for 2 months. The recognition memory compromised in DHA-depleted animals was also improved. Our results suggest a potential role of DHA in alleviating aging-associated cognitive decline by offsetting the loss of neurotransmission-regulating synaptic proteins involved in synaptic function. Published by Elsevier Inc.

Membrane-associated stress proteins: more than simply chaperones.

PubMed

Horváth, Ibolya; Multhoff, Gabriele; Sonnleitner, Alois; Vígh, László

2008-01-01

The protein- and/or lipid-mediated association of chaperone proteins to membranes is a widespread phenomenon and implicated in a number of physiological and pathological events that were earlier partially or completely overlooked. A temporary association of certain HSPs with membranes can re-establish the fluidity and bilayer stability and thereby restore the membrane functionality during stress conditions. The fluidity and microdomain organization of membranes are decisive factors in the perception and transduction of stresses into signals that trigger the activation of specific HS genes. Conversely, the membrane association of HSPs may result in the inactivation of membrane-perturbing signals, thereby switch off the heat shock response. Interactions between certain HSPs and specific lipid microdomains ("rafts") might be a previously unrecognized means for the compartmentalization of HSPs to specific signaling platforms, where key signaling proteins are known to be concentrated. Any modulations of the membranes, especially the raft-lipid composition of the cells can alter the extracellular release and thus the immuno-stimulatory activity of certain HSPs. Reliable techniques, allowing mapping of the composition and dynamics of lipid microdomains and simultaneously the spatio-temporal localization of HSPs in and near the plasma membrane can provide suitable means with which to address fundamental questions, such as how HSPs are transported to and translocated through the plasma membrane. The possession of such information is critical if we are to target the membrane association principles of HSPs for successful drug development in most various diseases.

Effect of a Vietnamese Cinnamomum cassia essential oil and its major component trans-cinnamaldehyde on the cell viability, membrane integrity, membrane fluidity, and proton motive force of Listeria innocua.

PubMed

Trinh, Nga-Thi-Thanh; Dumas, Emilie; Thanh, Mai Le; Degraeve, Pascal; Ben Amara, Chedia; Gharsallaoui, Adem; Oulahal, Nadia

2015-04-01

The antibacterial mechanism of a Cinnamomum cassia essential oil from Vietnam and of its main component (trans-cinnamaldehyde, 90% (m/m) of C. cassia essential oil) against a Listeria innocua strain was investigated to estimate their potential for food preservation. In the presence of C. cassia essential oil or trans-cinnamaldehyde at their minimal bactericidal concentration (2700 μg·mL(-1)), L. innocua cells fluoresced green after staining with Syto9® and propidium iodide, as observed by epifluorescence microscopy, suggesting that the perturbation of membrane did not cause large pore formation and cell lysis but may have introduced the presence of viable but nonculturable bacteria. Moreover, the fluidity, potential, and intracellular pH of the cytoplasmic membrane were perturbed in the presence of the essential oil or trans-cinnamaldehyde. However, these membrane perturbations were less severe in the presence of trans-cinnamaldehyde than in the presence of multicomponent C. cassia essential oil. This indicates that in addition to trans-cinnamaldehyde, other minor C. cassia essential oil components play a major role in its antibacterial activity against L. innocua cells.

Isolation of Synaptosomes, Synaptic Plasma Membranes, and Synaptic Junctional Complexes.

PubMed

Michaelis, Mary L; Jiang, Lei; Michaelis, Elias K

2017-01-01

Isolation of synaptic nerve terminals or synaptosomes provides an opportunity to study the process of neurotransmission at many levels and with a variety of approaches. For example, structural features of the synaptic terminals and the organelles within them, such as synaptic vesicles and mitochondria, have been elucidated with electron microscopy. The postsynaptic membranes are joined to the presynaptic "active zone" of transmitter release through cell adhesion molecules and remain attached throughout the isolation of synaptosomes. These "post synaptic densities" or "PSDs" contain the receptors for the transmitters released from the nerve terminals and can easily be seen with electron microscopy. Biochemical and cell biological studies with synaptosomes have revealed which proteins and lipids are most actively involved in synaptic release of neurotransmitters. The functional properties of the nerve terminals, such as responses to depolarization and the uptake or release of signaling molecules, have also been characterized through the use of fluorescent dyes, tagged transmitters, and transporter substrates. In addition, isolated synaptosomes can serve as the starting material for the isolation of relatively pure synaptic plasma membranes (SPMs) that are devoid of organelles from the internal environment of the nerve terminal, such as mitochondria and synaptic vesicles. The isolated SPMs can reseal and form vesicular structures in which transport of ions such as sodium and calcium, as well as solutes such as neurotransmitters can be studied. The PSDs also remain associated with the presynaptic membranes during isolation of SPM fractions, making it possible to isolate the synaptic junctional complexes (SJCs) devoid of the rest of the plasma membranes of the nerve terminals and postsynaptic membrane components. Isolated SJCs can be used to identify the proteins that constitute this highly specialized region of neurons. In this chapter, we describe the steps involved in isolating synaptosomes, SPMs, and SJCs from brain so that these preparations can be used with new technological advances to address many as yet unanswered questions about the synapse and its remarkable activities in neuronal cell communication.

Characterization of Bovine Brain ATPase

DTIC Science & Technology

1988-07-01

Experiment D . Only very small amounts of (3H)-ligand (0.8 fmole/mg protein) were observed to bind to the toxin as indicated by Experiments E and F. Since...B. Synaptic Membranes + 3H-Ligand + 7.6 Unlabelled Ltgand C. Toxin + Synaptic Membranes + 3H-Ligand 7.5 D . Toxin + Synaptic Membranes + 3H-Ligand...Europaeus Agglutinin L-Fucose SBA= Soy Bean Agglutinin D -Galactose LPA= Limulus Polyphemus Agglutinin N-Acetylgalactosamine Con-A= Concanavalin-A, D -Glucose

Interaction of nanoparticles with lipid membranes: a multiscale perspective

NASA Astrophysics Data System (ADS)

Montis, Costanza; Maiolo, Daniele; Alessandri, Ivano; Bergese, Paolo; Berti, Debora

2014-05-01

Freestanding lipid bilayers were challenged with 15 nm Au nanospheres either coated by a citrate layer or passivated by a protein corona. The effect of Au nanospheres on the bilayer morphology, permeability and fluidity presents strong differences or similarities, depending on the observation length scale, from the colloidal to the molecular domains. These findings suggest that the interaction between nanoparticles and lipid membranes should be conveniently treated as a multiscale phenomenon.Freestanding lipid bilayers were challenged with 15 nm Au nanospheres either coated by a citrate layer or passivated by a protein corona. The effect of Au nanospheres on the bilayer morphology, permeability and fluidity presents strong differences or similarities, depending on the observation length scale, from the colloidal to the molecular domains. These findings suggest that the interaction between nanoparticles and lipid membranes should be conveniently treated as a multiscale phenomenon. Electronic supplementary information (ESI) available: All the experimental details, figures and tables. See DOI: 10.1039/c4nr00838c

Characterization of Storage-Induced Red Blood Cell Hemolysis Using Raman Spectroscopy.

PubMed

Gautam, Rekha; Oh, Joo-Yeun; Marques, Marisa B; Dluhy, Richard A; Patel, Rakesh P

2018-06-11

The therapeutic efficacy and safety of stored red blood cells (RBCs) relies on minimal in-bag hemolysis. The accuracy of current methods of measuring hemolysis can suffer as a result of specimen collection and processing artefacts. To test whether Raman spectroscopy could be used to assess hemolysis. RBCs were stored for as long as 42 days. Raman spectra of RBCs were measured before and after washing, and hemolysis was measured in supernatant by visible spectroscopy. Raman spectra indicated increased concentrations of oxyhemoglobin (oxyHb) and methemoglobin (metHb), and decreased membrane fluidity with storage age. Changes in oxyHb and metHb were associated with the intraerythrocytic and extracellular fractions, respectively. Hemolysis increased in a storage age-dependent manner. Changes in Raman bands reflective of oxyHb, metHb, and RBC membranes correlated with hemolysis; the most statistically significant change was an increased intensity of metHb and decreased membrane fluidity. These data suggest that Raman spectroscopy may offer a new label-free modality to assess RBC hemolysis during cold storage.

In vitro analysis of the effect of alkyl-chain length of anionic surfactants on the skin by using a reconstructed human epidermal model.

PubMed

Yamaguchi, Fumiko; Watanabe, Shin-Ichi; Harada, Fusae; Miyake, Miyuki; Yoshida, Masaki; Okano, Tomomichi

2014-01-01

We investigated the effect of the alkyl-chain length of anionic surfactants on the skin using an in vitro model. The evaluated anionic surfactants were sodium alkyl sulfate (AS) and sodium fatty acid methyl ester sulfonate (MES), which had different alkyl-chain lengths (C8-C14). Skin tissue damage and permeability were examined using a reconstructed human epidermal model, LabCyte EPI-MODEL24. Skin tissue damage was examined by measuring cytotoxicity with an MTT assay. Liquid chromatography/tandem mass spectrometry (LC/MS-MS) and liquid chromatography/mass spectrometry (LC/MS) were used to detect surfactants that permeated into the assay medium through an epidermal model. To assess the permeation mechanism and cell damage caused by the surfactants through the epidermis, we evaluated the structural changes of Bovine Serum Albumin (BSA), used as a simple model protein, and the fluidity of 1,2-dipalmitoyl-sn-glycero-3-phosphpcholine (DPPC) liposome, which serves as one of the most abundant phospholipid models of living cell membranes in the epidermis. The effects of the surfactants on the proteins were measured using Circular Dichroism (CD) spectroscopy, while the effects on membrane fluidity were investigated by electron spin resonance (ESR) spectroscopy. ET50 (the 50% median effective time) increased as follows: C10 < C12 < C8 < C14 in AS and C8, C10 < C12 < C14 in MES. The order of permeation through the LabCyte EPI-MODEL24 was C10 > C12 > C14, for both AS and MES. For both AS and MES, the order parameter, which is the criteria for the microscopic viscosity of lipid bilayers, increased as follows: C10 < C12 < C14, which means the membrane fluidity is C10 > C12 > C14. It was determined that the difference in skin tissue damage in the LabCyte EPI-MODEL24 with C10 to C14 AS and MES was caused by the difference in permeation and cell membrane fluidity through the lipid bilayer path in the epidermis.

Membrane lipid physiology and toxin catabolism underlie ethanol and acetic acid tolerance in Drosophila melanogaster.

PubMed

Montooth, Kristi L; Siebenthall, Kyle T; Clark, Andrew G

2006-10-01

Drosophila melanogaster has evolved the ability to tolerate and utilize high levels of ethanol and acetic acid encountered in its rotting-fruit niche. Investigation of this phenomenon has focused on ethanol catabolism, particularly by the enzyme alcohol dehydrogenase. Here we report that survival under ethanol and acetic acid stress in D. melanogaster from high- and low-latitude populations is an integrated consequence of toxin catabolism and alteration of physical properties of cellular membranes by ethanol. Metabolic detoxification contributed to differences in ethanol tolerance between populations and acclimation temperatures via changes in both alcohol dehydrogenase and acetyl-CoA synthetase mRNA expression and enzyme activity. Independent of changes in ethanol catabolism, rapid thermal shifts that change membrane fluidity had dramatic effects on ethanol tolerance. Cold temperature treatments upregulated phospholipid metabolism genes and enhanced acetic acid tolerance, consistent with the predicted effects of restoring membrane fluidity. Phospholipase D was expressed at high levels in all treatments that conferred enhanced ethanol tolerance, suggesting that this lipid-mediated signaling enzyme may enhance tolerance by sequestering ethanol in membranes as phophatidylethanol. These results reveal new candidate genes underlying toxin tolerance and membrane adaptation to temperature in Drosophila and provide insight into how interactions between these phenotypes may underlie the maintenance of latitudinal clines in ethanol tolerance.

Inactivation of neurotensin by rat brain synaptic membranes. Cleavage at the Pro10-Tyr11 bond by endopeptidase 24.11 (enkephalinase) and a peptidase different from proline-endopeptidase.

PubMed

Checler, F; Emson, P C; Vincent, J P; Kitabgi, P

1984-11-01

It was shown previously that the tridecapeptide neurotensin is inactivated by rat brain synaptic membranes and that one of the primary inactivating cleavages occurs at the Pro10-Try11 peptide bond, leading to the formation of NT1-10 and NT11-13. The present study was designed to investigate the possibility that this cleavage was catalyzed by proline endopeptidase and/or endopeptidase 24.11 (enkephalinase). Purified rat brain synaptic membranes were found to contain a N-benzyloxycarbonyl-Gly-Pro-4-methyl-coumarinyl-7-amide-hydrolyzin g activity that was markedly inhibited (93%) by the proline endopeptidase inhibitor N-benzyloxycarbonyl-Pro-Prolinal and partially blocked (25%) by an antiproline endopeptidase antiserum. In contrast, the cleavage of neurotensin at the Pro10-Tyr11 bond by synaptic membranes was not affected by N-benzyloxycarbonyl-Pro-Prolinal and the antiserum. When the conversion of NT1-10 to NT1-8 by angiotensin converting enzyme was blocked by captopril and when the processing of NT11-13 by aminopeptidase(s) was inhibited by bestatin, it was found that thiorphan, a potent endopeptidase 24.11 inhibitor, partially decreased the formation of NT1-10 and NT11-13 by synaptic membranes. (1) proline endopeptidase, although it is present in synaptic membranes, is not involved in the cleavage of neurotensin at the Pro10-Tyr11 bond; (2) endopeptidase 24.11 only partially contributes to this cleavage; (3) there exists in rat brain synaptic membranes a peptidase different from proline endopeptidase and endopeptidase 24.11 that is mainly responsible for inactivating neurotensin by cleaving at the Pro10-Tyr11 bond.

Fine structure of synapses of the central nervous system in resinless sections.

PubMed

Cohen, R S; Wolosewick, J J; Becker, R P; Pappas, G D

1983-10-01

The cytoskeleton has been implicated in neuronal function, particularly in axonal transport, excitability at axonal membranes, and movement of synaptic vesicles at preganglionic endings. The present study demonstrates the presence of a pre- and postsynaptic cytoskeleton in resinless sections of CNS tissue by use of the polyethylene glycol (PEG) technique of Wolosewick (1980) viewed by conventional transmission EM, scanning transmission EM, and surface scanning EM. The PEG technique permits visualization of the cytoskeletal network unobscured by the electron scattering properties of epoxy embedment. In the presynaptic process, synaptic vesicles appear to be suspended in a filamentous network that is contiguous with the synaptic vesicle membrane and with the presynaptic plasma membrane and its dense material. In the postsynaptic process, the postsynaptic density (PSD) is seen in intimate contact with the postsynaptic membrane. En face images of the PSD in some synapses appear as a torus. Emanating from the filamentous web of the PSD are filaments which extend to the adjacent plasma membrane. We conclude that membranous synaptic elements are contiguous with a three-dimensional lattice network that is similar to that described in whole unembedded cells (Wolosewick and Porter, 1976). Moreover, the synaptic densities represent a specialized elaboration of the cytoskeleton.

Thermal Regulation of Membrane Lipid Fluidity by a Two-Component System in "Bacillus Subtilis"

ERIC Educational Resources Information Center

Bredeston, L. M.; Marciano, D.; Albanesi, D.; De Mendoza, D.; Delfino, J. M.

2011-01-01

This article describes a simple and robust laboratory exercise on the regulation of membrane unsaturated fatty acid composition in bacteria by a decrease in growth temperature. We take advantage of the well characterized Des pathway of "Bacillus subtilis", composed of a [delta]5-desaturase (encoded by the "des" gene) and the canonical…

Pathological levels of glucosylceramide change the biophysical properties of artificial and cell membranes.

PubMed

Varela, Ana R P; Ventura, Ana E; Carreira, Ana C; Fedorov, Aleksander; Futerman, Anthony H; Prieto, Manuel; Silva, Liana C

2016-12-21

Glucosylceramide (GlcCer) plays an active role in the regulation of various cellular events. Moreover, GlcCer is also a key modulator of membrane biophysical properties, which might be linked to the mechanism of its biological action. In order to understand the biophysical implications of GlcCer on membranes of living cells, we first studied the effect of GlcCer on artificial membranes containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), sphingomyelin (SM) and cholesterol (Chol). Using an array of biophysical methods, we demonstrate that at lower GlcCer/Chol ratios, GlcCer stabilizes SM/Chol-enriched liquid-ordered domains. However, upon decreasing the Chol content, GlcCer significantly increased membrane order through the formation of gel domains. Changes in pH disturbed the packing properties of GlcCer-containing membranes, leading to an increase in membrane fluidity and reduced membrane electronegativity. To address the biophysical impact of GlcCer in biological membranes, studies were performed in wild type and in fibroblasts treated with conduritol-B-epoxide (CBE), which causes intracellular GlcCer accumulation, and in fibroblasts from patients with type I Gaucher disease (GD). Decreased membrane fluidity was observed in cells containing higher levels of GlcCer, such as in CBE-treated and GD cells. Together, we demonstrate that elevated GlcCer levels change the biophysical properties of cellular membranes, which might compromise membrane-associated cellular events and be of relevance for understanding the pathology of diseases, such as GD, in which GlcCer accumulates at high levels.

Analysis of Lipids and Lipid Rafts in Borrelia.

PubMed

Toledo, Alvaro; Huang, Zhen; Benach, Jorge L; London, Erwin

2018-01-01

Lipid rafts are membrane microdomains that are involved in cellular processes such as protein trafficking and signaling processes, and which play a fundamental role in membrane fluidity and budding. The lipid composition of the membrane and the biochemical characteristics of the lipids found within rafts define the ability of cells to form microdomains and compartmentalize the membrane. In this chapter, we describe the biophysical, biochemical, and molecular approaches used to define and characterize lipid rafts in the Lyme disease agent, Borrelia burgdorferi.

Trans-cis isomerization of lipophilic dyes probing membrane microviscosity in biological membranes and in live cells.

PubMed

Chmyrov, Volodymyr; Spielmann, Thiemo; Hevekerl, Heike; Widengren, Jerker

2015-06-02

Membrane environment and fluidity can modulate the dynamics and interactions of membrane proteins and can thereby strongly influence the function of cells and organisms in general. In this work, we demonstrate that trans-cis isomerization of lipophilic dyes is a useful parameter to monitor packaging and fluidity of biomembranes. Fluorescence fluctuations, generated by trans-cis isomerization of the thiocarbocyanine dye Merocyanine 540 (MC540), were first analyzed by fluorescence correlation spectroscopy (FCS) in different alcohol solutions. Similar isomerization kinetics of MC540 in lipid vesicles could then also be monitored, and the influence of lipid polarity, membrane curvature, and cholesterol content was investigated. While no influence of membrane curvature and lipid polarity could be observed, a clear decrease in the isomerization rates could be observed with increasing cholesterol contents in the vesicle membranes. Finally, procedures to spatially map photoinduced and thermal isomerization rates on live cells by transient state (TRAST) imaging were established. On the basis of these procedures, MC540 isomerization was studied on live MCF7 cells, and TRAST images of the cells at different temperatures were found to reliably detect differences in the isomerization parameters. Our studies indicate that trans-cis isomerization is a useful parameter for probing membrane dynamics and that the TRAST imaging technique can provide spatial maps of photoinduced isomerization as well as both photoinduced and thermal back-isomerization, resolving differences in local membrane microviscosity in live cells.

Changes in the lipid composition of Bradyrhizobium cell envelope reveal a rapid response to water deficit involving lysophosphatidylethanolamine synthesis from phosphatidylethanolamine in outer membrane.

PubMed

Cesari, Adriana B; Paulucci, Natalia S; Biasutti, María A; Morales, Gustavo M; Dardanelli, Marta S

2018-06-02

We evaluate the behavior of the membrane of Bradyrhizobium sp. SEMIA6144 during adaptation to polyethylene glycol (PEG). A dehydrating effect on the morphology of the cell surface, as well as a fluidizing effect on the membrane was observed 10 min after PEG shock; however, the bacteria were able to restore optimal membrane fluidity. Shock for 1 h caused an increase of lysophosphatidylethanolamine in the outer membrane at the expense of phosphatidylcholine and phosphatidylethanolamine (PE), through an increase in phospholipase activity. The amount of lysophosphatidylethanolamine did not remain constant during PEG shock, but after 24 h the outer membrane was composed of large amounts of phosphatidylcholine and less amount of lysophosphatidylethanolamine similar to the control. The inner membrane composition was also modified after 1 h of shock, observing an increase of phosphatidylcholine at the expense of PE, the proportions of these phospholipids were then modified to reach 24 h of shock values similar to the control. Vesicles prepared with the lipids of cells exposed to 1 h shock presented higher rigidity compared to the control, indicating that changes in the composition of phospholipids after 1 h of shock restoring fluidity after the PEG effect and would allow cells to maintain surface morphology. Copyright © 2018 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Electronic paramagnetic resonance investigation of the activity of Origanum vulgare L. essential oil on the Listeria monocytogenes membrane.

PubMed

Serio, A; Chiarini, M; Tettamanti, E; Paparella, A

2010-08-01

To evaluate the effect of oregano essential oil on Listeria monocytogenes cytoplasmic membrane. Nitroxide free-radical Electron Paramagnetic Resonance was applied on L. monocytogenes after 30 min exposure to oregano essential oil concentrations ranging from 0 to 1.25%. The impact of essential oil on the number of viable cells was evaluated by plate count. Growth dynamics of survivors in BHI and TSB were evaluated by turbidometry. After exposure to essential oil concentrations up to 0.50%, the membrane fluidity was changed and its order increased. When L. monocytogenes was exposed to higher concentrations, membrane order parameters slightly returned to the values of untreated cells. However, when the cells were exposed to EO in the presence of sodium azide, which impairs energy metabolism, the membrane fluidity was progressively enhanced, even at the lowest EO concentration (0.25%). Microbiological analyses confirmed a progressive reduction of viable count, at increasing essential oil concentrations. Both in BHI and TSB, the Lag phase length increased in treated cells with respect to controls, suggesting a cell damage recovery. The combined approach including microbiological and EPR analyses provided relevant information on membrane modification and cell response to essential oils. EPR approach was demonstrated to be an effective and helpful tool to comprehend the modifications exerted by essential oil on the bacterial membrane.

Alteration by prolactin of surface charge and membrane fluidity of rat 13762 mammary ascites tumor cells.

PubMed

Zarkower, D A; Plank, L D; Kunze, E; Keith, A; Todd, P; Hymer, W C

1984-03-01

Intraperitoneal injection of ovine prolactin (100 micrograms/d) in Fischer 344 rats bearing transplantable 13762 mammary ascites tumor (MAT) cells modifies the surface charge density and membrane fluidity of the tumor cells. In each of five experiments the mean electrophoretic mobility (epm) of MAT cells taken from prolactin-treated rats was significantly lower than that of cells from nonhormone-treated controls. Prolactin concentrations were increased in vivo by (a) direct intraperitoneal injection of ovine prolactin; (b) subcutaneous implantation of diethylstilbestrol-containing silastic capsules to produce pituitary prolactin secreting tumors; or (c) a single subcutaneous injection of polyestradiol phosphate, a long-acting estrogen. In an effort to establish that the prolactin effect was a direct one, two in vivo protocols were used: (a) MAT cells were coincubated with anterior pituitary halves obtained from nontumor-bearing littermates; or (b) rat or ovine prolactin was added to serum-free culture media containing MAT cells. In both protocols, the epm of the prolactin-treated cells was significantly lower. The isoelectric focusing pH of whole cells was increased by prolactin treatment from 4.93 to 5.12, consistent with a reduction in the number of surface carboxyl groups. The fluidity of membranes of treated cells was drastically increased, as measured by spin-label probe rotation rates. These combined results imply that the hormone exerts its effect by stimulating events in the cell that lead to a reduction of the average density of carboxylic acid residues on the tumor cell surface.

Effect of antihypertensive agents - captopril and nifedipine - on the functional properties of rat heart mitochondria

PubMed Central

Kancirová, Ivana; Jašová, Magdaléna; Waczulíková, Iveta; Ravingerová, Táňa; Ziegelhöffer, Attila; Ferko, Miroslav

2016-01-01

Objective(s): Investigation of acute effect on cellular bioenergetics provides the opportunity to characterize the possible adverse effects of drugs more comprehensively. This study aimed to investigate the changes in biochemical and biophysical properties of heart mitochondria induced by captopril and nifedipine antihypertensive treatment. Materials and Methods: Male, 12-week-old Wistar rats in two experimental models (in vivo and in vitro) were used. In four groups, the effects of escalating doses of captopril, nifedipine and combination of captopril + nifedipine added to the incubation medium (in vitro) or administered per os to rat (in vivo) on mitochondrial ATP synthase activity and membrane fluidity were monitored. Results: In the in vitro model we observed a significant inhibitory effect of treatment on the ATP synthase activity (P<0.05) with nonsignificant differences in membrane fluidity. Decrease in the value of maximum reaction rate Vmax (P<0.05) without any change in the value of Michaelis-Menten constant Km, indicative of a noncompetitive inhibition, was presented. At the in vivo level, we did not demonstrate any significant changes in the ATP synthase activity and the membrane fluidity in rats receiving captopril, nifedipine, and combined therapy. Conclusion: In vitro kinetics study revealed that antihypertensive drugs (captopril and nifedipine) directly interact with mitochondrial ATP synthase. In vivo experiment did not prove any acute effect on myocardial bioenergetics and suggest that drugs do not enter cardiomyocyte and have no direct effect on mitochondria. PMID:27482342

High Cholesterol/Low Cholesterol: Effects in Biological Membranes: A Review.

PubMed

Subczynski, Witold K; Pasenkiewicz-Gierula, Marta; Widomska, Justyna; Mainali, Laxman; Raguz, Marija

2017-12-01

Lipid composition determines membrane properties, and cholesterol plays a major role in this determination as it regulates membrane fluidity and permeability, as well as induces the formation of coexisting phases and domains in the membrane. Biological membranes display a very diverse lipid composition, the lateral organization of which plays a crucial role in regulating a variety of membrane functions. We hypothesize that, during biological evolution, membranes with a particular cholesterol content were selected to perform certain functions in the cells of eukaryotic organisms. In this review, we discuss the major membrane properties induced by cholesterol, and their relationship to certain membrane functions.

Liposomes for Topical Use: A Physico-Chemical Comparison of Vesicles Prepared from Egg or Soy Lecithin

PubMed Central

Budai, Lívia; Kaszás, Nóra; Gróf, Pál; Lenti, Katalin; Maghami, Katayoon; Antal, István; Klebovich, Imre; Petrikovics, Ilona; Budai, Marianna

2013-01-01

Developments in nanotechnology and in the formulation of liposomal systems provide the opportunity for cosmetic dermatology to design novel delivery systems. Determination of their physico-chemical parameters has importance when developing a nano-delivery system. The present study highlights some technological aspects/characteristics of liposomes formulated from egg or soy lecithins for topical use. Alterations in the pH, viscosity, surface tension, and microscopic/macroscopic appearance of these vesicular systems were investigated. The chemical composition of the two types of lecithin was checked by mass spectrometry. Caffeine, as a model molecule, was encapsulated into multilamellar vesicles prepared from the two types of lecithin: then zeta potential, membrane fluidity, and encapsulation efficiency were compared. According to our observations, samples prepared from the two lecithins altered the pH in opposite directions: egg lecithin increased it while soy lecithin decreased it with increased lipid concentration. Our EPR spectroscopic results showed that the binding of caffeine did not change the membrane fluidity in the temperature range of possible topical use (measured between 2 and 50 °C). Combining our results on encapsulation efficiency for caffeine (about 30% for both lecithins) with those on membrane fluidity data, we concluded that the interaction of caffeine with the liposomal membrane does not change the rotational motion of the lipid molecules close to the head group region. In conclusion, topical use of egg lecithin for liposomal formulations can be preferred if there are no differences in the physico-chemical properties due to the encapsulated drugs, because the physiological effects of egg lecithin vesicles on skin are significantly better than that of soy lecithin liposomes. PMID:24482779

Membrane Fusion Involved in Neurotransmission: Glimpse from Electron Microscope and Molecular Simulation

PubMed Central

Yang, Zhiwei; Gou, Lu; Chen, Shuyu; Li, Na; Zhang, Shengli; Zhang, Lei

2017-01-01

Membrane fusion is one of the most fundamental physiological processes in eukaryotes for triggering the fusion of lipid and content, as well as the neurotransmission. However, the architecture features of neurotransmitter release machinery and interdependent mechanism of synaptic membrane fusion have not been extensively studied. This review article expounds the neuronal membrane fusion processes, discusses the fundamental steps in all fusion reactions (membrane aggregation, membrane association, lipid rearrangement and lipid and content mixing) and the probable mechanism coupling to the delivery of neurotransmitters. Subsequently, this work summarizes the research on the fusion process in synaptic transmission, using electron microscopy (EM) and molecular simulation approaches. Finally, we propose the future outlook for more exciting applications of membrane fusion involved in synaptic transmission, with the aid of stochastic optical reconstruction microscopy (STORM), cryo-EM (cryo-EM), and molecular simulations. PMID:28638320

Omega-3 Fatty Acid Supplementation for the Treatment of Children with Attention-Deficit/Hyperactivity Disorder Symptomatology: Systematic Review and Meta-Analysis

ERIC Educational Resources Information Center

Bloch, Michael H.; Qawasmi, Ahmad

2011-01-01

Objective: Several studies have demonstrated differences in omega-3 fatty acid composition in plasma and in erythrocyte membranes in patients with attention-deficit/hyperactivity disorder (ADHD) compared with unaffected controls. Omega-3 fatty acids have anti-inflammatory properties and can alter central nervous system cell membrane fluidity and…

The Septate Junction Protein Caspr is Required for Structural Support and Retention of KCNQ4 at Calyceal Synapses of Vestibular Hair Cells

PubMed Central

Sousa, Aurea D.; Andrade, Leonardo R.; Salles, Felipe T.; Pillai, Anilkumar M.; Buttermore, Elizabeth; Bhat, Manzoor A.; Kachar, Bechara

2009-01-01

The afferent innervation contacting the type I hair cells of the vestibular sensory epithelia form distinct calyceal synapses. The apposed pre- and post-synaptic membranes at this large area of synaptic contact are kept at a remarkably regular distance. Here, we show by freeze-fracture electron microscopy that a patterned alignment of proteins at the calyceal membrane resembles a type of intercellular junction that is rare in vertebrates, the septate junction (SJ). We found that a core molecular component of SJs, Caspr, colocalizes with the K+ channel KCNQ4 at the post-synaptic membranes of these calyceal synapses. Immunolabeling and ultrastructural analyses of Caspr knockout mice reveal that, in the absence of Caspr, the separation between the membranes of the hair cells and the afferent neurons is conspicuously irregular and often increased by an order of magnitude. In these mutants, KCNQ4 fails to cluster at the post-synaptic membrane and appears diffused along the entire calyceal membrane. Our results indicate that a septate-like junction provides structural support to calyceal synaptic contact with the vestibular hair cell, and that Caspr is required for the recruitment or retention of KCNQ4 at these synapses. PMID:19279247

Visualization of Synchronous or Asynchronous Release of Single Synaptic Vesicle in Active-Zone-Like Membrane Formed on Neuroligin-Coated Glass Surface.

PubMed

Funahashi, Junichiro; Tanaka, Hiromitsu; Hirano, Tomoo

2018-01-01

Fast repetitive synaptic transmission depends on efficient exocytosis and retrieval of synaptic vesicles around a presynaptic active zone. However, the functional organization of an active zone and regulatory mechanisms of exocytosis, endocytosis and reconstruction of release-competent synaptic vesicles have not been fully elucidated. By developing a novel visualization method, we attempted to identify the location of exocytosis of a single synaptic vesicle within an active zone and examined movement of synaptic vesicle protein synaptophysin (Syp) after exocytosis. Using cultured hippocampal neurons, we induced formation of active-zone-like membranes (AZLMs) directly adjacent and parallel to a glass surface coated with neuroligin, and imaged Syp fused to super-ecliptic pHluorin (Syp-SEP) after its translocation to the plasma membrane from a synaptic vesicle using total internal reflection fluorescence microscopy (TIRFM). An AZLM showed characteristic molecular and functional properties of a presynaptic active zone. It contained active zone proteins, cytomatrix at the active zone-associated structural protein (CAST), Bassoon, Piccolo, Munc13 and RIM, and showed an increase in intracellular Ca 2+ concentration upon electrical stimulation. In addition, single-pulse stimulation sometimes induced a transient increase of Syp-SEP signal followed by lateral spread in an AZLM, which was considered to reflect an exocytosis event of a single synaptic vesicle. The diffusion coefficient of Syp-SEP on the presynaptic plasma membrane after the membrane fusion was estimated to be 0.17-0.19 μm 2 /s, suggesting that Syp-SEP diffused without significant obstruction. Synchronous exocytosis just after the electrical stimulation tended to occur at multiple restricted sites within an AZLM, whereas locations of asynchronous release occurring later after the stimulation tended to be more scattered.

Phospholipid bilayer-perturbing properties underlying lysis induced by pH-sensitive cationic lysine-based surfactants in biomembranes.

PubMed

Nogueira, Daniele Rubert; Mitjans, Montserrat; Busquets, M Antonia; Pérez, Lourdes; Vinardell, M Pilar

2012-08-14

Amino acid-based surfactants constitute an important class of natural surface-active biomolecules with an unpredictable number of industrial applications. To gain a better mechanistic understanding of surfactant-induced membrane destabilization, we assessed the phospholipid bilayer-perturbing properties of new cationic lysine-based surfactants. We used erythrocytes as biomembrane models to study the hemolytic activity of surfactants and their effects on cells' osmotic resistance and morphology, as well as on membrane fluidity and membrane protein profile with varying pH. The antihemolytic capacity of amphiphiles correlated negatively with the length of the alkyl chain. Anisotropy measurements showed that the pH-sensitive surfactants, with the positive charge on the α-amino group of lysine, significantly increased membrane fluidity at acidic conditions. SDS-PAGE analysis revealed that surfactants induced significant degradation of membrane proteins in hypo-osmotic medium and at pH 5.4. By scanning electron microscopy examinations, we corroborated the interaction of surfactants with lipid bilayer. We found that varying the surfactant chemical structure is a way to modulate the positioning of the molecule inside bilayer and, thus, the overall effect on the membrane. Our work showed that pH-sensitive lysine-based surfactants significantly disturb the lipid bilayer of biomembranes especially at acidic conditions, which suggests that these compounds are promising as a new class of multifunctional bioactive excipients for active intracellular drug delivery.

PAQR-2 Regulates Fatty Acid Desaturation during Cold Adaptation in C. elegans

PubMed Central

Svensk, Emma; Ståhlman, Marcus; Andersson, Carl-Henrik; Johansson, Maja; Borén, Jan; Pilon, Marc

2013-01-01

C. elegans PAQR-2 is homologous to the insulin-sensitizing adiponectin receptors in mammals, and essential for adaptation to growth at 15°C, a low but usually acceptable temperature for this organism. By screening for novel paqr-2 suppressors, we identified mutations in genes involved in phosphatidylcholine synthesis (cept-1, pcyt-1 and sams-1) and fatty acid metabolism (ech-7, hacd-1, mdt-15, nhr-49 and sbp-1). We then show genetic evidence that paqr-2, phosphatidylcholines, sbp-1 and Δ9-desaturases form a cold adaptation pathway that regulates the increase in unsaturated fatty acids necessary to retain membrane fluidity at low temperatures. This model is supported by the observations that the paqr-2 suppressors normalize the levels of saturated fatty acids, and that low concentrations of detergents that increase membrane fluidity can rescue the paqr-2 mutant. PMID:24068966

PAQR-2 regulates fatty acid desaturation during cold adaptation in C. elegans.

PubMed

Svensk, Emma; Ståhlman, Marcus; Andersson, Carl-Henrik; Johansson, Maja; Borén, Jan; Pilon, Marc

2013-01-01

C. elegans PAQR-2 is homologous to the insulin-sensitizing adiponectin receptors in mammals, and essential for adaptation to growth at 15°C, a low but usually acceptable temperature for this organism. By screening for novel paqr-2 suppressors, we identified mutations in genes involved in phosphatidylcholine synthesis (cept-1, pcyt-1 and sams-1) and fatty acid metabolism (ech-7, hacd-1, mdt-15, nhr-49 and sbp-1). We then show genetic evidence that paqr-2, phosphatidylcholines, sbp-1 and Δ9-desaturases form a cold adaptation pathway that regulates the increase in unsaturated fatty acids necessary to retain membrane fluidity at low temperatures. This model is supported by the observations that the paqr-2 suppressors normalize the levels of saturated fatty acids, and that low concentrations of detergents that increase membrane fluidity can rescue the paqr-2 mutant.

Protons Regulate Vesicular Glutamate Transporters through an Allosteric Mechanism.

PubMed

Eriksen, Jacob; Chang, Roger; McGregor, Matt; Silm, Katlin; Suzuki, Toshiharu; Edwards, Robert H

2016-05-18

The quantal nature of synaptic transmission requires a mechanism to transport neurotransmitter into synaptic vesicles without promoting non-vesicular efflux across the plasma membrane. Indeed, the vesicular transport of most classical transmitters involves a mechanism of H(+) exchange, which restricts flux to acidic membranes such as synaptic vesicles. However, vesicular transport of the principal excitatory transmitter glutamate depends primarily on membrane potential, which would drive non-vesicular efflux, and the role of protons is unclear. Adapting electrophysiology to record currents associated with the vesicular glutamate transporters (VGLUTs), we characterize a chloride conductance that is gated by lumenal protons and chloride and supports glutamate uptake. Rather than coupling stoichiometrically to glutamate flux, lumenal protons and chloride allosterically activate vesicular glutamate transport. Gating by protons serves to inhibit what would otherwise be substantial non-vesicular glutamate efflux at the plasma membrane, thereby restricting VGLUT activity to synaptic vesicles. Copyright © 2016 Elsevier Inc. All rights reserved.

Differential roles of nonsynaptic and synaptic plasticity in operant reward learning-induced compulsive behavior.

PubMed

Sieling, Fred; Bédécarrats, Alexis; Simmers, John; Prinz, Astrid A; Nargeot, Romuald

2014-05-05

Rewarding stimuli in associative learning can transform the irregularly and infrequently generated motor patterns underlying motivated behaviors into output for accelerated and stereotyped repetitive action. This transition to compulsive behavioral expression is associated with modified synaptic and membrane properties of central neurons, but establishing the causal relationships between cellular plasticity and motor adaptation has remained a challenge. We found previously that changes in the intrinsic excitability and electrical synapses of identified neurons in Aplysia's central pattern-generating network for feeding are correlated with a switch to compulsive-like motor output expression induced by in vivo operant conditioning. Here, we used specific computer-simulated ionic currents in vitro to selectively replicate or suppress the membrane and synaptic plasticity resulting from this learning. In naive in vitro preparations, such experimental manipulation of neuronal membrane properties alone increased the frequency but not the regularity of feeding motor output found in preparations from operantly trained animals. On the other hand, changes in synaptic strength alone switched the regularity but not the frequency of feeding output from naive to trained states. However, simultaneously imposed changes in both membrane and synaptic properties reproduced both major aspects of the motor plasticity. Conversely, in preparations from trained animals, experimental suppression of the membrane and synaptic plasticity abolished the increase in frequency and regularity of the learned motor output expression. These data establish direct causality for the contributions of distinct synaptic and nonsynaptic adaptive processes to complementary facets of a compulsive behavior resulting from operant reward learning. Copyright © 2014 Elsevier Ltd. All rights reserved.

Effects of DHA-phospholipids, melatonin and tryptophan supplementation on erythrocyte membrane physico-chemical properties in elderly patients suffering from mild cognitive impairment.

PubMed

Cazzola, Roberta; Rondanelli, Mariangela; Faliva, Milena; Cestaro, Benvenuto

2012-12-01

A randomized, double-blind placebo-controlled clinical trial was carried out to assess the efficacy of a docosahexenoic acid (DHA)-phospholipids, melatonin and tryptophan supplemented diet in improving the erythrocyte oxidative stress, membrane fluidity and membrane-bound enzyme activities of elderly subjects suffering from mild cognitive impairment (MCI). These subjects were randomly assigned to the supplement group (11 subjects, 9F and 2M; age 85.3±5.3y) or placebo group (14-matched subjects, 11F and 3M; 86.1±6.5). The duration of the treatment was 12weeks. The placebo group showed no significant changes in erythrocyte membrane composition and function. The erythrocyte membranes of the supplement group showed a significant increase in eicosapentenoic acid, docosapentenoic acid and DHA concentrations and a significant decrease in arachidonic acid, malondialdehyde and lipofuscin levels. These changes in membrane composition resulted in an increase in the unsaturation index, membrane fluidity and acetylcholine esterase activity. Moreover, a significant increase in the ratio between reduced and oxidized glutathione was observed in the erythrocyte of the supplement group. Although this study is a preliminary investigation, we believe these findings to be of great speculative and interpretative interest to better understand the complex and multi-factorial mechanisms behind the possible links between diets, their functional components and possible molecular processes that contribute to increasing the risk of developing MCI and Alzheimer's. Copyright © 2012 Elsevier Inc. All rights reserved.

Synaptic Vesicle Endocytosis

PubMed Central

Saheki, Yasunori; De Camilli, Pietro

2012-01-01

Neurons can sustain high rates of synaptic transmission without exhausting their supply of synaptic vesicles. This property relies on a highly efficient local endocytic recycling of synaptic vesicle membranes, which can be reused for hundreds, possibly thousands, of exo-endocytic cycles. Morphological, physiological, molecular, and genetic studies over the last four decades have provided insight into the membrane traffic reactions that govern this recycling and its regulation. These studies have shown that synaptic vesicle endocytosis capitalizes on fundamental and general endocytic mechanisms but also involves neuron-specific adaptations of such mechanisms. Thus, investigations of these processes have advanced not only the field of synaptic transmission but also, more generally, the field of endocytosis. This article summarizes current information on synaptic vesicle endocytosis with an emphasis on the underlying molecular mechanisms and with a special focus on clathrin-mediated endocytosis, the predominant pathway of synaptic vesicle protein internalization. PMID:22763746

Physiological and biochemical effects of 17β estradiol in aging female rat brain.

PubMed

Kumar, Pardeep; Taha, Asia; Kale, R K; Cowsik, S M; Baquer, Najma Zaheer

2011-07-01

Aging in females and males is considered as the end of natural protection against age related diseases like osteoporosis, coronary heart disease, diabetes, Alzheimer's disease and Parkinson's disease. These changes increase during menopausal condition in females when the level of estradiol is decreased. The objective of this study was to observe the changes in activities of monoamine oxidase, glucose transporter-4 levels, membrane fluidity, lipid peroxidation levels and lipofuscin accumulation occurring in brains of female rats of 3 months (young), 12 months (adult) and 24 months (old) age groups, and to see whether these changes are restored to normal levels after exogenous administration of estradiol (0.1 μg/g body weight for 1 month). The results obtained in the present work revealed that normal aging was associated with significant increases in the activity of monoamine oxidase, lipid peroxidation levels and lipofuscin accumulation in the brains of aging female rats, and a decrease in glucose transporter-4 level and membrane fluidity. Our data showed that estradiol treatment significantly decreased monoamine oxidase activity, lipid peroxidation and lipofuscin accumulation in brain regions of aging rats, and a reversal of glucose transporter-4 levels and membrane fluidity was achieved, therefore it can be concluded from the present findings that estradiol's beneficial effects seemed to arise from its antilipofuscin, antioxidant and antilipidperoxidative effects, implying an overall anti-aging action. The results of this study will be useful for pharmacological modification of the aging process and applying new strategies for control of age related disorders. Copyright © 2011 Elsevier Inc. All rights reserved.

Mechanical Characterization of Hybrid Vesicles Based on Linear Poly(Dimethylsiloxane-b-Ethylene Oxide) and Poly(Butadiene-b-Ethylene Oxide) Block Copolymers

PubMed Central

Gaspard, Jeffery; Casey, Liam M.; Rozin, Matt; Munoz-Pinto, Dany J.; Silas, James A.; Hahn, Mariah S.

2016-01-01

Poly(dimethylsiloxane-ethylene oxide) (PDMS-PEO) and poly(butadiene-b-ethylene oxide) (PBd-PEO) are two block copolymers which separately form vesicles with disparate membrane permeabilities and fluidities. Thus, hybrid vesicles formed from both PDMS-PEO and PBd-PEO may ultimately allow for systematic, application-specific tuning of vesicle membrane fluidity and permeability. However, given the relatively low strength previously noted for comb-type PDMS-PEO vesicles, the mechanical robustness of the resulting hybrid vesicles must first be confirmed. Toward this end, we have characterized the mechanical behavior of vesicles formed from mixtures of linear PDMS-PEO and linear PBd-PEO using micropipette aspiration. Tension versus strain plots of pure PDMS12-PEO46 vesicles revealed a non-linear response in the high tension regime, in contrast to the approximately linear response of pure PBd33-PEO20 vesicles. Remarkably, the area expansion modulus, critical tension, and cohesive energy density of PDMS12-PEO46 vesicles were each significantly greater than for PBd33-PEO20 vesicles, although critical strain was not significantly different between these vesicle types. PDMS12-PEO46/PBd33-PEO20 hybrid vesicles generally displayed graded responses in between that of the pure component vesicles. Thus, the PDMS12-PEO46/PBd33-PEO20 hybrid vesicles retained or exceeded the strength and toughness characteristic of pure PBd-PEO vesicles, indicating that future assessment of the membrane permeability and fluidity of these hybrid vesicles may be warranted. PMID:26999148

Interaction of curcumin with 1,2-dioctadecanoyl-sn-glycero-3-phosphocholine liposomes: Intercalation of rhamnolipids enhances membrane fluidity, permeability and stability of drug molecule.

PubMed

Moussa, Zeinab; Chebl, Mazhar; Patra, Digambara

2017-01-01

Stability of curcumin in neutral and alkaline buffer conditions has been a serious concern for its medicinal applications. We demonstrate that the stability of curucmin can be improved in 1,2-Dioctadecanoyl-sn-glycero-3-phosphocholine (DSPC) liposomes. Curcumin strongly partition into liquid crystalline phase compared to solid gel phase of DSPC liposomes. Variation of fluorescence intensity of curcumin associated with liposomes with temperature successfully determines phase transition temperature of DSPC liposomes. However, at higher molar ratio curcumin can influence phase transition temperature by intercalating into deep hydrophobic layer of liposomes and facilitating fusion of two membrane phases. Rhamnolipids (RLs) are recently being applied for various biomedical applications. Here, we have explored new insight on intercalation of rhamnolipids with DSPC liposomes. Intercalation of rhamnolipids exceptionally increases partition of curcumin into solid gel phase of DSPC liposomes, whereas this increase is moderate in liquid crystalline phase. Fluorescence quenching study establishes that permeability and fluidity of the DSPC liposomes are enhanced in the presence of RLs. Membrane permeability and fluidity can be improved further by increasing the percentage of RLs in DSPC liposomes. The phase transition temperature of DSPC liposomes decreases with increase in percentage of RLs in DSPC liposomes by encouraging fusion between solid gel and liquid crystalline phases. Intercalation of RLs is found to further boost stability of drug, curcumin, in DSPC liposomes. Thus, mixing RLs with DSPC liposomes could potentially serve as a good candidate for drug delivery application. Copyright © 2016 Elsevier B.V. All rights reserved.

Neuromedin N: high affinity interaction with brain neurotensin receptors and rapid inactivation by brain synaptic peptidases.

PubMed

Checler, F; Vincent, J P; Kitabgi, P

1986-07-31

Neuromedin N (NN) is a novel neurotensin (NT)-like hexapeptide recently isolated from porcine spinal cord. NN competitively inhibited the binding of monoiodinated [Trp11]NT to rat brain synaptic membranes with a 19-fold lower potency than NT. In the presence of 1 mM 1,10-phenanthroline or 10 microM bestatin, the potency of NN relative to NT was increased about 5-fold. NN was readily degraded by rat brain synaptic membranes, and NN-(2-6) was the major degradation product. NN-(2-6) did not bind to NT receptors at concentrations up to 1 microM whether or not peptidase inhibitors were present in the binding assay. The rate of degradation by synaptic membranes was nearly 2.5 times higher for NN than for NT. NN degradation by membranes was totally prevented by 1,10-phenanthroline and markedly inhibited by bestatin. The presence of NN in the central nervous system, its high potency to interact with brain NT receptors and its rapid inactivation by brain synaptic peptidases make it a potential neurotransmitter candidate acting at the NT receptor.

In vivo voltage-dependent influences on summation of synaptic potentials in neurons of the lateral nucleus of the amygdala

PubMed Central

Rosenkranz, J. Amiel

2012-01-01

The amygdala has a fundamental role in driving affective behaviors in response to sensory cues. To accomplish this, neurons of the lateral nucleus (LAT) must integrate a large number of synaptic inputs. A wide range of factors influence synaptic integration, including membrane potential, voltage-gated ion channels and GABAergic inhibition. However, little is known about how these factors modulate integration of synaptic inputs in LAT neurons in vivo. The purpose of this study was to determine the voltage-dependent factors that modify in vivo integration of synaptic inputs in the soma of LAT neurons. In vivo intracellular recordings from anesthetized rats were used to measure post-synaptic potentials (PSPs) and clusters of PSPs across a range of membrane potentials. These studies found that the relationship between membrane potential and PSP clusters was sublinear, due to a reduction of cluster amplitude and area at depolarized membrane potentials. In combination with intracellular delivery of pharmacological agents, it was found that the voltage-dependent suppression of PSP clusters was sensitive to tetraethylammonium (TEA), but not cesium or a blocker of fast GABAergic inhibition. These findings indicate that integration of PSPs in LAT neurons in vivo is strongly modified by somatic membrane potential, likely through voltage-dependent TEA-sensitive potassium channels. Conditions that lead to a shift in membrane potential, or a modulation of the number or function of these ion channels will lead to a more uniform capacity for integration across voltages, and perhaps greatly facilitate amygdala-dependent behaviors. PMID:22989917

pH-induced change in cell susceptibility to butanol in a high butanol-tolerant bacterium, Enterococcus faecalis strain CM4A.

PubMed

Kanno, Manabu; Tamaki, Hideyuki; Mitani, Yasuo; Kimura, Nobutada; Hanada, Satoshi; Kamagata, Yoichi

2015-01-01

Though butanol is considered as a potential biofuel, its toxicity toward microorganisms is the main bottleneck for the biological butanol production. Recently, butanol-tolerant bacteria have been proposed as alternative butanol production hosts overcoming the end product inhibition. One remaining key issue to be addressed is how physicochemical properties such as pH and temperature affect microbial butanol tolerance during cultivation and fermentation. We investigated the pH effect on butanol tolerance of a high butanol-tolerant bacterium, Enterococcus faecalis strain CM4A. The strain grew over a broad pH range (pH 4.0 to 12.0) and preferred alkaline pH (pH 8.0 and 10.0) in the absence of butanol. However, in the presence of butanol, strain CM4A grew better under acidic and neutral pH conditions (pH 6.0 and 6.8). Membrane fatty acid analysis revealed that the cells exposed to butanol exhibited increased cyclopropane and saturated fatty acids, which contribute to butanol tolerance of the strain by decreasing membrane fluidity, more evidently at acidic and neutral pH than at alkaline pH. Meanwhile, the strain grown under alkaline pH without butanol increased short chain fatty acids, which is involved in increasing membrane fluidity for alkaline adaptation. Such a change was not observed in the cells grown under alkaline pH with butanol. These results suggested that strain CM4A simultaneously exposed to butanol and alkali stresses was not likely able to properly adjust membrane fluidity due to the opposite response to each stress and thereby showed low butanol tolerance under alkaline pH. Indeed, the cells exposed to butanol at alkaline pH showed an irregular shape with disrupted membrane structure under transmission electron microscopy observation, which also indicated the impact of butanol and alkali stresses on functioning of cellular membrane. The study clearly demonstrated the alkaline pH-induced increase of cell susceptibility to butanol in the tested strain. Our findings indicate the non-negligible impact of pH on microbial butanol tolerance, providing a new insight into efficient butanol production.

Remodeling of the postsynaptic plasma membrane during neural development.

PubMed

Tulodziecka, Karolina; Diaz-Rohrer, Barbara B; Farley, Madeline M; Chan, Robin B; Di Paolo, Gilbert; Levental, Kandice R; Waxham, M Neal; Levental, Ilya

2016-11-07

Neuronal synapses are the fundamental units of neural signal transduction and must maintain exquisite signal fidelity while also accommodating the plasticity that underlies learning and development. To achieve these goals, the molecular composition and spatial organization of synaptic terminals must be tightly regulated; however, little is known about the regulation of lipid composition and organization in synaptic membranes. Here we quantify the comprehensive lipidome of rat synaptic membranes during postnatal development and observe dramatic developmental lipidomic remodeling during the first 60 postnatal days, including progressive accumulation of cholesterol, plasmalogens, and sphingolipids. Further analysis of membranes associated with isolated postsynaptic densities (PSDs) suggests the PSD-associated postsynaptic plasma membrane (PSD-PM) as one specific location of synaptic remodeling. We analyze the biophysical consequences of developmental remodeling in reconstituted synaptic membranes and observe remarkably stable microdomains, with the stability of domains increasing with developmental age. We rationalize the developmental accumulation of microdomain-forming lipids in synapses by proposing a mechanism by which palmitoylation of the immobilized scaffold protein PSD-95 nucleates domains at the postsynaptic plasma membrane. These results reveal developmental changes in lipid composition and palmitoylation that facilitate the formation of postsynaptic membrane microdomains, which may serve key roles in the function of the neuronal synapse. © 2016 Tulodziecka et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Coupling of lipid membrane elasticity and in-plane dynamics

NASA Astrophysics Data System (ADS)

Tsang, Kuan-Yu; Lai, Yei-Chen; Chiang, Yun-Wei; Chen, Yi-Fan

2017-07-01

Biomembranes exhibit liquid and solid features concomitantly with their in-plane fluidity and elasticity tightly regulated by cells. Here, we present experimental evidence supporting the existence of the dynamics-elasticity correlations for lipid membranes and propose a mechanism involving molecular packing densities to explain them. This paper thereby unifies, at the molecular level, the aspects of the continuum mechanics long used to model the two membrane features. This ultimately may elucidate the universal physical principles governing the cellular phenomena involving biomembranes.

Liposomes composed of unsaturated lipids for membrane modification of human erythrocytes.

PubMed

Stoll, Christoph; Holovati, Jelena L; Acker, Jason P; Wolkers, Willem F

2011-01-01

Previous studies have shown that certain saturated lipids protect red blood cells (RBCs) during hypothermic storage but provide little protection during freezing or freeze-drying, whereas various unsaturated lipids destabilize RBCs during hypothermic storage but protect during freezing and freeze-drying. The protective effect of liposomes has been attributed to membrane modifications. We have previously shown that cholesterol exchange and lipid transfer between liposomes composed of saturated lipids and RBCs critically depends on the length of the lipid acyl chains. In this study the effect of unsaturated lipids with differences in their number of unsaturated bonds (18:0/18:1, 18:1/18:1, 18:2/18:2) on RBC membrane properties has been studied. RBCs were incubated in the presence of liposomes and both the liposomal and RBC fraction were analyzed by Fourier transform infrared spectroscopy (FTIR) after incubation. The liposomes caused an increase in RBC membrane conformational disorder at suprazero temperatures. The fluidizing effect of the liposomes on the RBC membranes, however, was found to be similar for the different lipids irrespective of their unsaturation level. The gel to liquid crystalline phase transition temperature of the liposomes increased after incubation with RBCs. RBC membrane fluidity increased linearly during the first 8 hours of incubation in the presence of liposomes. The increase in RBC membrane fluidity was found to be temperature dependent and displayed Arrhenius behaviour between 20 and 40°C, with an activation energy of 88 kJ mol⁻¹. Taken together, liposomes composed of unsaturated lipids increase RBC membrane conformational disorder, which could explain their cryoprotective action.

Polycyclic aromatic hydrocarbons in model bacterial membranes - Langmuir monolayer studies.

PubMed

Broniatowski, Marcin; Binczycka, Martyna; Wójcik, Aneta; Flasiński, Michał; Wydro, Paweł

2017-12-01

High molecular weight polycyclic aromatic hydrocarbons (HMW-PAHs) are persistent organic pollutants which due to their limited biodegradability accumulate in soils where their increased presence can lead to the impoverishment of the decomposer organisms. As very hydrophobic PAHs easily penetrate cellular membranes of soil bacteria and can be incorporated therein, changing the membrane fluidity and other functions which in consequence can lead to the death of the organism. The structure and size of PAH molecule can be crucial for its membrane activity; however the correlation between PAH structure and its interaction with phospholipids have not been investigated so far. In our studies we applied phospholipid Langmuir monolayers as model bacterial membranes and investigated how the incorporation of six structurally different PAH molecules change the membrane texture and physical properties. In our studies we registered surface pressure and surface potential isotherms upon the monolayer compression, visualized the monolayer texture with the application of Brewster angle microscopy and searched the ordering of the film-forming molecules with molecular resolution with the application of grazing incidence X-ray diffraction (GIXD) method. It turned out that the phospholipid-PAH interactions are strictly structure dependent. Four and five-ring PAHs of the angular or cluster geometry can be incorporated into the model membranes changing profoundly their textures and fluidity; whereas linear or large cluster PAHs cannot be incorporated and separate from the lipid matrix. The observed phenomena were explained based on structural similarities of the applied PAHs with membrane steroids and hopanoids. Copyright © 2017. Published by Elsevier B.V.

Effects of clary sage oil and its main components, linalool and linalyl acetate, on the plasma membrane of Candida albicans: an in vivo EPR study.

PubMed

Blaskó, Ágnes; Gazdag, Zoltán; Gróf, Pál; Máté, Gábor; Sárosi, Szilvia; Krisch, Judit; Vágvölgyi, Csaba; Makszin, Lilla; Pesti, Miklós

2017-02-01

The effects of clary sage (Salvia sclarea L.) oil (CS-oil), and its two main components, linalool (Lol) and linalyl acetate (LA), on cells of the eukaryotic human pathogen yeast Candida albicans were studied. Dynamic and thermodynamic properties of the plasma membrane were investigated by electron paramagnetic resonance (EPR) spectroscopy, with 5-doxylstearic acid (5-SASL) and 16-SASL as spin labels. The monitoring of the head group regions with 5-SASL revealed break-point frequency decrease in a temperature dependent manner of the plasma membrane between 9.55 and 13.15 °C in untreated, in CS-oil-, Lol- and LA-treated membranes. The results suggest a significant increase in fluidity of the treated plasma membranes close to the head groups. Comparison of the results observed with the two spin labels demonstrated that CS-oil and LA induced an increased level of fluidization at both depths of the plasma membrane. Whereas Lol treatment induced a less (1 %) ordered bilayer organization in the superficial regions and an increased (10 %) order of the membrane leaflet in deeper layers. Acute toxicity tests and EPR results indicated that both the apoptotic and the effects exerted on the plasma membrane fluidity depended on the composition and chemical structure of the examined materials. In comparison with the control, treatment with CS-oil, Lol or LA induced 13.0, 12.3 and 26.4 % loss respectively, of the metabolites absorbing at 260 nm, as a biological consequence of the plasma membrane fluidizing effects. Our results confirmed that clary sage oil causes plasma membrane perturbations which leads to cell apoptosis process.

Cytoplasmic membrane response to copper and nickel in Acidithiobacillus ferrooxidans.

PubMed

Mykytczuk, N C S; Trevors, J T; Ferroni, G D; Leduc, L G

2011-03-20

Metal tolerance has been found to vary among Acidithiobacillus ferrooxidans strains and this can impact the efficiency of biomining practices. To explain observed strain variability for differences in metal tolerance we examined the effects of Cu(2+) and Ni(2+) concentrations (1-200 mM) on cytoplasmic membrane properties of two A. ferrooxidans type strains (ATCC 23270 and 19859) and four strains isolated from AMD water around Sudbury, Ontario, Canada. Growth rate, membrane fluidity and phase, determined from the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), and fatty acid profiles indicated that three different modes of adaptation were present and could separate between strains showing moderate, or high metal tolerance from more sensitive strains. To compensate for the membrane ordering effects of the metals, significant remodelling of the membrane was used to either maintain homeoviscous adaptation in the moderately tolerant strains or to increase membrane fluidity in the sensitive strains. Shifts in the gel-to-liquid crystalline transition temperature in the moderately tolerant strains led to multiple phase transitions, increasing the potential for phase separation and compromised membrane integrity. The metal-tolerant strain however, was able to tolerate increases in membrane order without significant compensation via fatty acid composition. Our multivariate analyses show a common adaptive response which involves changes in the abundant 16:0 and 18:1 fatty acids. However, fatty acid composition and membrane properties showed no difference in response to either copper or nickel suggesting that adaptive response was non-specific and tolerance dependent. We demonstrate that strain variation can be evaluated using differences in membrane properties as intrinsic determinants of metal susceptibility. Copyright © 2010 Elsevier GmbH. All rights reserved.

Metabolism of Fructooligosaccharides in Lactobacillus plantarum ST-III via Differential Gene Transcription and Alteration of Cell Membrane Fluidity

PubMed Central

Chen, Chen; Zhao, Guozhong

2015-01-01

Although fructooligosaccharides (FOS) can selectively stimulate the growth and activity of probiotics and beneficially modulate the balance of intestinal microbiota, knowledge of the molecular mechanism for FOS metabolism by probiotics is still limited. Here a combined transcriptomic and physiological approach was used to survey the global alterations that occurred during the logarithmic growth of Lactobacillus plantarum ST-III using FOS or glucose as the sole carbon source. A total of 363 genes were differentially transcribed; in particular, two gene clusters were induced by FOS. Gene inactivation revealed that both of the clusters participated in the metabolism of FOS, which were transported across the membrane by two phosphotransferase systems (PTSs) and were subsequently hydrolyzed by a β-fructofuranosidase (SacA) in the cytoplasm. Combining the measurements of the transcriptome- and membrane-related features, we discovered that the genes involved in the biosynthesis of fatty acids (FAs) were repressed in cells grown on FOS; as a result, the FA profiles were altered by shortening of the carbon chains, after which membrane fluidity increased in response to FOS transport and utilization. Furthermore, incremental production of acetate was observed in both the transcriptomic and the metabolic experiments. Our results provided new insights into gene transcription, the production of metabolites, and membrane alterations that could explain FOS metabolism in L. plantarum. PMID:26319882

High-level iron mitigates fusaricidin-induced membrane damage and reduces membrane fluidity leading to enhanced drug resistance in Bacillus subtilis.

PubMed

Yu, Wen-Bang; Ye, Bang-Ce

2016-05-01

Fusaricidins are a class of cyclic lipopeptide antibiotics that have strong antifungal activities against plant pathogenic fungi and excellent bactericidal activities against Gram-positive bacteria. The mechanism through which fusaricidin exerts its action is not yet entirely clear. To investigate the mode of action of fusaricidin, we determined the physiological and transcriptional responses of Bacillus subtilis to fusaricidin treatment by using a systems-level approach. Our data show that fusaricidin rapidly induced the expression of σ(W) regulon and caused membrane damage in B. subtilis. We further demonstrated that ferric ions play multiple roles in the action of fusaricidin on B. subtilis. Iron deprivation blocked the formation of hydroxyl radical in the cells and significantly inhibited the bactericidal activity of fusaricidin. Conversely, high levels of iron (>2 mM) repressed the expression of BkdR regulon, resulting in a smaller cellular pool of branched-chain precursors for iso- and anteiso-branched fatty acids, which in turn led to a decrease in the proportion of branched-chain fatty acids in the membrane of B. subtilis. This change in membrane composition reduced its bilayer fluidity and increased its resistance to antimicrobial agents. In conclusion, our experiments uncovered some novel interactions and a synergism between cellular iron levels and drug resistance in Gram-positive bacteria. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Changes Caused by Fruit Extracts in the Lipid Phase of Biological and Model Membranes

PubMed Central

Pruchnik, Hanna; Oszmiański, Jan; Sarapuk, Janusz; Kleszczyńska, Halina

2010-01-01

The aim of the study was to determine changes incurred by polyphenolic compounds from selected fruits in the lipid phase of the erythrocyte membrane, in liposomes formed of erythrocyte lipids and phosphatidylcholine liposomes. In particular, the effect of extracts from apple, chokeberry, and strawberry on the red blood cell morphology, on packing order in the lipid hydrophilic phase, on fluidity of the hydrophobic phase, as well as on the temperature of phase transition in DPPC liposomes was studied. In the erythrocyte population, the proportions of echinocytes increased due to incorporation of polyphenolic compounds. Fluorimetry with a laurdan probe indicated increased packing density in the hydrophilic phase of the membrane in presence of polyphenolic extracts, the highest effect being observed for the apple extract. Using the fluorescence probes DPH and TMA-DPH, no effect was noted inside the hydrophobic phase of the membrane, as the lipid bilayer fluidity was not modified. The polyphenolic extracts slightly lowered the phase transition temperature of phosphatidylcholine liposomes. The studies have shown that the phenolic compounds contained in the extracts incorporate into the outer region of the erythrocyte membrane, affecting its shape and lipid packing order, which is reflected in the increasing number of echinocytes. The compounds also penetrate the outer part of the external lipid layer of liposomes formed of natural and DPPC lipids, changing its packing order. PMID:21423329

Specific interaction of postsynaptic densities with membrane rafts isolated from synaptic plasma membranes.

PubMed

Liu, Qian; Yao, Wei-Dong; Suzuki, Tatsuo

2013-06-01

Postsynaptic membrane rafts are believed to play important roles in synaptic signaling, plasticity, and maintenance. We recently demonstrated the presence, at the electron microscopic level, of complexes consisting of membrane rafts and postsynaptic densities (PSDs) in detergent-resistant membranes (DRMs) prepared from synaptic plasma membranes (SPMs) ( Suzuki et al., 2011 , J Neurochem, 119, 64-77). To further explore these complexes, here we investigated the nature of the binding between purified SPM-DRMs and PSDs in vitro. In binding experiments, we used SPM-DRMs prepared after treating SPMs with n-octyl-β-d-glucoside, because at concentrations of 1.0% or higher it completely separates SPM-DRMs and PSDs, providing substantially PSD-free unique SPM-DRMs as well as DRM-free PSDs. PSD binding to PSD-free DRMs was identified by mass spectrometry, Western blotting, and electron microscopy. PSD proteins were not incorporated into SPMs, and significantly less PSD proteins were incorporated into DRMs prepared from liver membranes, providing in vitro evidence that binding of PSDs to DRMs is specific and suggestion of the presence of specific interacting molecules. These specific interactions may have important roles in synaptic development, function, and plasticity in vivo. In addition, the binding system we developed may be a good tool to search for binding molecules and binding mechanisms between PSDs and rafts.

The in vitro comparative study of the effect of BPA, BPS, BPF and BPAF on human erythrocyte membrane; perturbations in membrane fluidity, alterations in conformational state and damage to proteins, changes in ATP level and Na+/K+ ATPase and AChE activities.

PubMed

Maćczak, Aneta; Duchnowicz, Piotr; Sicińska, Paulina; Koter-Michalak, Maria; Bukowska, Bożena; Michałowicz, Jaromir

2017-12-01

Bisphenols are massively used in the industry, and thus the exposure of biota including humans to these substances has been noted. In this study we have assessed the effect of BPA and its selected analogs, i.e. BPS, BPF and BPAF on membrane of human red blood cells, which is the first barrier that must be overcome by xenobiotics penetrating the cell, and is commonly utilized as a model in the investigation of the effect of different xenobiotics on various cell types. Red blood cells were incubated with BPA and its analogs in the concentrations ranging from 0.1 to 250 μg/ml for 4 h and 24 h. We have noted that the compounds studied altered membrane fluidity at its hydrophobic region, increased internal viscosity and osmotic fragility of the erythrocytes and altered conformational state of membrane proteins. Moreover, bisphenols examined increased thiol groups level, caused oxidative damage to membrane proteins, decreased ATP level, depleted the activity of Na+/K + ATPase and changed the activity of AChE in human red blood cells. It has been shown that the strongest changes were noted in cells treated with BPAF, while BPS caused the weakest (or none) alterations in the parameters studied. Copyright © 2017 Elsevier Ltd. All rights reserved.

IFITM proteins-cellular inhibitors of viral entry.

PubMed

Smith, Se; Weston, S; Kellam, P; Marsh, M

2014-02-01

Interferon inducible transmembrane (IFITM) proteins are a recently discovered family of cellular anti-viral proteins that restrict the replication of a number of enveloped and non-enveloped viruses. IFITM proteins are located in the plasma membrane and endosomal membranes, the main portals of entry for many viruses. Biochemical and membrane fusion studies suggest IFITM proteins have the ability to inhibit viral entry, possibly by modulating the fluidity of cellular membranes. Here we discuss the IFITM proteins, recent work on their mode of action, and future directions for research. Copyright © 2014 Elsevier B.V. All rights reserved.

Imaging synaptic vesicle recycling by staining and destaining vesicles with FM dyes.

PubMed

Hoopmann, Peer; Rizzoli, Silvio O; Betz, William J

2012-01-01

The synaptic vesicle is the essential organelle of the synapse. Many approaches for studying synaptic vesicle recycling have been devised, one of which, the styryl (FM) dye, is well suited for this purpose. FM dyes reversibly stain, but do not permeate, membranes; hence they can specifically label membrane-bound organelles. Their quantum yield is drastically higher when bound to membranes than when in aqueous solution. This protocol describes the imaging of synaptic vesicle recycling by staining and destaining vesicles with FM dyes. Nerve terminals are stimulated (electrically or by depolarization with high K(+)) in the presence of dye, their vesicles are then allowed to recycle, and finally dye is washed from the chamber. In neuromuscular junction (NMJ) preparations, movements of the muscle must be inhibited if imaging during stimulation is desired (e.g., by application of curare, a potent acetylcholine receptor inhibitor). The main characteristics of FM dyes are also reviewed here, as are recent FM dye monitoring techniques that have been used to investigate the kinetics of synaptic vesicle fusion.

A Novel Synaptic Vesicle Fusion Path in the Rat Cerebral Cortex: The “Saddle” Point Hypothesis

PubMed Central

Zampighi, Guido A.; Serrano, Raul; Vergara, Julio L.

2014-01-01

We improved freeze-fracture electron microscopy to study synapses in the neuropil of the rat cerebral cortex at ∼2 nm resolution and in three-dimensions. In the pre-synaptic axon, we found that “rods” assembled from short filaments protruding from the vesicle and the plasma membrane connects synaptic vesicles to the membrane of the active zone. We equated these “connector rods” to protein complexes involved in “docking” and “priming” vesicles to the active zone. Depending on their orientation, the “rods” define two synaptic vesicle-fusion paths: When parallel to the plasma membrane, the vesicles hemi-fuse anywhere (“randomly”) in the active zone following the conventional path anticipated by the SNARE hypothesis. When perpendicular to the plasma membrane, the vesicles hemi-fuse at the base of sharp crooks, called “indentations,” that are spaced 75–85 nm center-to-center, arranged in files and contained within gutters. They result from primary and secondary membrane curvatures that intersect at stationary inflection (“saddle”) points. Computer simulations indicate that this novel vesicle-fusion path evokes neurotransmitter concentration domains on the post-synaptic spine that are wider, shallower, and that reach higher average concentrations than the more conventional vesicle fusion path. In the post-synaptic spine, large (∼9× ∼15 nm) rectangular particles at densities of 72±10/ µm2 (170–240/spine) match the envelopes of the homotetrameric GluR2 AMPA-sensitive receptor. While these putative receptors join clusters, called the “post-synaptic domains,” the overwhelming majority of the rectangular particles formed bands in the “non-synaptic” plasma membrane of the spine. In conclusion, in the neuropil of the rat cerebral cortex, curvatures of the plasma membrane define a novel vesicle-fusion path that preconditions specific regions of the active zone for neurotransmitter release. We hypothesize that a change in the hybridization of the R-SNARE synaptobrevin from parallel to antiparallel swings the synapse into this novel vesicle-fusion path. PMID:24959848

[Status of vitamin A, vitamin B2, iron and an-oxidantive activity in anemic pregnant women in China].

PubMed

Yang, Fang; Ma, Ai-Guo; Zhang, Xiu-Zhen; Jiang, Dian-Chen

2006-05-01

To investigate the status of vitamin A(VA), vitamin B2 (VB2), iron and anoxidative function in anemic and non-anemic pregnant women. 426 anemic and 36 non-anemic pregnant women were included in the study. The survey of 24-hour's diet recall of pregnant women was made to evaluate intake of iron, VB2, folic acid, etc by the nutrition software provided by Beijing 301 hospital, iron and VA were measured by Radioimmunoassay (RIA) and by high-pressure liquid chromatography. VB2 status was detected using the assay for erythrocyte glutathione reductase (Egr; EC 1.6.4.2) activity. SOD and GSH-Px activities and MDA were determined using commercial kits. Peripheral blood erythrocyte membrane fluidity was detected by using 1,6-diphenyl-1,3,5-hexatriene as a probe, the degree of fluorescence polarization (P) at 25 degrees C of disrupted cells plasma membranes were compared for a variety of systems. Median intakes of protein and vitamin C met the current Chinese RNIs for pregnancy, whereas intakes of(VA) and VB2 were well below the recommendations. Intake of iron were above 90%, but the main sources of iron are vegetables. Plasma VA (1.25 micromol/L) and iron (20.57 microg/L) were lower, BGRAC (1.79) was higher than that in non-anemia group (VA 1.57 micromol/L, SF 33.16 microg/L, BGRAC 1.52). The level of plasma SOD (77.1U/ml) and the activity of GSH-Px (61.9U) were lower than those in non-anemia group (92.2U/ml, 71.6U, P < 0.05), while MDA (4.58 nmol/ml) level and erythrocyte membrane (P = 0.2622, eta = 2.7465) fluidity were higher than those non-anemia group(MDA = 3.78 nmol/ ml, P = 0.2360, eta = 2.3658). Plasam VA, VB2 and iron, antioxidantcapacity and erythrocyte membrane fluidity were decreased in the anemic pregnant women.

Oxidative Stress and Erythrocyte Membrane Alterations in Children with Autism: Correlation with Clinical Features.

PubMed

Ghezzo, Alessandro; Visconti, Paola; Abruzzo, Provvidenza M; Bolotta, Alessandra; Ferreri, Carla; Gobbi, Giuseppe; Malisardi, Gemma; Manfredini, Stefano; Marini, Marina; Nanetti, Laura; Pipitone, Emanuela; Raffaelli, Francesca; Resca, Federica; Vignini, Arianna; Mazzanti, Laura

2013-01-01

It has been suggested that oxidative stress may play a role in the pathogenesis of Autism Spectrum Disorders (ASD), but the literature reports somewhat contradictory results. To further investigate the issue, we evaluated a high number of peripheral oxidative stress parameters, and some related issues such as erythrocyte membrane functional features and lipid composition. Twenty-one autistic children (Au) aged 5 to 12 years, were gender and age-matched with 20 typically developing children (TD). Erythrocyte thiobarbituric acid reactive substances, urinary isoprostane and hexanoyl-lysine adduct levels were elevated in Au, thus confirming the occurrence of an imbalance of the redox status of Au, whilst other oxidative stress markers or associated parameters (urinary 8-oxo-dG, plasma radical absorbance capacity and carbonyl groups, erythrocyte superoxide dismutase and catalase activities) were unchanged. A very significant reduction of Na(+)/K(+)-ATPase activity (-66%, p<0.0001), a reduction of erythrocyte membrane fluidity and alteration in erythrocyte fatty acid membrane profile (increase in monounsaturated fatty acids, decrease in EPA and DHA-ω3 with a consequent increase in ω6/ω3 ratio) were found in Au compared to TD, without change in membrane sialic acid content. Some Au clinical features appear to be correlated with these findings; in particular, hyperactivity score appears to be related with some parameters of the lipidomic profile and membrane fluidity. Oxidative stress and erythrocyte membrane alterations may play a role in the pathogenesis of ASD and prompt the development of palliative therapeutic protocols. Moreover, the marked decrease in NKA could be potentially utilized as a peripheral biomarker of ASD.

Cannabinoid receptor signalling in neurodegenerative diseases: a potential role for membrane fluidity disturbance

PubMed Central

Maccarrone, M; Bernardi, G; Agrò, A Finazzi; Centonze, D

2011-01-01

Type-1 cannabinoid receptor (CB1) is the most abundant G-protein-coupled receptor (GPCR) in the brain. CB1 and its endogenous agonists, the so-called ‘endocannabinoids (eCBs)’, belong to an ancient neurosignalling system that plays important functions in neurodegenerative and neuroinflammatory disorders like Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and multiple sclerosis. For this reason, research on the therapeutic potential of drugs modulating the endogenous tone of eCBs is very intense. Several GPCRs reside within subdomains of the plasma membranes that contain high concentrations of cholesterol: the lipid rafts. Here, the hypothesis that changes in membrane fluidity alter function of the endocannabinoid system, as well as progression of particular neurodegenerative diseases, is described. To this end, the impact of membrane cholesterol on membrane properties and hence on neurodegenerative diseases, as well as on CB1 signalling in vitro and on CB1-dependent neurotransmission within the striatum, is discussed. Overall, present evidence points to the membrane environment as a critical regulator of signal transduction triggered by CB1, and calls for further studies aimed at better clarifying the contribution of membrane lipids to eCBs signalling. The results of these investigations might be exploited also for the development of novel therapeutics able to combat disorders associated with abnormal activity of CB1. LINKED ARTICLES This article is part of a themed issue on Cannabinoids in Biology and Medicine. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.163.issue-7 PMID:21323908

Bile salts stimulate mucin secretion by cultured dog gallbladder epithelial cells independent of their detergent effect.

PubMed

Klinkspoor, J H; Yoshida, T; Lee, S P

1998-05-15

1. Bile salts stimulate mucin secretion by the gallbladder epithelium. We have investigated whether this stimulatory effect is due to a detergent effect of bile salts. 2. The bile salts taurocholic acid (TC) and tauroursodeoxycholic acid (TUDC) and the detergents Triton X-100 (12.5-400 microM) and Tween-20 (0.1-3.2 mM) were applied to monolayers of cultured dog gallbladder epithelial cells. Mucin secretion was studied by measuring the secretion of [3H]N-acetyl-d-glucosamine-labelled glycoproteins. We also attempted to alter the fluidity of the apical membrane of the cells through extraction of cholesterol with beta-cyclodextrin (2.5-15 mM). The effect on TUDC-induced mucin secretion was studied. Cell viability was assessed by measuring lactate dehydrogenase (LDH) leakage or 51Cr release. 3. In contrast with the bile salts, the detergents were not able to cause an increase in mucin secretion without causing concomitant cell lysis. Concentrations of detergent that increased mucin release (>100 microM Triton X-100, >0.8 mM Tween-20), caused increased LDH release. Incubation with beta-cyclodextrin resulted in effective extraction of cholesterol without causing an increase in 51Cr release. However, no effect of the presumed altered membrane fluidity on TUDC (10 mM)-induced mucin secretion was observed. 4. The stimulatory effect of bile salts on mucin secretion by gallbladder epithelial cells is not affected by the fluidity of the apical membrane of the cells and also cannot be mimicked by other detergents. We conclude that the ability of bile salts to cause mucin secretion by the gallbladder epithelium is not determined by their detergent properties.

Rice mutants deficient in ω-3 fatty acid desaturase (FAD8) fail to acclimate to cold temperatures.

PubMed

Tovuu, Altanzaya; Zulfugarov, Ismayil S; Wu, Guangxi; Kang, In Soon; Kim, Choongrak; Moon, Byoung Yong; An, Gynheung; Lee, Choon-Hwan

2016-12-01

To investigate the role of ω-3 fatty acid (FA) desaturase (FAD8) during cold acclimation in higher plants, we characterized three independent T-DNA insertional knock-out mutants of OsFAD8 from rice (Oryza sativa L.). At room temperature (28 °C), osfad8 plants exhibited significant alterations in fatty acid (FA) unsaturation for all four investigated plastidic lipid classes. During a 5-d acclimation period at 4 °C, further changes in FA unsaturation in both wild-type (WT) and mutant plants varied according to the type of lipid. We also monitored the fluidity of the thylakoid membrane using a threshold temperature to represent the change in fluorescence. The values were altered significantly by both FAD8 mutation and cold acclimation, suggesting that factors other than FAD8 are involved in C18 FA unsaturation and fluctuations in membrane fluidity. Similarly, significant changes were noted for both the mutant and WT samples in terms of their FA compositions as well as activities related to photosystem (PS) I, PSII, and photoprotection. This included the development of non-photochemical quenching and increased zeaxanthin accumulation. Despite the relatively small changes in FA composition during cold acclimation, cold-inducible FAD8 knock-out mutants displayed strong differences in photoprotective activities and a further drop in membrane fluidity. The mutants were more sensitive than WT to short-term low-temperature stress that resulted in increased production of reactive oxygen species after 5 d of chilling. Taken together, our findings suggest that FA unsaturation by OsFAD8 is crucial for the acclimation of higher plants to low-temperature stress. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Targeting synaptic dysfunction in Alzheimer's disease by administering a specific nutrient combination.

PubMed

van Wijk, Nick; Broersen, Laus M; de Wilde, Martijn C; Hageman, Robert J J; Groenendijk, Martine; Sijben, John W C; Kamphuis, Patrick J G H

2014-01-01

Synapse loss and synaptic dysfunction are pathological processes already involved in the early stages of Alzheimer's disease (AD). Synapses consist principally of neuronal membranes, and the neuronal and synaptic losses observed in AD have been linked to the degeneration and altered composition and structure of these membranes. Consequently, synapse loss and membrane-related pathology provide viable targets for intervention in AD. The specific nutrient combination Fortasyn Connect (FC) is designed to ameliorate synapse loss and synaptic dysfunction in AD by addressing distinct nutritional needs believed to be present in these patients. This nutrient combination comprises uridine, docosahexaenoic acid, eicosapentaenoic acid, choline, phospholipids, folic acid, vitamins B12, B6, C, and E, and selenium, and is present in Souvenaid, a medical food intended for use in early AD. It has been hypothesized that FC counteracts synaptic loss and reduces membrane-related pathology in AD by providing nutritional precursors and cofactors that act together to support neuronal membrane formation and function. Preclinical studies formed the basis of this hypothesis which is being validated in a broad clinical study program investigating the potential of this nutrient combination in AD. Memory dysfunction is one key early manifestation in AD and is associated with synapse loss. The clinical studies to date show that the FC-containing medical food improves memory function and preserves functional brain network organization in mild AD compared with controls, supporting the hypothesis that this intervention counteracts synaptic dysfunction. This review provides a comprehensive overview of basic scientific studies that led to the creation of FC and of its effects in various preclinical models.

Phase transitions in methyl parben doped dipalmitoyl phosphatidylethanolamine vesicles

NASA Astrophysics Data System (ADS)

Panicker, Lata

2013-02-01

Influence of the preservative, methyl paraben (MPB), on the thermal properties of dipalmitoyl phosphatidylethanolamine (DPPE) vesicles was investigated using DSC. DSC measurement of the lipid acyl chain melting transition in DPPE membrane doped with MPB, showed MPB concentration dependant modifications in the membrane thermal properties. The interesting findings are: (1) the presence of parabens increases the membrane fluidity. (2) the MPB molecules seem to be present in the aqueous bilayer interfacial region intercalated between the neighboring lipid polar headgroup (3) high concentration of MPB favored formation of crystalline and glassy phases.

Specific binding of 15 HETE to lymphocytes. Effects on the fluidity of plasmatic membranes.

PubMed

Mexmain, S; Gualde, N; Aldigier, J C; Motta, C; Chable-Rabinovitch, H; Rigaud, M

1984-01-01

Specific binding of mouse lymphocytes for 15 HETE was examined by incubating cells with [14C]-15 HETE, 1 X 10(-8) to 1 X 10(-10)M. It was observed that the specific binding of radiolabeled 15 HETE is a function of time, of temperature and is modified by Ca2+ and dithiothreitol. When a fluorescent probe was embedded in the phospholipid core of the lymphocyte membrane and its motion analysed by fluorescence polarization, it was observed that 15 HETE increases the viscosity of the plasmatic membrane.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Towata, Tomomi; Division of Hematopoiesis, Center for AIDS Research, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811; Komizu, Yuji

Primary effusion lymphoma (PEL) is an aggressive neoplasm caused by human herpes virus-8 infection, and is generally resistant to chemotherapy. Hybrid liposomes, composed of dimyristoylphosphatidylcholine (DMPC) and polyoxyethylene (21) dodecyl ether (C{sub 12}(EO){sub 21}) (HL-21), were rapidly accumulated in the membrane of PEL cells. HL-21 also increased membrane fluidity of PEL cells, and induced caspase-3 activation along with cell death. These results suggest that HL-21 should be an effective and attractive regent for PEL treatment.

Interaction of nanoparticles with lipid membranes: a multiscale perspective.

PubMed

Montis, Costanza; Maiolo, Daniele; Alessandri, Ivano; Bergese, Paolo; Berti, Debora

2014-06-21

Freestanding lipid bilayers were challenged with 15 nm Au nanospheres either coated by a citrate layer or passivated by a protein corona. The effect of Au nanospheres on the bilayer morphology, permeability and fluidity presents strong differences or similarities, depending on the observation length scale, from the colloidal to the molecular domains. These findings suggest that the interaction between nanoparticles and lipid membranes should be conveniently treated as a multiscale phenomenon.

Interactions of plaunotol with bacterial membranes.

PubMed

Koga, T; Watanabe, H; Kawada, H; Takahashi, K; Utsui, Y; Domon, H; Ishii, C; Narita, T; Yasuda, H

1998-08-01

Plaunotol, a cytoprotective antiulcer agent, has a bactericidal effect against Helicobacter pylori, which may result from interaction of this compound with the bacterial cell membrane. The purpose of the present study was to confirm that plaunotol interacts with the H. pylori membrane. Membrane fluidities were measured using two stearic acid spin labels, namely 5-doxyl-stearic acid (in which the nitroxide group is located in the upper portion of the bacterial cell membrane) and 16-doxyl-stearic acid methyl ester (in which the nitroxide group is located deeper in the bacterial cell membrane), by means of electron spin resonance. The membrane fluidities of plaunotol-treated cells were significantly increased in the measurements made using the two spin labels. We also attempted to isolate plaunotol-resistant H. pylori in vitro by two different methods. To assess the level of resistance that could be reached, H. pylori was passaged five times on an agar plate containing subinhibitory concentrations of plaunotol or metronidazole. To measure the rate of development of resistance, H. pylori was grown with subinhibitory concentrations (0.25 x MIC) of plaunotol or metronidazole, and quantitatively plated on to medium containing 4 x MIC of the compounds. This treatment was repeated once more. No plaunotol-resistant colonies were selected by the two methods. H. pylori developed resistance to metronidazole easily and at a relatively high rate. The mechanism by which plaunotol directly fluidizes and destroys the H. pylori membrane might make it difficult for this organism to develop resistance to plaunotol. It was confirmed that the bactericidal effects of plaunotol were also shown against Staphylococcus aureus, Streptococcus pneumoniae, Neisseria gonorrhoeae, Moraxella catarrhalis and Haemophilus influenzae. No such effect was seen against Escherichia coli and Pseudomonas aeruginosa.

Dietary n-6:n-3 ratio and Vitamin E improve motility characteristics in association with membrane properties of boar spermatozoa.

PubMed

Liu, Qing; Zhou, Yuan-Fei; Duan, Run-Jia; Wei, Hong-Kui; Peng, Jian; Jiang, Si-Wen

2017-01-01

This study was aimed to evaluate the effects of dietary n-6:n-3 ratio and Vitamin E on the membrane properties and motility characteristics of spermatozoa in boars. Forty Duroc boars were randomly distributed in a 2 × 2 factorial design with two n-6:n-3 ratios (14.4 and 6.6) and two Vitamin E levels (200 and 400 mg kg-1 ). During 16 weeks of treatment, fresh semen was collected at weeks 0, 8, 12, and 16 for measurements of motility characteristics, contents of fatty acids, membrane properties (membrane fluidity and membrane integrity), and lipid peroxidation of the spermatozoa. The semen was diluted in Beltsville Thawing Solution (BTS) extender and stored at 17°C, and the sperm motility was assessed at 12, 36, 72, and 120 h of storage. The 6.6 n-6:n-3 dietary ratio increased the contents of n-3 polyunsaturated fatty acids (PUFAs) and docosahexaenoic acid (DHA) and improved the membrane integrity and membrane fluidity of the spermatozoa, resulting in notably increased total motility, sperm progressive motility, and velocity parameters of fresh semen. Feeding diet with Vitamin E (400 mg kg-1 ) prevented sperm lipid peroxidation, and resulted in higher total motility and sperm progressive motility in fresh and liquid stored semen. In conclusion, the adjustment of n-6:n-3 ratio (6.6) and supply of Vitamin E (400 mg kg-1 ) successfully improved sperm motility characteristics and thus may be beneficial to the fertility of boars, which might be due to the modification of the physical and functional properties of spermatozoa membrane in response to dietary supplementation.

Dietary n-6:n-3 ratio and Vitamin E improve motility characteristics in association with membrane properties of boar spermatozoa

PubMed Central

Liu, Qing; Zhou, Yuan-Fei; Duan, Run-Jia; Wei, Hong-Kui; Peng, Jian; Jiang, Si-Wen

2017-01-01

This study was aimed to evaluate the effects of dietary n-6:n-3 ratio and Vitamin E on the membrane properties and motility characteristics of spermatozoa in boars. Forty Duroc boars were randomly distributed in a 2 × 2 factorial design with two n-6:n-3 ratios (14.4 and 6.6) and two Vitamin E levels (200 and 400 mg kg−1). During 16 weeks of treatment, fresh semen was collected at weeks 0, 8, 12, and 16 for measurements of motility characteristics, contents of fatty acids, membrane properties (membrane fluidity and membrane integrity), and lipid peroxidation of the spermatozoa. The semen was diluted in Beltsville Thawing Solution (BTS) extender and stored at 17°C, and the sperm motility was assessed at 12, 36, 72, and 120 h of storage. The 6.6 n-6:n-3 dietary ratio increased the contents of n-3 polyunsaturated fatty acids (PUFAs) and docosahexaenoic acid (DHA) and improved the membrane integrity and membrane fluidity of the spermatozoa, resulting in notably increased total motility, sperm progressive motility, and velocity parameters of fresh semen. Feeding diet with Vitamin E (400 mg kg−1) prevented sperm lipid peroxidation, and resulted in higher total motility and sperm progressive motility in fresh and liquid stored semen. In conclusion, the adjustment of n-6:n-3 ratio (6.6) and supply of Vitamin E (400 mg kg−1) successfully improved sperm motility characteristics and thus may be beneficial to the fertility of boars, which might be due to the modification of the physical and functional properties of spermatozoa membrane in response to dietary supplementation. PMID:26763547

SV2 frustrating exocytosis at the semi-diffusor synapse.

PubMed

Vautrin, Jean

2009-04-01

Presynaptic exocytosis is the mechanism commonly believed to release transmitters by diffusion through a pore opening during vesicular membrane fusion with the plasmalemma, but evidence suggesting that exocytosis and transmitter release are two separate steps of synaptic transmission is accumulating. Vesicular glycoconjugates such as Synaptic Vesicle Protein 2 (SV2) proteoglycans and gangliosides retain transmitters in a nondiffusible form and are transported to the synaptic cleft where they contribute forming a dense synaptomatrix. Transmitters are permanently present in synaptic clefts and readily releasable transmitter is easily accessible from the outer side of the presynaptic membrane suggesting that synaptomatrix glycoconjugates prevent immediate release after PKC-dependent exocytosis. The calcium sensor synaptotagmin is also present at the presynaptic plasma membrane and binds SV2 suggesting a direct coupling between the calcium transient and transmitter release from the synaptomatrix. A quantitative coupling of the cytosolic calcic transient to transmitter release from the synaptomatrix explains better complexity and plasticity of miniature postsynaptic signals hitherto difficult to account for in exocytic terms. This alternative representation of synaptic transmission in which the same components of the synaptomatrix support adhesion and signaling functions may cast new lights on synaptic diseases such as Alzheimer's disease. Copyright 2008 Wiley-Liss, Inc.

IFITM Proteins Restrict Viral Membrane Hemifusion

PubMed Central

Golfetto, Ottavia; Bungart, Brittani; Li, Minghua; Ding, Shilei; He, Yuxian; Liang, Chen; Lee, James C.; Gratton, Enrico; Cohen, Fredric S.; Liu, Shan-Lu

2013-01-01

The interferon-inducible transmembrane (IFITM) protein family represents a new class of cellular restriction factors that block early stages of viral replication; the underlying mechanism is currently not known. Here we provide evidence that IFITM proteins restrict membrane fusion induced by representatives of all three classes of viral membrane fusion proteins. IFITM1 profoundly suppressed syncytia formation and cell-cell fusion induced by almost all viral fusion proteins examined; IFITM2 and IFITM3 also strongly inhibited their fusion, with efficiency somewhat dependent on cell types. Furthermore, treatment of cells with IFN also markedly inhibited viral membrane fusion and entry. By using the Jaagsiekte sheep retrovirus envelope and influenza A virus hemagglutinin as models for study, we showed that IFITM-mediated restriction on membrane fusion is not at the steps of receptor- and/or low pH-mediated triggering; instead, the creation of hemifusion was essentially blocked by IFITMs. Chlorpromazine (CPZ), a chemical known to promote the transition from hemifusion to full fusion, was unable to rescue the IFITM-mediated restriction on fusion. In contrast, oleic acid (OA), a lipid analog that generates negative spontaneous curvature and thereby promotes hemifusion, virtually overcame the restriction. To explore the possible effect of IFITM proteins on membrane molecular order and fluidity, we performed fluorescence labeling with Laurdan, in conjunction with two-photon laser scanning and fluorescence-lifetime imaging microscopy (FLIM). We observed that the generalized polarizations (GPs) and fluorescence lifetimes of cell membranes expressing IFITM proteins were greatly enhanced, indicating higher molecularly ordered and less fluidized membranes. Collectively, our data demonstrated that IFITM proteins suppress viral membrane fusion before the creation of hemifusion, and suggested that they may do so by reducing membrane fluidity and conferring a positive spontaneous curvature in the outer leaflets of cell membranes. Our study provides novel insight into the understanding of how IFITM protein family restricts viral membrane fusion and infection. PMID:23358889

Neutralizing antibodies decrease the envelope fluidity of HIV-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harada, Shinji; Monde, Kazuaki; Tanaka, Yuetsu

2008-01-05

For successful penetration of HIV-1, the formation of a fusion pore may be required in order to accumulate critical numbers of fusion-activated gp41 with the help of fluidization of the plasma membrane and viral envelope. An increase in temperature to 40 {sup o}C after viral adsorption at 25 {sup o}C enhanced the infectivity by 1.4-fold. The enhanced infectivity was inhibited by an anti-CXCR4 peptide, T140, and anti-V3 monoclonal antibodies (0.5{beta} and 694/98-D) by post-attachment neutralization, but not by non-neutralizing antibodies (670-30D and 246-D) specific for the C5 of gp120 and cluster I of gp41, respectively. Anti-HLA-II and an anti-HTLV-I gp46more » antibody, LAT27, neutralized the molecule-carrying HIV-1{sub C-2(MT-2)}. The anti-V3 antibodies suppressed the fluidity of the HIV-1{sub C-2} envelope, whereas the non-neutralizing antibodies did not. The anti-HLA-II antibody decreased the envelope fluidity of HIV-1{sub C-2(MT-2)}, but not that of HIV-1{sub C-2}. Therefore, fluidity suppression by these antibodies represents an important neutralization mechanism, in addition to inhibition of viral attachment.« less

Radixin regulates synaptic GABAA receptor density and is essential for reversal learning and short-term memory

PubMed Central

Hausrat, Torben J.; Muhia, Mary; Gerrow, Kimberly; Thomas, Philip; Hirdes, Wiebke; Tsukita, Sachiko; Heisler, Frank F.; Herich, Lena; Dubroqua, Sylvain; Breiden, Petra; Feldon, Joram; Schwarz, Jürgen R; Yee, Benjamin K.; Smart, Trevor G.; Triller, Antoine; Kneussel, Matthias

2015-01-01

Neurotransmitter receptor density is a major variable in regulating synaptic strength. Receptors rapidly exchange between synapses and intracellular storage pools through endocytic recycling. In addition, lateral diffusion and confinement exchanges surface membrane receptors between synaptic and extrasynaptic sites. However, the signals that regulate this transition are currently unknown. GABAA receptors containing α5-subunits (GABAAR-α5) concentrate extrasynaptically through radixin (Rdx)-mediated anchorage at the actin cytoskeleton. Here we report a novel mechanism that regulates adjustable plasma membrane receptor pools in the control of synaptic receptor density. RhoA/ROCK signalling regulates an activity-dependent Rdx phosphorylation switch that uncouples GABAAR-α5 from its extrasynaptic anchor, thereby enriching synaptic receptor numbers. Thus, the unphosphorylated form of Rdx alters mIPSCs. Rdx gene knockout impairs reversal learning and short-term memory, and Rdx phosphorylation in wild-type mice exhibits experience-dependent changes when exposed to novel environments. Our data suggest an additional mode of synaptic plasticity, in which extrasynaptic receptor reservoirs supply synaptic GABAARs. PMID:25891999

Med15B Regulates Acid Stress Response and Tolerance in Candida glabrata by Altering Membrane Lipid Composition

PubMed Central

Qi, Yanli; Liu, Hui; Yu, Jiayin; Chen, Xiulai

2017-01-01

ABSTRACT Candida glabrata is a promising producer of organic acids. To elucidate the physiological function of the Mediator tail subunit Med15B in the response to low-pH stress, we constructed a deletion strain, C. glabrata med15BΔ, and an overexpression strain, C. glabrata HTUΔ/CgMED15B. Deletion of MED15B caused biomass production, glucose consumption rate, and cell viability to decrease by 28.3%, 31.7%, and 26.5%, respectively, compared with those of the parent (HTUΔ) strain at pH 2.0. Expression of lipid metabolism-related genes was significantly downregulated in the med15BΔ strain, whereas key genes of ergosterol biosynthesis showed abnormal upregulation. This caused the proportion of C18:1 fatty acids, the ratio of unsaturated to saturated fatty acids (UFA/SFA), and the total phospholipid content to decrease by 11.6%, 27.4%, and 37.6%, respectively. Cells failed to synthesize fecosterol and ergosterol, leading to the accumulation and a 60.3-fold increase in the concentration of zymosterol. Additionally, cells showed reductions of 69.2%, 11.6%, and 21.8% in membrane integrity, fluidity, and H+-ATPase activity, respectively. In contrast, overexpression of Med15B increased the C18:1 levels, total phospholipids, ergosterol content, and UFA/SFA by 18.6%, 143.5%, 94.5%, and 18.7%, respectively. Membrane integrity, fluidity, and H+-ATPase activity also increased by 30.2%, 6.9%, and 51.8%, respectively. Furthermore, in the absence of pH buffering, dry weight of cells and pyruvate concentrations were 29.3% and 61.2% higher, respectively, than those of the parent strain. These results indicated that in C. glabrata, Med15B regulates tolerance toward low pH via transcriptional regulation of acid stress response genes and alteration in lipid composition. IMPORTANCE This study explored the role of the Mediator tail subunit Med15B in the metabolism of Candida glabrata under acidic conditions. Overexpression of MED15B enhanced yeast tolerance to low pH and improved biomass production, cell viability, and pyruvate yield. Membrane lipid composition data indicated that Med15B might play a critical role in membrane integrity, fluidity, and H+-ATPase activity homeostasis at low pH. Thus, controlling membrane composition may serve to increase C. glabrata productivity at low pH. PMID:28710262

MODEL AND CELL MEMBRANE PARTITIONING OF PERFLUOROOCTANESULFONATE IS INDEPENDENT OF THE LIPID CHAIN LENGTH

PubMed Central

Xie, Wei; Ludewig, Gabriele; Wang, Kai; Lehmler, Hans-Joachim

2009-01-01

Perfluorooctanesulfonic acid (PFOS) is a persistent environmental pollutant that may cause adverse health effects in humans and animals by interacting with and disturbing of the normal properties of biological lipid assemblies. To gain further insights into these interactions, we investigated the effect of PFOS potassium salt on dimyristoyl- (DMPC), dipalmitoyl- (DPPC) and distearoylphosphatidylcholine (DSPC) model membranes using fluorescence anisotropy measurements and differential scanning calorimetry (DSC) and on the cell membrane of HL-60 human leukemia cells and freshly isolated rat alveolar macrophages using fluorescence anisotropy measurements. PFOS caused a concentration-dependent decrease of the main phase transition temperature (Tm) and an increased peak width (ΔTw) in both the fluorescence anisotropy and the DSC experiments, with a rank order DMPC > DPPC > DSPC. PFOS caused a fluidization of the gel phase of all phosphatidylcholines investigated, but had the opposite effect on the liquid crystalline phase. The apparent partition coefficients of PFOS between the phosphatidylcholine bilayer and the bulk aqueous phase were largely independent of the phosphatidylcholine chain length and ranged from 4.4 × 104 to 8.8 × 104. PFOS also significantly increased the fluidity of membranes of cells. These findings suggest that PFOS readily partitions into lipid assemblies, independent of their composition, and may cause adverse biological effects by altering their fluidity in a manner that depends on the membrane cooperativity and state (e.g., gel versus liquid crystalline phase) of the lipid assembly. PMID:19932010

Composite S-layer lipid structures

PubMed Central

Schuster, Bernhard; Sleytr, Uwe B.

2010-01-01

Designing and utilization of biomimetic membrane systems generated by bottom-up processes is a rapidly growing scientific and engineering field. Elucidation of the supramolecular construction principle of archaeal cell envelopes composed of S-layer stabilized lipid membranes led to new strategies for generating highly stable functional lipid membranes at meso- and macroscopic scale. In this review, we provide a state of the art survey how S-layer proteins, lipids, and polysaccharides may be used as basic building blocks for the assembly of S-layer supported lipid membranes. These biomimetic membrane systems are distinguished by a nanopatterned fluidity, enhanced stability and longevity and thus, provide a dedicated reconstitution matrix for membrane-active peptides and transmembrane proteins. Exciting areas for application of composite S-layer membrane systems concern sensor systems involving specific membrane functions. PMID:19303933

Zinc transporter-1 concentrates at the postsynaptic density of hippocampal synapses.

PubMed

Sindreu, Carlos; Bayés, Álex; Altafaj, Xavier; Pérez-Clausell, Jeús

2014-03-07

Zinc concentrates at excitatory synapses, both at the postsynaptic density and in a subset of glutamatergic boutons. Zinc can modulate synaptic plasticity, memory formation and nociception by regulating transmitter receptors and signal transduction pathways. Also, intracellular zinc accumulation is a hallmark of degenerating neurons in several neurological disorders. To date, no single zinc extrusion mechanism has been directly localized to synapses. Based on the presence of a canonical PDZ I motif in the Zinc Transporter-1 protein (ZnT1), we hypothesized that ZnT1 may be targeted to synaptic compartments for local control of cytosolic zinc. Using our previously developed protocol for the co-localization of reactive zinc and synaptic proteins, we further asked if ZnT1 expression correlates with presynaptic zinc content in individual synapses. Here we demonstrate that ZnT1 is a plasma membrane protein that is enriched in dendritic spines and in biochemically isolated synaptic membranes. Hippocampal CA1 synapses labelled by postembedding immunogold showed over a 5-fold increase in ZnT1 concentration at synaptic junctions compared with extrasynaptic membranes. Subsynaptic analysis revealed a peak ZnT1 density on the postsynaptic side of the synapse, < 10 nm away from the postsynaptic membrane. ZnT1 was found in the vast majority of excitatory synapses regardless of the presence of vesicular zinc in presynaptic boutons. Our study has identified ZnT1 as a novel postsynaptic density protein, and it may help elucidate the role of zinc homeostasis in synaptic function and disease.

Heat Stress Dictates Microbial Lipid Composition along a Thermal Gradient in Marine Sediments

PubMed Central

Sollich, Miriam; Yoshinaga, Marcos Y.; Häusler, Stefan; Price, Roy E.; Hinrichs, Kai-Uwe; Bühring, Solveig I.

2017-01-01

Temperature exerts a first-order control on microbial populations, which constantly adjust the fluidity and permeability of their cell membrane lipids to minimize loss of energy by ion diffusion across the membrane. Analytical advances in liquid chromatography coupled to mass spectrometry have allowed the detection of a stunning diversity of bacterial and archaeal lipids in extreme environments such as hot springs, hydrothermal vents and deep subsurface marine sediments. Here, we investigated a thermal gradient from 18 to 101°C across a marine sediment field and tested the hypothesis that cell membrane lipids provide a major biochemical basis for the bioenergetics of archaea and bacteria under heat stress. This paper features a detailed lipidomics approach with the focus on membrane lipid structure-function. Membrane lipids analyzed here include polar lipids of bacteria and polar and core lipids of archaea. Reflecting the low permeability of their ether-linked isoprenoids, we found that archaeal polar lipids generally dominate over bacterial lipids in deep layers of the sediments influenced by hydrothermal fluids. A close examination of archaeal and bacterial lipids revealed a membrane quandary: not only low permeability, but also increased fluidity of membranes are required as a unified property of microbial membranes for energy conservation under heat stress. For instance, bacterial fatty acids were composed of longer chain lengths in concert with higher degree of unsaturation while archaea modified their tetraethers by incorporation of additional methyl groups at elevated sediment temperatures. It is possible that these configurations toward a more fluidized membrane at elevated temperatures are counterbalanced by the high abundance of archaeal glycolipids and bacterial sphingolipids, which could reduce membrane permeability through strong intermolecular hydrogen bonding. Our results provide a new angle for interpreting membrane lipid structure-function enabling archaea and bacteria to survive and grow in hydrothermal systems. PMID:28878741

Polyhedral protein cages encase synaptic vesicles and participate in their attachment to the active zone.

PubMed

Zampighi, G A; Fisher, R S

1997-08-01

In an effort to elucidate the interactions between synaptic vesicles and the membrane of the active zone, we have investigated the structure of interneuronal asymmetric synapses in the neocortex of adult rats using thin-sectioning, freeze-fracture, and negative staining electron microscopy. We identified three subtypes of spherical synaptic vesicles. Type I were agranular vesicles of 47.5 +/- 3.8 nm (mean SD, n = 24) in diameter usually seen aggregated in clusters in the presynaptic bouton. Type II synaptic vesicles were composed of a approximately 45-nm-diameter lipid bilayer sphere encased in a cage 77 +/- 4.6 nm (mean SD, n = 42) in diameter. The cage was composed of open-faced pentamers 20-22 nm/side arranged as a regular polyhedron. Type II caged vesicles were found in clusters at the boutons, adhered to the active zone, and were also present in axons. Type III synaptic vesicles appeared as electron-dense spheres 60-75 nm in diameter abutted to the membrane of the active zone. Clathrin-coated vesicles and pits of 116.6 +/- 9 nm (mean SD, n = 14) in diameter were also present in both the pre- and postsynaptic sides. Freeze-fracture showed that some intrinsic membrane proteins in the active zone were arranged as pentamers exhibiting the same dimension of those forming cages (approximately 22 nm/side). From these data, we concluded that: (a) the presynaptic bouton contains a heterogeneous population of "caged" and "plain" synaptic vesicles and (b) type II synaptic vesicles bind to receptors in the active zone. Therefore, current models of transmitter release should take into account the substantial heterogeneity of the vesicle population and the binding of vesicular cages to the membrane of the active zone.

Oxidative Stress and Erythrocyte Membrane Alterations in Children with Autism: Correlation with Clinical Features

PubMed Central

Visconti, Paola; Bolotta, Alessandra; Ferreri, Carla; Gobbi, Giuseppe; Malisardi, Gemma; Manfredini, Stefano; Marini, Marina; Nanetti, Laura; Pipitone, Emanuela; Raffaelli, Francesca; Resca, Federica; Mazzanti, Laura

2013-01-01

It has been suggested that oxidative stress may play a role in the pathogenesis of Autism Spectrum Disorders (ASD), but the literature reports somewhat contradictory results. To further investigate the issue, we evaluated a high number of peripheral oxidative stress parameters, and some related issues such as erythrocyte membrane functional features and lipid composition. Twenty-one autistic children (Au) aged 5 to 12 years, were gender and age-matched with 20 typically developing children (TD). Erythrocyte thiobarbituric acid reactive substances, urinary isoprostane and hexanoyl-lysine adduct levels were elevated in Au, thus confirming the occurrence of an imbalance of the redox status of Au, whilst other oxidative stress markers or associated parameters (urinary 8-oxo-dG, plasma radical absorbance capacity and carbonyl groups, erythrocyte superoxide dismutase and catalase activities) were unchanged. A very significant reduction of Na+/K+-ATPase activity (−66%, p<0.0001), a reduction of erythrocyte membrane fluidity and alteration in erythrocyte fatty acid membrane profile (increase in monounsaturated fatty acids, decrease in EPA and DHA-ω3 with a consequent increase in ω6/ω3 ratio) were found in Au compared to TD, without change in membrane sialic acid content. Some Au clinical features appear to be correlated with these findings; in particular, hyperactivity score appears to be related with some parameters of the lipidomic profile and membrane fluidity. Oxidative stress and erythrocyte membrane alterations may play a role in the pathogenesis of ASD and prompt the development of palliative therapeutic protocols. Moreover, the marked decrease in NKA could be potentially utilized as a peripheral biomarker of ASD. PMID:23840462

Sources of protons and a role for bicarbonate in inhibitory feedback from horizontal cells to cones in Ambystoma tigrinum retina.

PubMed

Warren, Ted J; Van Hook, Matthew J; Supuran, Claudiu T; Thoreson, Wallace B

2016-11-15

In the vertebrate retina, photoreceptors influence the signalling of neighbouring photoreceptors through lateral-inhibitory interactions mediated by horizontal cells (HCs). These interactions create antagonistic centre-surround receptive fields important for detecting edges and generating chromatically opponent responses in colour vision. The mechanisms responsible for inhibitory feedback from HCs involve changes in synaptic cleft pH that modulate photoreceptor calcium currents. However, the sources of synaptic protons involved in feedback and the mechanisms for their removal from the cleft when HCs hyperpolarize to light remain unknown. Our results indicate that Na + -H + exchangers are the principal source of synaptic cleft protons involved in HC feedback but that synaptic cleft alkalization during light-evoked hyperpolarization of HCs also involves changes in bicarbonate transport across the HC membrane. In addition to delineating processes that establish lateral inhibition in the retina, these results contribute to other evidence showing the key role for pH in regulating synaptic signalling throughout the nervous system. Lateral-inhibitory feedback from horizontal cells (HCs) to photoreceptors involves changes in synaptic cleft pH accompanying light-evoked changes in HC membrane potential. We analysed HC to cone feedback by studying surround-evoked light responses of cones and by obtaining paired whole cell recordings from cones and HCs in salamander retina. We tested three potential sources for synaptic cleft protons: (1) generation by extracellular carbonic anhydrase (CA), (2) release from acidic synaptic vesicles and (3) Na + /H + exchangers (NHEs). Neither antagonizing extracellular CA nor blocking loading of protons into synaptic vesicles eliminated feedback. However, feedback was eliminated when extracellular Na + was replaced with choline and significantly reduced by an NHE inhibitor, cariporide. Depriving NHEs of intracellular protons by buffering HC cytosol with a pH 9.2 pipette solution eliminated feedback, whereas alkalinizing the cone cytosol did not, suggesting that HCs are a major source for protons in feedback. We also examined mechanisms for changing synaptic cleft pH in response to changes in HC membrane potential. Increasing the trans-membrane proton gradient by lowering the extracellular pH from 7.8 to 7.4 to 7.1 strengthened feedback. While maintaining constant extracellular pH with 1 mm HEPES, removal of bicarbonate abolished feedback. Elevating intracellular bicarbonate levels within HCs prevented this loss of feedback. A bicarbonate transport inhibitor, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), also blocked feedback. Together, these results suggest that NHEs are the primary source of extracellular protons in HC feedback but that changes in cleft pH accompanying changes in HC membrane voltage also require bicarbonate flux across the HC membrane. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Sources of protons and a role for bicarbonate in inhibitory feedback from horizontal cells to cones in Ambystoma tigrinum retina

PubMed Central

Warren, Ted J.; Van Hook, Matthew J.; Supuran, Claudiu T.

2016-01-01

Key points In the vertebrate retina, photoreceptors influence the signalling of neighbouring photoreceptors through lateral‐inhibitory interactions mediated by horizontal cells (HCs). These interactions create antagonistic centre‐surround receptive fields important for detecting edges and generating chromatically opponent responses in colour vision.The mechanisms responsible for inhibitory feedback from HCs involve changes in synaptic cleft pH that modulate photoreceptor calcium currents. However, the sources of synaptic protons involved in feedback and the mechanisms for their removal from the cleft when HCs hyperpolarize to light remain unknown.Our results indicate that Na+–H+ exchangers are the principal source of synaptic cleft protons involved in HC feedback but that synaptic cleft alkalization during light‐evoked hyperpolarization of HCs also involves changes in bicarbonate transport across the HC membrane.In addition to delineating processes that establish lateral inhibition in the retina, these results contribute to other evidence showing the key role for pH in regulating synaptic signalling throughout the nervous system. Abstract Lateral‐inhibitory feedback from horizontal cells (HCs) to photoreceptors involves changes in synaptic cleft pH accompanying light‐evoked changes in HC membrane potential. We analysed HC to cone feedback by studying surround‐evoked light responses of cones and by obtaining paired whole cell recordings from cones and HCs in salamander retina. We tested three potential sources for synaptic cleft protons: (1) generation by extracellular carbonic anhydrase (CA), (2) release from acidic synaptic vesicles and (3) Na+/H+ exchangers (NHEs). Neither antagonizing extracellular CA nor blocking loading of protons into synaptic vesicles eliminated feedback. However, feedback was eliminated when extracellular Na+ was replaced with choline and significantly reduced by an NHE inhibitor, cariporide. Depriving NHEs of intracellular protons by buffering HC cytosol with a pH 9.2 pipette solution eliminated feedback, whereas alkalinizing the cone cytosol did not, suggesting that HCs are a major source for protons in feedback. We also examined mechanisms for changing synaptic cleft pH in response to changes in HC membrane potential. Increasing the trans‐membrane proton gradient by lowering the extracellular pH from 7.8 to 7.4 to 7.1 strengthened feedback. While maintaining constant extracellular pH with 1 mm HEPES, removal of bicarbonate abolished feedback. Elevating intracellular bicarbonate levels within HCs prevented this loss of feedback. A bicarbonate transport inhibitor, 4,4′‐diisothiocyano‐2,2′‐stilbenedisulfonic acid (DIDS), also blocked feedback. Together, these results suggest that NHEs are the primary source of extracellular protons in HC feedback but that changes in cleft pH accompanying changes in HC membrane voltage also require bicarbonate flux across the HC membrane. PMID:27345444

The Nanoscale Observation of the Three-Dimensional Structures of Neurosynapses, Membranous Conjunctions Between Cultured Hippocampal Neurons and Their Significance in the Development of Epilepsy.

PubMed

Sun, Lan; Jiang, Shuang; Tang, Xianhua; Zhang, Yingge; Qin, Luye; Jiang, Xia; Yu, Albert Cheung Hoi

2016-12-01

The nanoscale three-dimensional structures of neurosynapses are unknown, and the neuroanatomical basis of epilepsy remains to be elucidated. Here, we studied the nanoscale three-dimensional synapses between hippocampal neurons, and membranous conjunctions between neurons were found with atomic force microscopy (AFM) and confirmed by transmission electron microscope (TEM), and their pathophysiological significance was primarily investigated. The neurons and dendrites were marked by MAP-2, axons by neurofilament 200, and synapses by synapsin I immunological staining. In the synapsin I-positive neurite ends of the neurons positively stained with MAP-2 and neurofilament 200, neurosynapses with various nanoscale morphology and structure could be found by AFM. The neurosynapses had typical three-dimensional structures of synaptic triplet including the presynaptic neurite end, synaptic cleft of 30 ∼ 40 in chemical synapses and 2 ∼ 6 nm in electrical ones, the postsynaptic neurite or dendrite spine, the typical neurite end button, the distinct pre- and postsynaptic membranes, and the obvious thickening of the postsynaptic membranes or neurites. Some membranous connections including membrane-like junctions (MLJ) and fiber-tube links (FTL) without triplet structures and cleft were found between neurons. The development frequencies of the two membranous conjunctions increased while those of the synaptic conjunctions decreased between the neurons from Otx1 knock-out mice in comparison with those between the neurons from normal mice. These results suggested that the neuroanatomical basis of Otx1 knock-out epilepsy is the combination of the decreased synaptic conjunctions and the increased membranous conjunctions.

SNARE interactions in membrane trafficking: a perspective from mammalian central synapses.

PubMed

Kavalali, Ege T

2002-10-01

SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are a large family of proteins that are present on all organelles involved in intracellular vesicle trafficking and secretion. The interaction of complementary SNAREs found on opposing membranes presents an attractive lock-and-key mechanism, which may underlie the specificity of vesicle trafficking. Moreover, formation of the tight complex between a vesicle membrane SNARE and corresponding target membrane SNAREs could drive membrane fusion. In synapses, this tight complex, also referred to as the synaptic core complex, is essential for neurotransmitter release. However, recent observations in knockout mice lacking major synaptic SNAREs challenge the prevailing notion on the executive role of these proteins in fusion and open up several questions about their exact role(s) in neurotransmitter release. Persistence of a form of regulated neurotransmitter release in these mutant mice also raises the possibility that other cognate or non-cognate SNAREs may partially compensate for the loss of a particular SNARE. Future analysis of SNARE function in central synapses will also have implications for the role of these molecules in other vesicle trafficking events such as endocytosis and vesicle replenishment. Such analysis can provide a molecular basis for synaptic processes including certain forms of short-term synaptic plasticity. Copyright 2002 Wiley Periodicals, Inc.

Zinc transporter-1 concentrates at the postsynaptic density of hippocampal synapses

PubMed Central

2014-01-01

Background Zinc concentrates at excitatory synapses, both at the postsynaptic density and in a subset of glutamatergic boutons. Zinc can modulate synaptic plasticity, memory formation and nociception by regulating transmitter receptors and signal transduction pathways. Also, intracellular zinc accumulation is a hallmark of degenerating neurons in several neurological disorders. To date, no single zinc extrusion mechanism has been directly localized to synapses. Based on the presence of a canonical PDZ I motif in the Zinc Transporter-1 protein (ZnT1), we hypothesized that ZnT1 may be targeted to synaptic compartments for local control of cytosolic zinc. Using our previously developed protocol for the co-localization of reactive zinc and synaptic proteins, we further asked if ZnT1 expression correlates with presynaptic zinc content in individual synapses. Findings Here we demonstrate that ZnT1 is a plasma membrane protein that is enriched in dendritic spines and in biochemically isolated synaptic membranes. Hippocampal CA1 synapses labelled by postembedding immunogold showed over a 5-fold increase in ZnT1 concentration at synaptic junctions compared with extrasynaptic membranes. Subsynaptic analysis revealed a peak ZnT1 density on the postsynaptic side of the synapse, < 10 nm away from the postsynaptic membrane. ZnT1 was found in the vast majority of excitatory synapses regardless of the presence of vesicular zinc in presynaptic boutons. Conclusions Our study has identified ZnT1 as a novel postsynaptic density protein, and it may help elucidate the role of zinc homeostasis in synaptic function and disease. PMID:24602382

LL5beta: a regulator of postsynaptic differentiation identified in a screen for synaptically enriched transcripts at the neuromuscular junction.

PubMed

Kishi, Masashi; Kummer, Terrance T; Eglen, Stephen J; Sanes, Joshua R

2005-04-25

In both neurons and muscle fibers, specific mRNAs are concentrated beneath and locally translated at synaptic sites. At the skeletal neuromuscular junction, all synaptic RNAs identified to date encode synaptic components. Using microarrays, we compared RNAs in synapse-rich and -free regions of muscles, thereby identifying transcripts that are enriched near synapses and that encode soluble membrane and nuclear proteins. One gene product, LL5beta, binds to both phosphoinositides and a cytoskeletal protein, filamin, one form of which is concentrated at synaptic sites. LL5beta is itself associated with the cytoplasmic face of the postsynaptic membrane; its highest levels border regions of highest acetylcholine receptor (AChR) density, which suggests a role in "corraling" AChRs. Consistent with this idea, perturbing LL5beta expression in myotubes inhibits AChR aggregation. Thus, a strategy designed to identify novel synaptic components led to identification of a protein required for assembly of the postsynaptic apparatus.

Electron microscopic immunocytochemical study of somatostatin neurons in the periventricular nucleus of the rat hypothalamus with special reference to their relationships with homologous neuronal processes.

PubMed

Alonso, G; Tapia-Arancibia, L; Assenmacher, I

1985-10-01

The neurons containing somatostatin in the rat periventricular nucleus were studied by using a modified electron microscopic immunocytochemical technique that improves both the penetration of immunoreagents into unembedded immunostained tissues and the preservation of ultrastructural morphology. Inside perikarya and dendrites, immunostaining was not only associated with neurosecretory granules but also with ribosomes and saccules of the cis face of the Golgi apparatus. In the axonal profiles found in this region the labeling was observed both on neurosecretory granule cores and on the limiting membrane of small synaptic-like vesicles. Throughout the periventricular nucleus, both non-synaptic and synaptic relationships were shown between labeled neurons. Non-synaptic relationships mainly consisted of direct apposition of the membranes of neighboring neurons by dendrosomatic, somasomatic or dendrodendritic contacts. These labeled perikarya and dendrites were also synaptically contacted by labeled axonal endings containing numerous aggregated synaptic-like vesicles. The physiological significance of the synaptic and non-synaptic relationships between somatostatinergic neurons is discussed in terms of possible synchronization between homologous neurons of the somatostatin neuroendocrine system and control of these neurons by a central ultra-short loop feedback mechanism.

Influence of different surfactants on the physicochemical properties of elastic liposomes.

PubMed

Barbosa, R M; Severino, P; Preté, P S C; Santana, M H A

2017-05-01

Elastic liposomes are capable to improve drug transport through the skin by acting as penetration enhancers due to the high fluidity and elasticity of the liposome membranes. Therefore, elastic liposomes were prepared and characterized to facilitate the transdermal transport of bioactive molecules. Liposomes consisted of dimyristoylphosphatidylcholine (DMPC) as the structural component, with different surfactants derived from lauric acid as elastic components: C 12 E 5 (polyoxyethylene-5-lauryl ether), PEG4L (polyethyleneglycol-4-lauryl ester), PEG4DL (polyethylene glycol-4-dilauryl ester), PEG8L (polyethylene glycol-8-lauryl ester) and PEG8DL (polyethylene glycol-8-dilauryl ester). The elastic liposomes were characterized in terms of their phospholipid content, mean diameter, size distribution, elasticity and stability during storage, as well as their ability to incorporate surfactant and permeate through 50 nm pore size membranes. The results showed that the phospholipid phase transition temperature, the fluidity of the lipid bilayer resulting from incorporation of the surfactant and the preservation of particle integrity were factors determining the performance of the elastic liposomes in permeating through nanoporous membranes. The best results were obtained using DMPC combined with the surfactants PEG8L or PEG8DL. The findings demonstrate the potential of using elastic liposomes for transdermal administration of drugs.

Mechanical coupling of microtubule-dependent motor teams during peroxisome transport in Drosophila S2 cells.

PubMed

De Rossi, María Cecilia; Wetzler, Diana E; Benseñor, Lorena; De Rossi, María Emilia; Sued, Mariela; Rodríguez, Daniela; Gelfand, Vladimir; Bruno, Luciana; Levi, Valeria

2017-12-01

Intracellular transport requires molecular motors that step along cytoskeletal filaments actively dragging cargoes through the crowded cytoplasm. Here, we explore the interplay of the opposed polarity motors kinesin-1 and cytoplasmic dynein during peroxisome transport along microtubules in Drosophila S2 cells. We used single particle tracking with nanometer accuracy and millisecond time resolution to extract quantitative information on the bidirectional motion of organelles. The transport performance was studied in cells expressing a slow chimeric plus-end directed motor or the kinesin heavy chain. We also analyzed the influence of peroxisomes membrane fluidity in methyl-β-ciclodextrin treated cells. The experimental data was also confronted with numerical simulations of two well-established tug of war scenarios. The velocity distributions of retrograde and anterograde peroxisomes showed a multimodal pattern suggesting that multiple motor teams drive transport in either direction. The chimeric motors interfered with the performance of anterograde transport and also reduced the speed of the slowest retrograde team. In addition, increasing the fluidity of peroxisomes membrane decreased the speed of the slowest anterograde and retrograde teams. Our results support the existence of a crosstalk between opposed-polarity motor teams. Moreover, the slowest teams seem to mechanically communicate with each other through the membrane to trigger transport. Copyright © 2017 Elsevier B.V. All rights reserved.

Kinetics of apoptotic markers in exogeneously induced apoptosis of EL4 cells.

PubMed

Jessel, Robert; Haertel, Steffen; Socaciu, Carmen; Tykhonova, Svetlana; Diehl, Horst A

2002-01-01

We investigated the time-dependence of apoptotic events in EL4 cells by monitoring plasma membrane changes in correlation to DNA fragmentation and cell shrinkage. We applied three apoptosis inducers (staurosporine, tubericidine and X-rays) and we looked at various markers to follow the early-to-late apoptotic events: phospholipid translocation (identified through annexin V-fluorescein assay and propidium iodide), lipid package (via merocyanine assay), membrane fluidity and anisotropy (via fluorescent measurements), DNA fragmentation by the fluorescence-labeling test and cell size measurements. The different apoptotic inducers caused different reactions of the cells: staurosporine induced apoptosis most rapidly in a high number of cells, tubercidine triggered apoptosis only in the S phase cells, while X-rays caused a G2/M arrest and subsequently apoptosis. Loss of lipid asymmetry is promptly detectable after one hour of incubation time. The phosphatidylserine translocation, decrease of lipid package and anisotropy, and the increase of membrane fluidity appeared to be based on the same process of lipid asymmetry loss. Therefore, the DNA fragmentation and the cell shrinkage appear to be parallel and independent processes running on different time scales but which are kinetically inter-related. The results indicate different signal steps to apoptosis dependent on inducer characteristics but the kinetics of "early-to-late" apoptosis appears to be a fixed program.

Salt-induced changes in lipid composition and membrane fluidity of halophilic yeast-like melanized fungi.

PubMed

Turk, Martina; Méjanelle, Laurence; Sentjurc, Marjeta; Grimalt, Joan O; Gunde-Cimerman, Nina; Plemenitas, Ana

2004-02-01

The halophilic melanized yeast-like fungi Hortaea werneckii, Phaeotheca triangularis, and the halotolerant Aureobasidium pullulans, isolated from salterns as their natural environment, were grown at different NaCl concentrations and their membrane lipid composition and fluidity were examined. Among sterols, besides ergosterol, which was the predominant one, 23 additional sterols were identified. Their total content did not change consistently or significantly in response to raised NaCl concentrations in studied melanized fungi. The major phospholipid classes were phosphatidylcholine and phosphatidylethanolamine, followed by anionic phospholipids. The most abundant fatty acids in phospholipids contained C16 and C18 chain lengths with a high percentage of C18:2Delta9,12. Salt stress caused an increase in the fatty acid unsaturation in the halophilic H. werneckii and halotolerant A. pullulans but a slight decrease in halophilic P. triangularis. All the halophilic fungi maintained their sterol-to-phospholipid ratio at a significantly lower level than did the salt-sensitive Saccharomyces cerevisiae and halotolerant A. pullulans. Electron paramagnetic resonance (EPR) spectroscopy measurements showed that the membranes of all halophilic fungi were more fluid than those of the halotolerant A. pullulans and salt-sensitive S. cerevisiae, which is in good agreement with the lipid composition observed in this study.

Molecular Machines Determining the Fate of Endocytosed Synaptic Vesicles in Nerve Terminals

PubMed Central

Fassio, Anna; Fadda, Manuela; Benfenati, Fabio

2016-01-01

The cycle of a synaptic vesicle (SV) within the nerve terminal is a step-by-step journey with the final goal of ensuring the proper synaptic strength under changing environmental conditions. The SV cycle is a precisely regulated membrane traffic event in cells and, because of this, a plethora of membrane-bound and cytosolic proteins are devoted to assist SVs in each step of the journey. The cycling fate of endocytosed SVs determines both the availability for subsequent rounds of release and the lifetime of SVs in the terminal and is therefore crucial for synaptic function and plasticity. Molecular players that determine the destiny of SVs in nerve terminals after a round of exo-endocytosis are largely unknown. Here we review the functional role in SV fate of phosphorylation/dephosphorylation of SV proteins and of small GTPases acting on membrane trafficking at the synapse, as they are emerging as key molecules in determining the recycling route of SVs within the nerve terminal. In particular, we focus on: (i) the cyclin-dependent kinase-5 (cdk5) and calcineurin (CN) control of the recycling pool of SVs; (ii) the role of small GTPases of the Rab and ADP-ribosylation factor (Arf) families in defining the route followed by SV in their nerve terminal cycle. These regulatory proteins together with their synaptic regulators and effectors, are molecular nanomachines mediating homeostatic responses in synaptic plasticity and potential targets of drugs modulating the efficiency of synaptic transmission. PMID:27242505

Molecular Machines Determining the Fate of Endocytosed Synaptic Vesicles in Nerve Terminals.

PubMed

Fassio, Anna; Fadda, Manuela; Benfenati, Fabio

2016-01-01

The cycle of a synaptic vesicle (SV) within the nerve terminal is a step-by-step journey with the final goal of ensuring the proper synaptic strength under changing environmental conditions. The SV cycle is a precisely regulated membrane traffic event in cells and, because of this, a plethora of membrane-bound and cytosolic proteins are devoted to assist SVs in each step of the journey. The cycling fate of endocytosed SVs determines both the availability for subsequent rounds of release and the lifetime of SVs in the terminal and is therefore crucial for synaptic function and plasticity. Molecular players that determine the destiny of SVs in nerve terminals after a round of exo-endocytosis are largely unknown. Here we review the functional role in SV fate of phosphorylation/dephosphorylation of SV proteins and of small GTPases acting on membrane trafficking at the synapse, as they are emerging as key molecules in determining the recycling route of SVs within the nerve terminal. In particular, we focus on: (i) the cyclin-dependent kinase-5 (cdk5) and calcineurin (CN) control of the recycling pool of SVs; (ii) the role of small GTPases of the Rab and ADP-ribosylation factor (Arf) families in defining the route followed by SV in their nerve terminal cycle. These regulatory proteins together with their synaptic regulators and effectors, are molecular nanomachines mediating homeostatic responses in synaptic plasticity and potential targets of drugs modulating the efficiency of synaptic transmission.

One Cycle Fuels Another: The Energetics of Neurotransmitter Release.

PubMed

Silm, Katlin; Edwards, Robert H

2017-02-08

In this issue of Neuron, Ashrafi et al. (2017) show that activity induces translocation of the insulin-regulated glucose transporter GLUT4 to the plasma membrane, where it sustains the ATP production required for synaptic vesicle cycling. However, translocation occurs from presynaptic membranes other than synaptic vesicles and involves a distinct molecular mechanism. Copyright © 2017 Elsevier Inc. All rights reserved.

Selective effect of cell membrane on synaptic neurotransmission

NASA Astrophysics Data System (ADS)

Postila, Pekka A.; Vattulainen, Ilpo; Róg, Tomasz

2016-01-01

Atomistic molecular dynamics simulations were performed with 13 non-peptidic neurotransmitters (NTs) in three different membrane environments. The results provide compelling evidence that NTs are divided into membrane-binding and membrane-nonbinding molecules. NTs adhere to the postsynaptic membrane surface whenever the ligand-binding sites of their synaptic receptors are buried in the lipid bilayer. In contrast, NTs that have extracellular ligand-binding sites do not have a similar tendency to adhere to the membrane surface. This finding is a seemingly simple yet important addition to the paradigm of neurotransmission, essentially dividing it into membrane-independent and membrane-dependent mechanisms. Moreover, the simulations also indicate that the lipid composition especially in terms of charged lipids can affect the membrane partitioning of NTs. The revised paradigm, highlighting the importance of cell membrane and specific lipids for neurotransmission, should to be of interest to neuroscientists, drug industry and the general public alike.

In vitro effects of fermented papaya (Carica papaya, L.) on platelets obtained from patients with type 2 diabetes.

PubMed

Raffaelli, F; Nanetti, L; Montecchiani, G; Borroni, F; Salvolini, E; Faloia, E; Ferretti, G; Mazzanti, L; Vignini, A

2015-02-01

Oxidative stress is associated with insulin resistance pathogenesis, insulin secretion deficiency, and complication onset. Fermented papaya preparation (FPP), a dietary supplement obtained by fermentation of the papaya fruit, may be used as an antioxidant in the prevention of diabetic complications. Platelets from 30 patients with type 2 diabetes mellitus (DM 2) and 15 healthy subjects were analyzed to evaluate the in vitro effects of FPP incubation. Na(+)/K(+)-adenosine triphosphatase (ATPase) activity, membrane fluidity, total antioxidant capacity (TAC), superoxide dismutase (SOD) activity, and conjugated diene levels were determined. In vitro FPP incubation improved platelet function, by enhancing Na(+)/K(+)-ATPase activity and membrane fluidity, and ameliorated the antioxidant system functionality, through an increase in TAC and SOD activity and a parallel decrease in conjugated diene levels in patients with DM 2. Our data suggest that the incubation with FPP may have a protective effect on platelets from patients with DM 2, by preventing the progression of oxidative damage associated with diabetes and its complications. Copyright © 2014 Elsevier B.V. All rights reserved.

Fatty acids rehabilitated long-term neurodegenerative: like symptoms in olfactory bulbectomized rats.

PubMed

Yehuda, Shlomo; Rabinovitz, Sharon

2015-05-01

Our previous study demonstrated that an olfactory bulbectomy in rats induced short-term, multifaceted, devastating Alzheimer's-like effects, which included cognitive impairment, hyperactivity, hyperthermia, and increased levels of homocysteine and pro-inflammatory cytokines, including IL-17A. In addition, the rats exhibited an increase in the hyperphosphorylation of brain Tau proteins and in the number of neurofibrillary tangles. Here, we examined the long-term effects of the surgery and found that olfactory bulbectomy also rendered the rats to become anemic with brain iron overload. Additionally, a significant reduction in the membrane fluidity index in frontal cortex synaptosomes was found. Treatment with a mixture of n - 3/n - 6 of fatty acids restored the unwanted effect. The beneficial effects of fatty acids are mediated via the effects of fatty acids on the neuronal membrane structure and fluidity. These findings are similar to Alzheimer's symptoms, which suggest this model can be used as an animal model for Alzheimer's disease. We recommend using this model to scan potential new anti-Alzheimer's drugs.

The direct anti-MRSA effect of emodin via damaging cell membrane.

PubMed

Liu, Ming; Peng, Wei; Qin, Rongxin; Yan, Zifei; Cen, Yanyan; Zheng, Xinchuan; Pan, Xichun; Jiang, Weiwei; Li, Bin; Li, Xiaoli; Zhou, Hong

2015-09-01

Methicillin-resistant Staphylococcus aureus (MRSA) has become an important bacterium for nosocomial infection. Only a few antibiotics can be effective against MRSA. Therefore, searching for new drugs against MRSA is important. Herein, anti-MRSA activities of emodin and its mechanisms were investigated. Firstly, in vitro antimicrobial activity was investigated by minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and time-growth curve, and multipassage resistance testing was performed. Secondly, protection of emodin on mice survival and blood bacterial load in mice challenged with lethal or sublethal dose of MRSA were investigated. Subsequently, the influences of emodin on the bacterial morphology, messenger RNA (mRNA) expressions related to cell wall synthesis and lysis, β-lactamase activity, drug accumulation, membrane fluidity, and integrity were performed to investigate its mechanisms. Lastly, in vitro cytotoxicity assay were performed using the 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) method. The results showed MICs and MBCs of emodin against MRSA252 and 36 clinical MRSA strains were among 2-8 and 4-32 μg/mL, respectively. There was no MIC increase for emodin during 20 passages. In vivo, emodin dose-dependently protected mice challenged with lethal dose of MRSA and decreased bacterial load in mice challenged with sublethal dose of MRSA. Morphology observation showed emodin might disrupt cell wall and membrane of MRSA. Although emodin had no influence on genes related to cell wall synthesis and lysis as well as β-lactamase activity and drug accumulation, emodin reduced membrane fluidity and disrupted membrane integrity. Based on the fact that emodin had no significant cytotoxicity against mammalian cells, it could be further investigated as a membrane-damage bactericide against MRSA in the future.

Fermentation Temperature Modulates Phosphatidylethanolamine and Phosphatidylinositol Levels in the Cell Membrane of Saccharomyces cerevisiae

PubMed Central

Henderson, Clark M.; Zeno, Wade F.; Lerno, Larry A.; Longo, Marjorie L.

2013-01-01

During alcoholic fermentation, Saccharomyces cerevisiae is exposed to a host of environmental and physiological stresses. Extremes of fermentation temperature have previously been demonstrated to induce fermentation arrest under growth conditions that would otherwise result in complete sugar utilization at “normal” temperatures and nutrient levels. Fermentations were carried out at 15°C, 25°C, and 35°C in a defined high-sugar medium using three Saccharomyces cerevisiae strains with diverse fermentation characteristics. The lipid composition of these strains was analyzed at two fermentation stages, when ethanol levels were low early in stationary phase and in late stationary phase at high ethanol concentrations. Several lipids exhibited dramatic differences in membrane concentration in a temperature-dependent manner. Principal component analysis (PCA) was used as a tool to elucidate correlations between specific lipid species and fermentation temperature for each yeast strain. Fermentations carried out at 35°C exhibited very high concentrations of several phosphatidylinositol species, whereas at 15°C these yeast strains exhibited higher levels of phosphatidylethanolamine and phosphatidylcholine species with medium-chain fatty acids. Furthermore, membrane concentrations of ergosterol were highest in the yeast strain that experienced stuck fermentations at all three temperatures. Fluorescence anisotropy measurements of yeast cell membrane fluidity during fermentation were carried out using the lipophilic fluorophore diphenylhexatriene. These measurements demonstrate that the changes in the lipid composition of these yeast strains across the range of fermentation temperatures used in this study did not significantly affect cell membrane fluidity. However, the results from this study indicate that fermenting S. cerevisiae modulates its membrane lipid composition in a temperature-dependent manner. PMID:23811519

A role for the thermal environment in defining co-stimulation requirements for CD4+ T cell activation

PubMed Central

Zynda, Evan R; Grimm, Melissa J; Yuan, Min; Zhong, Lingwen; Mace, Thomas A; Capitano, Maegan; Ostberg, Julie R; Lee, Kelvin P; Pralle, Arnd; Repasky, Elizabeth A

2015-01-01

Maintenance of normal core body temperature is vigorously defended by long conserved, neurovascular homeostatic mechanisms that assist in heat dissipation during prolonged, heat generating exercise or exposure to warm environments. Moreover, during febrile episodes, body temperature can be significantly elevated for at least several hours at a time. Thus, as blood cells circulate throughout the body, physiologically relevant variations in surrounding tissue temperature can occur; moreover, shifts in core temperature occur during daily circadian cycles. This study has addressed the fundamental question of whether the threshold of stimulation needed to activate lymphocytes is influenced by temperature increases associated with physiologically relevant increases in temperature. We report that the need for co-stimulation of CD4+ T cells via CD28 ligation for the production of IL-2 is significantly reduced when cells are exposed to fever-range temperature. Moreover, even in the presence of sufficient CD28 ligation, provision of extra heat further increases IL-2 production. Additional in vivo and in vitro data (using both thermal and chemical modulation of membrane fluidity) support the hypothesis that the mechanism by which temperature modulates co-stimulation is linked to increases in membrane fluidity and membrane macromolecular clustering in the plasma membrane. Thermally-regulated changes in plasma membrane organization in response to physiological increases in temperature may assist in the geographical control of lymphocyte activation, i.e., stimulating activation in lymph nodes rather than in cooler surface regions, and further, may temporarily and reversibly enable CD4+ T cells to become more quickly and easily activated during times of infection during fever. PMID:26131730

Customizing semen preservation protocols for individual dogs and individual species: sperm preservation beyond the state of the art.

PubMed

Farstad, W

2012-12-01

Sperm quality can be variable in morphometric and physiological attributes between males of different species, between males within species subtypes reared under different environmental conditions, between ejaculates of the same male or even between sperm populations within an ejaculate. Clinical semen evaluation is based on evaluation of whole ejaculates, which is not a chemically or physiologically well-defined entity, rather a collection of heterogeneous subpopulations giving different measurements and possessing different fertilizing potential. Identification of subpopulations with different motility patterns is important as well as characterizing the subtle structural changes underlying the motility differences observed. The ability to identify populations of sperm responding rapidly or failing to progress through the capacitation process may have clinical applications. Studies of lipid-phase fluidity of sperm membranes, mathematical modelling of membrane ion transport, role of modifying components and detergent-resistant microdomains are of particular interest. When customizing extenders to ejaculates from cryosensitive males or species, a thorough knowledge of species sperm membrane physiology and an assessment of the individual ejaculate's sperm populations are necessary. Structural differences have been found in sperm membranes between fox species with different cryosurvival potential of their spermatozoa. Supplementation of lipids and detergents in cryoextenders may influence membrane fluidity of the surviving spermatozoa in a species-dependent manner and influence capacitation. Immobilization of sperm prior to cryopreservation with subsequent slow release of sperm in the female genital tract may be a way to prolong the fertile life of sperm. In canids with a long oocyte maturation time, delayed capacitation may be beneficial. © 2012 Blackwell Verlag GmbH.

The extent of the temperature-induced membrane remodeling in two closely related Bordetella species reflects their adaptation to diverse environmental niches

PubMed Central

Seydlova, Gabriela; Beranova, Jana; Bibova, Ilona; Dienstbier, Ana; Drzmisek, Jakub; Masin, Jiri; Fiser, Radovan; Konopasek, Ivo; Vecerek, Branislav

2017-01-01

Changes in environmental temperature represent one of the major stresses faced by microorganisms as they affect the function of the cytoplasmic membrane. In this study, we have analyzed the thermal adaptation in two closely related respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica. Although B. pertussis represents a pathogen strictly adapted to the human body temperature, B. bronchiseptica causes infection in a broad range of animals and survives also outside of the host. We applied GC-MS to determine the fatty acids of both Bordetella species grown at different temperatures and analyzed the membrane fluidity by fluorescence anisotropy measurement. In parallel, we also monitored the effect of growth temperature changes on the expression and production of several virulence factors. In response to low temperatures, B. pertussis adapted its fatty acid composition and membrane fluidity to a considerably lesser extent when compared with B. bronchiseptica. Remarkably, B. pertussis maintained the production of virulence factors at 24 °C, whereas B. bronchiseptica cells resumed the production only upon temperature upshift to 37 °C. This growth temperature-associated differential modulation of virulence factor production was linked to the phosphorylation state of transcriptional regulator BvgA. The observed differences in low-temperature adaptation between B. pertussis and B. bronchiseptica may result from selective adaptation of B. pertussis to the human host. We propose that the reduced plasticity of the B. pertussis membranes ensures sustained production of virulence factors at suboptimal temperatures and may play an important role in the transmission of the disease. PMID:28348085

Towards reconstitution of membrane fusion mediated by SNAREs and other synaptic proteins

PubMed Central

Brunger, Axel T.; Cipriano, Daniel J.; Diao, Jiajie

2015-01-01

Abstract Proteoliposomes have been widely used for in vitro studies of membrane fusion mediated by synaptic proteins. Initially, such studies were made with large unsynchronized ensembles of vesicles. Such ensemble assays limited the insights into the SNARE-mediated fusion mechanism that could be obtained from them. Single particle microscopy experiments can alleviate many of these limitations but they pose significant technical challenges. Here we summarize various approaches that have enabled studies of fusion mediated by SNAREs and other synaptic proteins at a single-particle level. Currently available methods are described and their advantages and limitations are discussed. PMID:25788028

Different dynamin blockers interfere with distinct phases of synaptic endocytosis during stimulation in motoneurones

PubMed Central

Linares-Clemente, Pedro; Rozas, José L; Mircheski, Josif; García-Junco-Clemente, Pablo; Martínez-López, José A; Nieto-González, José L; Vázquez, M Eugenio; Pintado, C Oscar; Fernández-Chacón, Rafael

2015-01-01

Key points Neurotransmitter release requires a tight coupling between synaptic vesicle exocytosis and endocytosis with dynamin being a key protein in that process. We used imaging techniques to examine the time course of endocytosis at mouse motor nerve terminals expressing synaptopHluorin, a genetically encoded reporter of the synaptic vesicle cycle. We separated two sequential phases of endocytosis taking place during the stimulation train: early and late endocytosis. Freshly released synaptic vesicle proteins are preferentially retrieved during the early phase, which is very sensitive to dynasore, an inhibitor of dynamin GTPase activity. Synaptic vesicle proteins pre-existing at the plasma membrane before the stimulation are preferentially retrieved during the late phase, which is very sensitive to myristyl trimethyl ammonium bromide (MitMAB), an inhibitor of the dynamin–phospholipid interaction. Abstract Synaptic endocytosis is essential at nerve terminals to maintain neurotransmitter release by exocytosis. Here, at the neuromuscular junction of synaptopHluorin (spH) transgenic mice, we have used imaging to study exo- and endocytosis occurring simultaneously during nerve stimulation. We observed two endocytosis components, which occur sequentially during stimulation. The early component of endocytosis apparently internalizes spH molecules freshly exocytosed. This component was sensitive to dynasore, a blocker of dynamin 1 GTPase activity. In contrast, this early component was resistant to myristyl trimethyl ammonium bromide (MiTMAB), a competitive agent that blocks dynamin binding to phospholipid membranes. The late component of endocytosis is likely to internalize spH molecules that pre-exist at the plasma membrane before stimulation starts. This component was blocked by MiTMAB, perhaps by impairing the binding of dynamin or other key endocytic proteins to phospholipid membranes. Our study suggests the co-existence of two sequential synaptic endocytosis steps taking place during stimulation that are susceptible to pharmacological dissection: an initial step, preferentially sensitive to dynasore, that internalizes vesicular components immediately after they are released, and a MiTMAB-sensitive step that internalizes vesicular components pre-existing at the plasma membrane surface. In addition, we report that post-stimulus endocytosis also has several components with different sensitivities to dynasore and MiTMAB. PMID:25981717

Different dynamin blockers interfere with distinct phases of synaptic endocytosis during stimulation in motoneurones.

PubMed

Linares-Clemente, Pedro; Rozas, José L; Mircheski, Josif; García-Junco-Clemente, Pablo; Martínez-López, José A; Nieto-González, José L; Vázquez, M Eugenio; Pintado, C Oscar; Fernández-Chacón, Rafael

2015-07-01

Neurotransmitter release requires a tight coupling between synaptic vesicle exocytosis and endocytosis with dynamin being a key protein in that process. We used imaging techniques to examine the time course of endocytosis at mouse motor nerve terminals expressing synaptopHluorin, a genetically encoded reporter of the synaptic vesicle cycle. We separated two sequential phases of endocytosis taking place during the stimulation train: early and late endocytosis. Freshly released synaptic vesicle proteins are preferentially retrieved during the early phase, which is very sensitive to dynasore, an inhibitor of dynamin GTPase activity. Synaptic vesicle proteins pre-existing at the plasma membrane before the stimulation are preferentially retrieved during the late phase, which is very sensitive to myristyl trimethyl ammonium bromide (MitMAB), an inhibitor of the dynamin-phospholipid interaction. Synaptic endocytosis is essential at nerve terminals to maintain neurotransmitter release by exocytosis. Here, at the neuromuscular junction of synaptopHluorin (spH) transgenic mice, we have used imaging to study exo- and endocytosis occurring simultaneously during nerve stimulation. We observed two endocytosis components, which occur sequentially during stimulation. The early component of endocytosis apparently internalizes spH molecules freshly exocytosed. This component was sensitive to dynasore, a blocker of dynamin 1 GTPase activity. In contrast, this early component was resistant to myristyl trimethyl ammonium bromide (MiTMAB), a competitive agent that blocks dynamin binding to phospholipid membranes. The late component of endocytosis is likely to internalize spH molecules that pre-exist at the plasma membrane before stimulation starts. This component was blocked by MiTMAB, perhaps by impairing the binding of dynamin or other key endocytic proteins to phospholipid membranes. Our study suggests the co-existence of two sequential synaptic endocytosis steps taking place during stimulation that are susceptible to pharmacological dissection: an initial step, preferentially sensitive to dynasore, that internalizes vesicular components immediately after they are released, and a MiTMAB-sensitive step that internalizes vesicular components pre-existing at the plasma membrane surface. In addition, we report that post-stimulus endocytosis also has several components with different sensitivities to dynasore and MiTMAB. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

A sphingolipid mechanism for behavioral extinction.

PubMed

Huston, Joseph P; Kornhuber, Johannes; Mühle, Christiane; Japtok, Lukasz; Komorowski, Mara; Mattern, Claudia; Reichel, Martin; Gulbins, Erich; Kleuser, Burkhard; Topic, Bianca; De Souza Silva, Maria A; Müller, Christian P

2016-05-01

Reward-dependent instrumental behavior must continuously be re-adjusted according to environmental conditions. Failure to adapt to changes in reward contingencies may incur psychiatric disorders like anxiety and depression. When an expected reward is omitted, behavior undergoes extinction. While extinction involves active re-learning, it is also accompanied by emotional behaviors indicative of frustration, anxiety, and despair (extinction-induced depression). Here, we report evidence for a sphingolipid mechanism in the extinction of behavior. Rapid extinction, indicating efficient re-learning, coincided with a decrease in the activity of the enzyme acid sphingomyelinase (ASM), which catalyzes turnover of sphingomyelin to ceramide, in the dorsal hippocampus of rats. The stronger the decline in ASM activity, the more rapid was the extinction. Sphingolipid-focused lipidomic analysis showed that this results in a decline of local ceramide species in the dorsal hippocampus. Ceramides shape the fluidity of lipid rafts in synaptic membranes and by that way can control neural plasticity. We also found that aging modifies activity of enzymes and ceramide levels in selective brain regions. Aging also changed how the chronic treatment with corticosterone (stress) or intranasal dopamine modified regional enzyme activity and ceramide levels, coinciding with rate of extinction. These data provide first evidence for a functional ASM-ceramide pathway in the brain involved in the extinction of learned behavior. This finding extends the known cellular mechanisms underlying behavioral plasticity to a new class of membrane-located molecules, the sphingolipids, and their regulatory enzymes, and may offer new treatment targets for extinction- and learning-related psychopathological conditions. Sphingolipids are common lipids in the brain which form lipid domains at pre- and postsynaptic membrane compartments. Here we show a decline in dorsal hippocampus ceramide species together with a reduction of acid sphingomyelinase activity during extinction of conditioned behavior in rats. This reduction was associated with expression of re-learning-related behavior, but not with emotional behaviors. Read the Editorial Highlight for this article on page 485. © 2016 International Society for Neurochemistry.

Calcium channel dynamics limit synaptic release in response to prosthetic stimulation with sinusoidal waveforms

PubMed Central

Freeman, Daniel K.; Jeng, Jed S.; Kelly, Shawn K.; Hartveit, Espen; Fried, Shelley I.

2011-01-01

Extracellular electric stimulation with sinusoidal waveforms has been shown to allow preferential activation of individual types of retinal neurons by varying stimulus frequency. It is important to understand the mechanisms underlying this frequency dependence as a step towards improving methods of preferential activation. In order to elucidate these mechanisms, we implemented a morphologically realistic model of a retinal bipolar cell and measured the response to extracellular stimulation with sinusoidal waveforms. We compared the frequency response of a passive membrane model to the kinetics of voltage-gated calcium channels that mediate synaptic release. The passive electrical properties of the membrane exhibited lowpass filtering with a relatively high cutoff frequency (nominal value = 717 Hz). This cutoff frequency was dependent on intra-axonal resistance, with shorter and wider axons yielding higher cutoff frequencies. However, we found that the cutoff frequency of bipolar cell synaptic release was primarily limited by the relatively slow opening kinetics of Land T-type calcium channels. The cutoff frequency of calcium currents depended nonlinearly on stimulus amplitude, but remained lower than the cutoff frequency of the passive membrane model for a large range of membrane potential fluctuations. These results suggest that while it may be possible to modulate the membrane potential of bipolar cells over a wide range of stimulus frequencies, synaptic release will only be initiated at the lower end of this range. PMID:21628768

[Effects of ursodeoxycholic acid on the liver plasma membrane fluidity, hepatic glutathione concentration, hepatic estrogen receptors and progesterone receptors in pregnant rats with ethinylestradiol and progesterone induced intrahepatic cholestasis].

PubMed

Shi, Qing-yun; Kong, Bei-hua; Ma, Kai-dong; Zhang, Xiang-li; Jiang, Sen

2003-11-01

To explore the effects of ursodeoxycholic acid (UDCA) on the fluidity of hepatic plasma membrane, glutathione concentration in liver, hepatic estrogen receptors and progesterone receptors in pregnant rats with ethinylestradiol and progesterone induced intrahepatic cholestasis. sixty clean SD pregnant rats were selected and divided into three groups at random. Since the 13th day of pregnancy after taking blood, normal group was injected subcutaneously with refined vegetable oil 2.5 ml x kg(-1) x d(-1). Control group and treatment group were injected subcutaneously with the solution of progesterone 75 mg x kg(-1) x d(-1) and 17-alpha-ethynylestradio 1.25 mg x kg(-1) x d(-1) till the 17th day. Since the 17th day control group, normal group were fedwish 0.9% natriichloridi solution 5 ml x kg(-1) x d(-1); Treatment group was fedwish UDCA 50 mg x kg(-1) x d(-1) every day. On the 21th day, all rats were killed. Then the livers were collected for study. Membrane fluidity was measured by fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe. Glutathione concentration was measured by 5,5'-dithionbis (2-nitrobenzoic acid) (DTNB). Estrogen receptors and progesterone receptors were measured by flow cytometry. (1) Hepatic plasma membrane fluidity and glutathione (GSH) concentration: significantly lower level of GSH concentration and higher fluorescence polarization (P) were detected in control group (GSH: 1.13 +/- 0.03, P: 0.149 +/- 0.008) in comparison with normal group (GSH: 2.11 +/- 0.07, P: 0.132 +/- 0.004, P < 0.05). However, Significantly higher level of GSH concentration and lower fluorescence polarization were detected in treatment group (GSH: 1.82 +/- 0.04, P: 0.141 +/- 0.006) in comparison with control group (P < 0.05). The level of GSH concentration and fluorescence polarization were no difference between treatment group and normal group. Hepatic estrogen receptors (ER) and progesterone receptors (PR): The expression of ER and PR in control group (ER: 89.4 +/- 8.4, PR: 112.3 +/- 11.6) were higher than that of other two groups (P < 0.05). The expression of ER and PR in treatment group (ER: 56.4 +/- 7.5, PR: 70.1 +/- 9.3) were lower than that of control group (P < 0.05). But there was no difference between treatment group and normal group (ER: 39.5 +/- 7.3, PR: 59.6 +/- 7.4; P > 0.05). Ursodeoxycholic acid may be effective drug in treatment intrahepatic cholestasis of pregnancy.

Degradation of substance P by membrane peptidases in the rat substantia nigra: effect of selective inhibitors.

PubMed

Oblin, A; Danse, M J; Zivkovic, B

1988-01-11

The hydrolysis of substance P by membrane peptidases prepared from the rat substantia nigra was studied in the presence of selective inhibitors. Substance P degradation by synaptic and mitochondrial membranes was completely inhibited by 1,10-phenanthroline (1 mM), a non-specific metallopeptidase inhibitor. Captopril and bestatine, selective inhibitors of angiotensin converting enzyme and aminopeptidases respectively, were without effects. However, phosphoramidon (1 microM), a putative 'enkephalinase' inhibitor, selectively inhibited substance P degradation by synaptic membranes. These results suggest that a phosphoramidon-sensitive endopeptidase may be the principal enzyme responsible for substance P degradation in substantia nigra.

Factors regulating the abundance and localization of synaptobrevin in the plasma membrane

PubMed Central

Dittman, Jeremy S.; Kaplan, Joshua M.

2006-01-01

After synaptic vesicle fusion, vesicle proteins must be segregated from plasma membrane proteins and recycled to maintain a functional vesicle pool. We monitored the distribution of synaptobrevin, a vesicle protein required for exocytosis, in Caenorhabditis elegans motor neurons by using a pH-sensitive synaptobrevin GFP fusion protein, synaptopHluorin. We estimated that 30% of synaptobrevin was present in the plasma membrane. By using a panel of endocytosis and exocytosis mutants, we found that the majority of surface synaptobrevin derives from fusion of synaptic vesicles and that, in steady state, synaptobrevin equilibrates throughout the axon. The surface synaptobrevin was enriched near active zones, and its spatial extent was regulated by the clathrin adaptin AP180. These results suggest that there is a plasma membrane reservoir of synaptobrevin that is supplied by the synaptic vesicle cycle and available for retrieval throughout the axon. The size of the reservoir is set by the relative rates of exo- and endocytosis. PMID:16844789

Mover Is a Homomeric Phospho-Protein Present on Synaptic Vesicles

PubMed Central

Kremer, Thomas; Hoeber, Jan; Kiran Akula, Asha; Urlaub, Henning; Islinger, Markus; Kirsch, Joachim; Dean, Camin; Dresbach, Thomas

2013-01-01

With remarkably few exceptions, the molecules mediating synaptic vesicle exocytosis at active zones are structurally and functionally conserved between vertebrates and invertebrates. Mover was found in a yeast-2-hybrid assay using the vertebrate-specific active zone scaffolding protein bassoon as a bait. Peptides of Mover have been reported in proteomics screens for self-interacting proteins, phosphorylated proteins, and synaptic vesicle proteins, respectively. Here, we tested the predictions arising from these screens. Using flotation assays, carbonate stripping of peripheral membrane proteins, mass spectrometry, immunogold labelling of purified synaptic vesicles, and immuno-organelle isolation, we found that Mover is indeed a peripheral synaptic vesicle membrane protein. In addition, by generating an antibody against phosphorylated Mover and Western blot analysis of fractionated rat brain, we found that Mover is a bona fide phospho-protein. The localization of Mover to synaptic vesicles is phosphorylation dependent; treatment with a phosphatase caused Mover to dissociate from synaptic vesicles. A yeast-2-hybrid screen, co-immunoprecipitation and cell-based optical assays of homomerization revealed that Mover undergoes homophilic interaction, and regions within both the N- and C- terminus of the protein are required for this interaction. Deleting a region required for homomeric interaction abolished presynaptic targeting of recombinant Mover in cultured neurons. Together, these data prove that Mover is associated with synaptic vesicles, and implicate phosphorylation and multimerization in targeting of Mover to synaptic vesicles and presynaptic sites. PMID:23723986

Anchoring and Synaptic stability of PSD-95 is driven by ephrin-B3

PubMed Central

Hruska, Martin; Henderson, Nathan T.; Xia, Nan L.; Le Marchand, Sylvain J.; Dalva, Matthew B.

2015-01-01

Summary Organization of signaling complexes at excitatory synapses by Membrane Associated Guanylate Kinase (MAGUK) proteins regulates synapse development, plasticity, senescence, and disease. Post-translational modification of MAGUK family proteins can drive their membrane localization, yet it is unclear how these intracellular proteins are targeted to sites of synaptic contact. Here we show using super-resolution imaging, biochemical approaches, and in vivo models that the trans-synaptic organizing protein, ephrin-B3, controls the synaptic localization and stability of PSD-95 and links these events to changes in neuronal activity via negative regulation of a novel MAPK-dependent phosphorylation site on ephrin-B3 (S332). Unphosphorylated ephrin-B3 is enriched at synapses, interacts directly with and stabilizes PSD-95 at synapses. Activity induced phosphorylation of S332 disperses ephrin-B3 from synapses, prevents the interaction with, and enhances the turnover of PSD-95. Thus, ephrin-B3 specifies the synaptic localization of PSD-95 and likely links the synaptic stability of PSD-95 to changes in neuronal activity. PMID:26479588

Anchoring and synaptic stability of PSD-95 is driven by ephrin-B3.

PubMed

Hruska, Martin; Henderson, Nathan T; Xia, Nan L; Le Marchand, Sylvain J; Dalva, Matthew B

2015-11-01

Organization of signaling complexes at excitatory synapses by membrane-associated guanylate kinase (MAGUK) proteins regulates synapse development, plasticity, senescence and disease. Post-translational modification of MAGUK family proteins can drive their membrane localization, yet it is unclear how these intracellular proteins are targeted to sites of synaptic contact. Here we show using super-resolution imaging, biochemical approaches and in vivo models that the trans-synaptic organizing protein ephrin-B3 controls the synaptic localization and stability of PSD-95 and links these events to changes in neuronal activity via negative regulation of a newly identified mitogen-associated protein kinase (MAPK)-dependent phosphorylation site on ephrin-B3, Ser332. Unphosphorylated ephrin-B3 was enriched at synapses, and interacted directly with and stabilized PSD-95 at synapses. Activity-induced phosphorylation of Ser332 dispersed ephrin-B3 from synapses, prevented the interaction with PSD-95 and enhanced the turnover of PSD-95. Thus, ephrin-B3 specifies the synaptic localization of PSD-95 and likely links the synaptic stability of PSD-95 to changes in neuronal activity.

APP is cleaved by Bace1 in pre-synaptic vesicles and establishes a pre-synaptic interactome, via its intracellular domain, with molecular complexes that regulate pre-synaptic vesicles functions.

PubMed

Del Prete, Dolores; Lombino, Franco; Liu, Xinran; D'Adamio, Luciano

2014-01-01

Amyloid Precursor Protein (APP) is a type I membrane protein that undergoes extensive processing by secretases, including BACE1. Although mutations in APP and genes that regulate processing of APP, such as PSENs and BRI2/ITM2B, cause dementias, the normal function of APP in synaptic transmission, synaptic plasticity and memory formation is poorly understood. To grasp the biochemical mechanisms underlying the function of APP in the central nervous system, it is important to first define the sub-cellular localization of APP in synapses and the synaptic interactome of APP. Using biochemical and electron microscopy approaches, we have found that APP is localized in pre-synaptic vesicles, where it is processed by Bace1. By means of a proteomic approach, we have characterized the synaptic interactome of the APP intracellular domain. We focused on this region of APP because in vivo data underline the central functional and pathological role of the intracellular domain of APP. Consistent with the expression of APP in pre-synaptic vesicles, the synaptic APP intracellular domain interactome is predominantly constituted by pre-synaptic, rather than post-synaptic, proteins. This pre-synaptic interactome of the APP intracellular domain includes proteins expressed on pre-synaptic vesicles such as the vesicular SNARE Vamp2/Vamp1 and the Ca2+ sensors Synaptotagmin-1/Synaptotagmin-2, and non-vesicular pre-synaptic proteins that regulate exocytosis, endocytosis and recycling of pre-synaptic vesicles, such as target-membrane-SNAREs (Syntaxin-1b, Syntaxin-1a, Snap25 and Snap47), Munc-18, Nsf, α/β/γ-Snaps and complexin. These data are consistent with a functional role for APP, via its carboxyl-terminal domain, in exocytosis, endocytosis and/or recycling of pre-synaptic vesicles.

Combined Diffusion Tensor Imaging and Transverse Relaxometry in Early-Onset Bipolar Disorder

ERIC Educational Resources Information Center

Gonenc, Atilla; Frazier, Jean A.; Crowley, David J.; Moore, Constance M.

2010-01-01

Objective: Transverse relaxation time (T2) imaging provides the opportunity to examine membrane fluidity, which can affect a number of cellular functions. The objective of the present work was to examine T2 abnormalities in children with unmodified DSM-IV-TR bipolar disorder (BD) in bilateral cingulate-paracingulate (CPC) white matter. Method: A…

Measuring the diffusion coefficient of ganglioside on cell membrane by fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Dong, Shiqing; You, Minghai; Chen, Jianling; Zhou, Jie; Xie, Shusen; Yang, Hongqin

2017-06-01

The fluidity of proteins and lipids on cell membrane plays an important role in cell’s physiological functions. Fluorescence correlation spectroscopy (FCS) is an effective technique to detect the rapid dynamic behaviors of proteins and/or lipids in living cells. In this study, we used the rhodamine6G solution to optimize the FCS system. And, cholera toxin B subunit (CT-B) was used to label ganglioside on living Hela cell membranes. The diffusion time and coefficients of ganglioside can be obtained through fitting the autocorrelation curve based on the model of two-dimensional cell membrane. The results showed that the diffusion coefficients of ganglioside distributed within a wide range. It revealed the lateral diffusion of lipids on cell membrane was inhomogeneous, which was due to different microstructures of cytoplasmic membrane. The study provides a helpful method for further studying the dynamic characteristics of proteins and lipids molecules on living cell membrane.

Synaptic Polarity Depends on Phosphatidylinositol Signaling Regulated by myo-Inositol Monophosphatase in Caenorhabditis elegans

PubMed Central

Kimata, Tsubasa; Tanizawa, Yoshinori; Can, Yoko; Ikeda, Shingo; Kuhara, Atsushi; Mori, Ikue

2012-01-01

Although neurons are highly polarized, how neuronal polarity is generated remains poorly understood. An evolutionarily conserved inositol-producing enzyme myo-inositol monophosphatase (IMPase) is essential for polarized localization of synaptic molecules in Caenorhabditis elegans and can be inhibited by lithium, a drug for bipolar disorder. The synaptic defect of IMPase mutants causes defects in sensory behaviors including thermotaxis. Here we show that the abnormalities of IMPase mutants can be suppressed by mutations in two enzymes, phospholipase Cβ or synaptojanin, which presumably reduce the level of membrane phosphatidylinositol 4,5-bisphosphate (PIP2). We also found that mutations in phospholipase Cβ conferred resistance to lithium treatment. Our results suggest that reduction of PIP2 on plasma membrane is a major cause of abnormal synaptic polarity in IMPase mutants and provide the first in vivo evidence that lithium impairs neuronal PIP2 synthesis through inhibition of IMPase. We propose that the PIP2 signaling regulated by IMPase plays a novel and fundamental role in the synaptic polarity. PMID:22446320

Haemato-protective influence of dietary fenugreek (Trigonella foenum-graecum L.) seeds is potentiated by onion (Allium cepa L.) in streptozotocin-induced diabetic rats.

PubMed

Pradeep, Seetur R; Srinivasan, Krishnapura

2018-02-01

We have recently reported the beneficial modulation of metabolic abnormalities and oxidative stress in diabetic rats by dietary fenugreek seeds and onion. This investigation evaluated the protective influence of dietary fenugreek seeds (100 g kg -1 ) and onion (30 g kg -1 ) on erythrocytes of streptozotocin-induced diabetic rats, through modulation of reduced haematological indices and antisickling potency. This study also evaluated the altered erythrocyte membrane lipid profile and beneficial countering of increased lipid peroxidation, osmotic fragility, along with reduced membrane fluidity and deformability, nitric oxide production and echinocyte formation. Dietary fenugreek seeds and onion appeared to counter the deformity and fragility of erythrocytes partially in diabetic rats by their antioxidant potential and hypocholesterolemic property. The antisickling potency of these spices was accomplished by a substantial decrease in echinocyte population and AGEs in diabetic rats. Further insight into the factors that might have reduced the fluidity of erythrocytes in diabetic rats revealed changes in the cholesterol: phospholipid ratio, fatty acid profile, and activities of membrane-bound enzymes. Dietary fenugreek seeds and onion offered a beneficial protective effect to the red blood cells, the effect being higher with fenugreek + onion. This is the first report on the hemato-protective influence of a nutraceutical food component in diabetic situation. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Terpenes increase the lipid dynamics in the Leishmania plasma membrane at concentrations similar to their IC50 values.

PubMed

Camargos, Heverton Silva; Moreira, Rodrigo Alves; Mendanha, Sebastião Antonio; Fernandes, Kelly Souza; Dorta, Miriam Leandro; Alonso, Antonio

2014-01-01

Although many terpenes have shown antitumor, antibacterial, antifungal, and antiparasitic activity, the mechanism of action is not well established. Electron paramagnetic resonance (EPR) spectroscopy of the spin-labeled 5-doxyl stearic acid revealed remarkable fluidity increases in the plasma membrane of terpene-treated Leishmania amazonensis promastigotes. For an antiproliferative activity assay using 5×10(6) parasites/mL, the sesquiterpene nerolidol and the monoterpenes (+)-limonene, α-terpineol and 1,8-cineole inhibited the growth of the parasites with IC50 values of 0.008, 0.549, 0.678 and 4.697 mM, respectively. The IC50 values of these terpenes increased as the parasite concentration used in the cytotoxicity assay increased, and this behavior was examined using a theoretical treatment of the experimental data. Cytotoxicity tests with the same parasite concentration as in the EPR experiments revealed a correlation between the IC50 values of the terpenes and the concentrations at which they altered the membrane fluidity. In addition, the terpenes induced small amounts of cell lysis (4-9%) at their respective IC50 values. For assays with high cell concentrations (2×10(9) parasites/mL), the incorporation of terpene into the cell membrane was very fast, and the IC50 values observed for 24 h and 5 min-incubation periods were not significantly different. Taken together, these results suggest that terpene cytotoxicity is associated with the attack on the plasma membrane of the parasite. The in vitro cytotoxicity of nerolidol was similar to that of miltefosine, and nerolidol has high hydrophobicity; thus, nerolidol might be used in drug delivery systems, such as lipid nanoparticles to treat leishmaniasis.

Terpenes Increase the Lipid Dynamics in the Leishmania Plasma Membrane at Concentrations Similar to Their IC50 Values

PubMed Central

Camargos, Heverton Silva; Moreira, Rodrigo Alves; Mendanha, Sebastião Antonio; Fernandes, Kelly Souza; Dorta, Miriam Leandro; Alonso, Antonio

2014-01-01

Although many terpenes have shown antitumor, antibacterial, antifungal, and antiparasitic activity, the mechanism of action is not well established. Electron paramagnetic resonance (EPR) spectroscopy of the spin-labeled 5-doxyl stearic acid revealed remarkable fluidity increases in the plasma membrane of terpene-treated Leishmania amazonensis promastigotes. For an antiproliferative activity assay using 5×106 parasites/mL, the sesquiterpene nerolidol and the monoterpenes (+)-limonene, α-terpineol and 1,8-cineole inhibited the growth of the parasites with IC50 values of 0.008, 0.549, 0.678 and 4.697 mM, respectively. The IC50 values of these terpenes increased as the parasite concentration used in the cytotoxicity assay increased, and this behavior was examined using a theoretical treatment of the experimental data. Cytotoxicity tests with the same parasite concentration as in the EPR experiments revealed a correlation between the IC50 values of the terpenes and the concentrations at which they altered the membrane fluidity. In addition, the terpenes induced small amounts of cell lysis (4–9%) at their respective IC50 values. For assays with high cell concentrations (2×109 parasites/mL), the incorporation of terpene into the cell membrane was very fast, and the IC50 values observed for 24 h and 5 min-incubation periods were not significantly different. Taken together, these results suggest that terpene cytotoxicity is associated with the attack on the plasma membrane of the parasite. The in vitro cytotoxicity of nerolidol was similar to that of miltefosine, and nerolidol has high hydrophobicity; thus, nerolidol might be used in drug delivery systems, such as lipid nanoparticles to treat leishmaniasis. PMID:25101672

Changes in membrane biophysical properties induced by the Budesonide/Hydroxypropyl-β-cyclodextrin complex.

PubMed

Dos Santos, Andreia G; Bayiha, Jules César; Dufour, Gilles; Cataldo, Didier; Evrard, Brigitte; Silva, Liana C; Deleu, Magali; Mingeot-Leclercq, Marie-Paule

2017-10-01

Budesonide (BUD), a poorly soluble anti-inflammatory drug, is used to treat patients suffering from asthma and COPD (Chronic Obstructive Pulmonary Disease). Hydroxypropyl-β-cyclodextrin (HPβCD), a biocompatible cyclodextrin known to interact with cholesterol, is used as a drug-solubilizing agent in pharmaceutical formulations. Budesonide administered as an inclusion complex within HPβCD (BUD:HPβCD) required a quarter of the nominal dose of the suspension formulation and significantly reduced neutrophil-induced inflammation in a COPD mouse model exceeding the effect of each molecule administered individually. This suggests the role of lipid domains enriched in cholesterol for inflammatory signaling activation. In this context, we investigated the effect of BUD:HPβCD on the biophysical properties of membrane lipids. On cellular models (A549, lung epithelial cells), BUD:HPβCD extracted cholesterol similarly to HPβCD. On large unilamellar vesicles (LUVs), by using the fluorescent probes diphenylhexatriene (DPH) and calcein, we demonstrated an increase in membrane fluidity and permeability induced by BUD:HPβCD in vesicles containing cholesterol. On giant unilamellar vesicles (GUVs) and lipid monolayers, BUD:HPβCD induced the disruption of cholesterol-enriched raft-like liquid ordered domains as well as changes in lipid packing and lipid desorption from the cholesterol monolayers, respectively. Except for membrane fluidity, all these effects were enhanced when HPβCD was complexed with budesonide as compared with HPβCD. Since cholesterol-enriched domains have been linked to membrane signaling including pathways involved in inflammation processes, we hypothesized the effects of BUD:HPβCD could be partly mediated by changes in the biophysical properties of cholesterol-enriched domains. Copyright © 2017 Elsevier B.V. All rights reserved.

Heat stress dictates microbial lipid composition in hydrothermal marine sediments

NASA Astrophysics Data System (ADS)

Sollich, M.; Yoshinaga, M. Y.; Häusler, S.; Hinrichs, K. U.; Bühring, S. I.

2016-02-01

Abundant and diverse microbial communities inhabit hydrothermal marine sediments. Since ion permeability of membranes increases with temperature archaea and bacteria that use proton/sodium as coupling ions for bioenergetics must constantly adjust their cytoplasmic membrane permeability, which in turn is mostly controlled by the lipid composition. Here, we investigated a thermal gradient across a marine sediment field (ranging from 18 to over 100°C) and tested the concept that membrane lipids provide a major biochemical basis for cellular bioenergetics of archaea and bacteria under stressful conditions. Reflecting the lower ion permeability of the ether-linked isoprenoidal lipids, we found that archaea dominate over bacteria in sediments of >50 °C. Moreover, a detailed examination of the molecular lipid species revealed a quandary: low membrane permeability concomitantly with increased fluidity is required for energy conservation of both archaea and bacteria under heat stress. For instance, bacterial fatty acids were found to increase chain length in concert with a higher degree of unsaturation at elevated sediment temperatures while archaeal tetraethers were observed to show a higher degree of bulking (e.g. methylation and H-shaped) and fluidity (i.e. cyclization) under elevated temperatures. In addition, our data indicate that strong intermolecular hydrogen bonding at the headgroup level of archaeal glycolipids and bacterial sphingolipids may provide ideal membrane stability to attain the required balance between low permeability and a more fluidized configuration. For example, sphingolipids may stabilize bacterial phospholipids into lipid domains, enabling bacteria to thrive in heated sediments under unfavorable thermodynamic conditions. The scientific marriage of lipidomics and bioenergetics described here provides a new dimension for understanding microbial life in natural environments.

The extent of the temperature-induced membrane remodeling in two closely related Bordetella species reflects their adaptation to diverse environmental niches.

PubMed

Seydlova, Gabriela; Beranova, Jana; Bibova, Ilona; Dienstbier, Ana; Drzmisek, Jakub; Masin, Jiri; Fiser, Radovan; Konopasek, Ivo; Vecerek, Branislav

2017-05-12

Changes in environmental temperature represent one of the major stresses faced by microorganisms as they affect the function of the cytoplasmic membrane. In this study, we have analyzed the thermal adaptation in two closely related respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica Although B. pertussis represents a pathogen strictly adapted to the human body temperature, B. bronchiseptica causes infection in a broad range of animals and survives also outside of the host. We applied GC-MS to determine the fatty acids of both Bordetella species grown at different temperatures and analyzed the membrane fluidity by fluorescence anisotropy measurement. In parallel, we also monitored the effect of growth temperature changes on the expression and production of several virulence factors. In response to low temperatures, B. pertussis adapted its fatty acid composition and membrane fluidity to a considerably lesser extent when compared with B. bronchiseptica Remarkably, B. pertussis maintained the production of virulence factors at 24 °C, whereas B. bronchiseptica cells resumed the production only upon temperature upshift to 37 °C. This growth temperature-associated differential modulation of virulence factor production was linked to the phosphorylation state of transcriptional regulator BvgA. The observed differences in low-temperature adaptation between B. pertussis and B. bronchiseptica may result from selective adaptation of B. pertussis to the human host. We propose that the reduced plasticity of the B. pertussis membranes ensures sustained production of virulence factors at suboptimal temperatures and may play an important role in the transmission of the disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Macroscopic neural mass model constructed from a current-based network model of spiking neurons.

PubMed

Umehara, Hiroaki; Okada, Masato; Teramae, Jun-Nosuke; Naruse, Yasushi

2017-02-01

Neural mass models (NMMs) are efficient frameworks for describing macroscopic cortical dynamics including electroencephalogram and magnetoencephalogram signals. Originally, these models were formulated on an empirical basis of synaptic dynamics with relatively long time constants. By clarifying the relations between NMMs and the dynamics of microscopic structures such as neurons and synapses, we can better understand cortical and neural mechanisms from a multi-scale perspective. In a previous study, the NMMs were analytically derived by averaging the equations of synaptic dynamics over the neurons in the population and further averaging the equations of the membrane-potential dynamics. However, the averaging of synaptic current assumes that the neuron membrane potentials are nearly time invariant and that they remain at sub-threshold levels to retain the conductance-based model. This approximation limits the NMM to the non-firing state. In the present study, we newly propose a derivation of a NMM by alternatively approximating the synaptic current which is assumed to be independent of the membrane potential, thus adopting a current-based model. Our proposed model releases the constraint of the nearly constant membrane potential. We confirm that the obtained model is reducible to the previous model in the non-firing situation and that it reproduces the temporal mean values and relative power spectrum densities of the average membrane potentials for the spiking neurons. It is further ensured that the existing NMM properly models the averaged dynamics over individual neurons even if they are spiking in the populations.

Cholesterol Regulates Multiple Forms of Vesicle Endocytosis at a Mammalian Central Synapse

PubMed Central

Yue, Hai-Yuan; Xu, Jianhua

2015-01-01

Endocytosis in synapses sustains neurotransmission by recycling vesicle membrane and maintaining the homeostasis of synaptic membrane. A role of membrane cholesterol in synaptic endocytosis remains controversial because of conflicting observations, technical limitations in previous studies, and potential interference from nonspecific effects after cholesterol manipulation. Furthermore, it is unclear whether cholesterol participates in distinct forms of endocytosis that function under different activity levels. In this study, applying the whole-cell membrane capacitance measurement to monitor endocytosis in real time at the rat calyx of Held terminals, we found that disrupting cholesterol with dialysis of cholesterol oxidase (COase) or methyl-β-cyclodextrin (MCD) impaired three different forms of endocytosis, i.e., slow endocytosis, rapid endocytosis, and endocytosis of the retrievable membrane that exists at the surface before stimulation. The effects were observed when disruption of cholesterol was mild enough not to change Ca2+ channel current or vesicle exocytosis, indicative of stringent cholesterol requirement in synaptic endocytosis. Extracting cholesterol with high concentrations of MCD reduced exocytosis, mainly by decreasing the readily releasable pool (RRP) and the vesicle replenishment after RRP depletion. Our study suggests that cholesterol is an important, universal regulator in multiple forms of vesicle endocytosis at mammalian central synapses. PMID:25893258

Modification of membrane cholesterol and its impact on frozen-thawed chicken sperm characteristics.

PubMed

Partyka, Agnieszka; Bonarska-Kujawa, Dorota; Sporniak, Marta; Strojecki, Maciej; Niżański, Wojciech

2016-10-01

This study was conducted to determine the changes in chicken sperm plasma membranes fluidity and polarity as lipid packing arrangement induced by cholesterol-loaded cyclodextrin (CLC) and 2-hydroxypropyl-β-cyclodextrin (HBCD) and how sperm cryopreservation outcomes are improved by these changes. Treatment with 2 mg HBCD supported the highest (P < 0.01) percentage of viable spermatozoa compared with the control and CLCs groups after cryopreservation. The percentage of post-thaw progressive and rapid sperm motility was highest in 2 mg HBCD (P < 0.01). After thawing, sperm treated with 1 or 2 mg CLC showed the highest anisotropy at 5, 21, 25 and 40°C (P < 0.01). At 25°C, the lowest anisotropy was observed in the thawed semen from the control group. The highest value (P < 0.01) of generalized polarization (GP) (0.5) at 5°C was observed in the 1 mg CLC treated sample. After 2 h of incubation, the highest percentage of viable spermatozoa was observed in the HBCD group in relation to the other treatments (P < 0.01). Exposure to 1 mg or 2 mg of CLC significantly decreased the percentage of live spermatozoa after thawing (P < 0.01). In conclusion, HBCD appears to play a role in the modification of sperm membranes, increasing their fluidity and preventing them against membrane phase transition to gel, thus minimizing freezing-thaw sperm damage. HBCD treatment enhances chicken sperm viability and motility after cryopreservation and subsequent storage. This novel procedure may be useful for improving the technology for cryopreservation of fowl spermatozoa.

Tolerance to chitosan by Trichoderma species is associated with low membrane fluidity.

PubMed

Zavala-González, Ernesto A; Lopez-Moya, Federico; Aranda-Martinez, Almudena; Cruz-Valerio, Mayra; Lopez-Llorca, Luis Vicente; Ramírez-Lepe, Mario

2016-07-01

The effect of chitosan on growth of Trichoderma spp., a cosmopolitan genus widely exploited for their biocontrol properties was evaluated. Based on genotypic (ITS of 18S rDNA) characters, four isolates of Trichoderma were identified as T. pseudokoningii FLM16, T. citrinoviride FLM17, T. harzianum EZG47, and T. koningiopsis VSL185. Chitosan reduces radial growth of Trichoderma isolates in concentration-wise manner. T. koningiopsis VSL185 was the most chitosan tolerant isolate in all culture media amended with chitosan (0.5-2.0 mg ml(-1) ). Minimal Inhibitory Concentration (MIC) and Minimal Fungicidal Concentration (MFC) were determined showing that T. koningiopsis VSL185 displays higher chitosan tolerance with MIC value >2000 μg ml(-1) while for other Trichoderma isolates MIC values were around 10 μg ml(-1) . Finally, free fatty acid composition reveals that T. koningiopsis VSL185, chitosan tolerant isolate, displays lower linolenic acid (C18:3) content than chitosan sensitive Trichoderma isolates. Our findings suggest that low membrane fluidity is associated with chitosan tolerance in Trichoderma spp. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Aluminum and temperature alteration of cell membrane permeability of Quercus rubra

DOE Office of Scientific and Technical Information (OSTI.GOV)

Junping Chen; Sucoff, E.I.; Stadelmann, E.J.

1991-06-01

Al toxicity is the major factor limiting plant growth in acid soils. This report extends research on Al-induced changes in membrane behavior of intact root cortex cells of Northern red oak (Quercus rubra). Membrane permeability was determined by the plasmometric method for individual intact cells at temperatures from 2 or 4 to 35 C. Al (0.37 millimolar) significantly increased membrane permeability to urea and monoethyl urea and decreased permeability to water. Al significantly altered the activation energy required to transport water (+ 32%), urea (+ 9%), and monoethyl urea ({minus}7%) across cell membranes. Above 9 C, Al increased the lipidmore » partiality of the cell membranes; below 7 C, Al decreased it. Al narrowed by 6 C the temperature range over which plasmolysis occurred without membrane damage. These changes in membrane behavior are explainable if Al reduced membrane lipid fluidity and kink frequency and increases packing density and the occurrence of straight lipid chains.« less

Alteration in lipid composition of plasma membranes of sensitive and resistant Guerin carcinoma cells due to the action of free and liposomal form of cisplatin.

PubMed

Naleskina, L A; Todor, I N; Nosko, M M; Lukianova, N Y; Pivnyuk, V M; Chekhun, V F

2013-09-01

To study in vivo changes of lipid composition of plasma membranes of sensitive and resistant to cisplatin Guerin carcinoma cells under influence of free and liposomal cisplatin forms. The isolation of plasma membranes from parental (sensitive) and resistant to cisplatin Guerin carcinoma cells was by differential ultracentrifugation in sucrose density gradient. Lipids were detected by method of thin-layer chromatography. It was determined that more effective action of cisplatin liposomal form on resistant cells is associated with essential abnormalities of conformation of plasma membrane due to change of lipid components and architectonics of rafts. It results in the increase of membrane fluidity. Reconstructions in lipid composition of plasma membranes of cisplatin-resistant Guerin carcinoma cells provide more intensive delivery of drug into the cells, increase of its concentration and more effective interaction with cellular structural elements.

Engineering membrane and cell-wall programs for tolerance to toxic chemicals: Beyond solo genes.

PubMed

Sandoval, Nicholas R; Papoutsakis, Eleftherios T

2016-10-01

Metabolite toxicity in microbes, particularly at the membrane, remains a bottleneck in the production of fuels and chemicals. Under chemical stress, native adaptation mechanisms combat hyper-fluidization by modifying the phospholipids in the membrane. Recent work in fluxomics reveals the mechanism of how membrane damage negatively affects energy metabolism while lipidomic and transcriptomic analyses show that strains evolved to be tolerant maintain membrane fluidity under stress through a variety of mechanisms such as incorporation of cyclopropanated fatty acids, trans-unsaturated fatty acids, and upregulation of cell wall biosynthesis genes. Engineered strains with modifications made in the biosynthesis of fatty acids, peptidoglycan, and lipopolysaccharide have shown increased tolerance to exogenous stress as well as increased production of desired metabolites of industrial importance. We review recent advances in elucidation of mechanisms or toxicity and tolerance as well as efforts to engineer the bacterial membrane and cell wall. Copyright © 2016 Elsevier Ltd. All rights reserved.

Structure-Function Relationship of Transporters in the Glutamate-Glutamine Cycle of the Central Nervous System.

PubMed

Hayashi, Mariko Kato

2018-04-12

Many kinds of transporters contribute to glutamatergic excitatory synaptic transmission. Glutamate is loaded into synaptic vesicles by vesicular glutamate transporters to be released from presynaptic terminals. After synaptic vesicle release, glutamate is taken up by neurons or astrocytes to terminate the signal and to prepare for the next signal. Glutamate transporters on the plasma membrane are responsible for transporting glutamate from extracellular fluid to cytoplasm. Glutamate taken up by astrocyte is converted to glutamine by glutamine synthetase and transported back to neurons through glutamine transporters on the plasma membranes of the astrocytes and then on neurons. Glutamine is converted back to glutamate by glutaminase in the neuronal cytoplasm and then loaded into synaptic vesicles again. Here, the structures of glutamate transporters and glutamine transporters, their conformational changes, and how they use electrochemical gradients of various ions for substrate transport are summarized. Pharmacological regulations of these transporters are also discussed.

Fusion competent synaptic vesicles persist upon active zone disruption and loss of vesicle docking

PubMed Central

Wang, Shan Shan H.; Held, Richard G.; Wong, Man Yan; Liu, Changliang; Karakhanyan, Aziz; Kaeser, Pascal S.

2016-01-01

In a nerve terminal, synaptic vesicle docking and release are restricted to an active zone. The active zone is a protein scaffold that is attached to the presynaptic plasma membrane and opposed to postsynaptic receptors. Here, we generated conditional knockout mice removing the active zone proteins RIM and ELKS, which additionally led to loss of Munc13, Bassoon, Piccolo, and RIM-BP, indicating disassembly of the active zone. We observed a near complete lack of synaptic vesicle docking and a strong reduction in vesicular release probability and the speed of exocytosis, but total vesicle numbers, SNARE protein levels, and postsynaptic densities remained unaffected. Despite loss of the priming proteins Munc13 and RIM and of docked vesicles, a pool of releasable vesicles remained. Thus, the active zone is necessary for synaptic vesicle docking and to enhance release probability, but releasable vesicles can be localized distant from the presynaptic plasma membrane. PMID:27537483

Influence of Glucose Deprivation on Membrane Potentials of Plasma Membranes, Mitochondria and Synaptic Vesicles in Rat Brain Synaptosomes.

PubMed

Hrynevich, Sviatlana V; Pekun, Tatyana G; Waseem, Tatyana V; Fedorovich, Sergei V

2015-06-01

Hypoglycemia can cause neuronal cell death similar to that of glutamate-induced cell death. In the present paper, we investigated the effect of glucose removal from incubation medium on changes of mitochondrial and plasma membrane potentials in rat brain synaptosomes using the fluorescent dyes DiSC3(5) and JC-1. We also monitored pH gradients in synaptic vesicles and their recycling by the fluorescent dye acridine orange. Glucose deprivation was found to cause an inhibition of K(+)-induced Ca(2+)-dependent exocytosis and a shift of mitochondrial and plasma membrane potentials to more positive values. The sensitivity of these parameters to the energy deficit caused by the removal of glucose showed the following order: mitochondrial membrane potential > plasma membrane potential > pH gradient in synaptic vesicles. The latter was almost unaffected by deprivation compared with the control. The pH-dependent dye acridine orange was used to investigate synaptic vesicle recycling. However, the compound's fluorescence was shown to be enhanced also by the mixture of mitochondrial toxins rotenone (10 µM) and oligomycin (5 µg/mL). This means that acridine orange can presumably be partially distributed in the intermembrane space of mitochondria. Glucose removal from the incubation medium resulted in a 3.7-fold raise of acridine orange response to rotenone + oligomycin suggesting a dramatic increase in the mitochondrial pH gradient. Our results suggest that the biophysical characteristics of neuronal presynaptic endings do not favor excessive non-controlled neurotransmitter release in case of hypoglycemia. The inhibition of exocytosis and the increase of the mitochondrial pH gradient, while preserving the vesicular pH gradient, are proposed as compensatory mechanisms.

Rapid synaptic vesicle endocytosis in cone photoreceptors of salamander retina

PubMed Central

Van Hook, Matthew J.; Thoreson, Wallace B.

2013-01-01

Following synaptic vesicle exocytosis, neurons retrieve the fused membrane by a process of endocytosis in order to provide a supply of vesicles for subsequent release and maintain the presynaptic active zone. Rod and cone photoreceptors use a specialized structure called the synaptic ribbon that enables them to sustain high rates of neurotransmitter release. They must also employ mechanisms of synaptic vesicle endocytosis capable of keeping up with release. While much is known about endocytosis at another retinal ribbon synapse, that of the goldfish Mb1 bipolar cell, less is known about endocytosis in photoreceptors. We used capacitance recording techniques to measure vesicle membrane fusion and retrieval in photoreceptors from salamander retinal slices. We found that application of brief depolarizing steps (<100 ms) to cones evoked exocytosis followed by rapid endocytosis with a time constant ~250 ms. In some cases, the capacitance trace overshot the baseline, indicating excess endocytosis. Calcium had no effect on the time constant, but enhanced excess endocytosis resulting in a faster rate of membrane retrieval. Surprisingly, endocytosis was unaffected by blockers of dynamin, suggesting that cone endocytosis is dynamin-independent. This contrasts with synaptic vesicle endocytosis in rods, which was inhibited by the dynamin inhibitor dynasore and GTPγS introduced through the patch pipette, suggesting that the two photoreceptor types employ distinct pathways for vesicle retrieval. The fast kinetics of synaptic vesicle endocytosis in photoreceptors likely enables these cells to maintain a high rate of transmitter release, allowing them to faithfully signal changes in illumination to second-order neurons. PMID:23238726

The effects of oxygen on the evolution of microbial membranes

NASA Technical Reports Server (NTRS)

Jahnke, L. L.

1991-01-01

One prokaryote, Methylococcus capsulatus, synthesizes both hopanoids and sterols and, thus, provides a unique opportunity to study the evolution of membrane function. When M. capsulatus was grown at different temperatures, lipid analysis of the whole cells showed that both sterol and unsaturated fatty acid levels decreased at higher growth temperatures; sterol concentrations were 0.116 micro mole/micro mole phospholipid at 30 C and 0.025 micro mole/mirco mole phospholipid at 45 C, while the saturated to unsaturated fatty acid ratio increased from 0.397 to 1.475. Hopane polyol levels were constant over this range; however, methylation of the A-ring decreased markedly in cells grown at 30 C. These results imply that sterol and hopane molecules are required for enhancement of some specific membrane function, potentially by modulating membrane fluidity.

Fragile X Mental Retardation Protein Regulates Activity-Dependent Membrane Trafficking and Trans-Synaptic Signaling Mediating Synaptic Remodeling

PubMed Central

Sears, James C.; Broadie, Kendal

2018-01-01

Fragile X syndrome (FXS) is the leading monogenic cause of autism and intellectual disability. The disease arises through loss of fragile X mental retardation protein (FMRP), which normally exhibits peak expression levels in early-use critical periods, and is required for activity-dependent synaptic remodeling during this transient developmental window. FMRP canonically binds mRNA to repress protein translation, with targets that regulate cytoskeleton dynamics, membrane trafficking, and trans-synaptic signaling. We focus here on recent advances emerging in these three areas from the Drosophila disease model. In the well-characterized central brain mushroom body (MB) olfactory learning/memory circuit, FMRP is required for activity-dependent synaptic remodeling of projection neurons innervating the MB calyx, with function tightly restricted to an early-use critical period. FMRP loss is phenocopied by conditional removal of FMRP only during this critical period, and rescued by FMRP conditional expression only during this critical period. Consistent with FXS hyperexcitation, FMRP loss defects are phenocopied by heightened sensory experience and targeted optogenetic hyperexcitation during this critical period. FMRP binds mRNA encoding Drosophila ESCRTIII core component Shrub (human CHMP4 homolog) to restrict Shrub translation in an activity-dependent mechanism only during this same critical period. Shrub mediates endosomal membrane trafficking, and perturbing Shrub expression is known to interfere with neuronal process pruning. Consistently, FMRP loss and Shrub overexpression targeted to projection neurons similarly causes endosomal membrane trafficking defects within synaptic boutons, and genetic reduction of Shrub strikingly rescues Drosophila FXS model defects. In parallel work on the well-characterized giant fiber (GF) circuit, FMRP limits iontophoretic dye loading into central interneurons, demonstrating an FMRP role controlling core neuronal properties through the activity-dependent repression of translation. In the well-characterized Drosophila neuromuscular junction (NMJ) model, developmental synaptogenesis and activity-dependent synaptic remodeling both require extracellular matrix metalloproteinase (MMP) enzymes interacting with the heparan sulfate proteoglycan (HSPG) glypican dally-like protein (Dlp) to restrict trans-synaptic Wnt signaling, with FXS synaptogenic defects alleviated by both MMP and HSPG reduction. This new mechanistic axis spanning from activity to FMRP to HSPG-dependent MMP regulation modulates activity-dependent synaptogenesis. We discuss future directions for these mechanisms, and intersecting research priorities for FMRP in glial and signaling interactions. PMID:29375303

The effects of membrane cholesterol and simvastatin on red blood cell deformability and ATP release.

PubMed

Forsyth, Alison M; Braunmüller, Susanne; Wan, Jiandi; Franke, Thomas; Stone, Howard A

2012-05-01

It is known that deformation of red blood cells (RBCs) is linked to ATP release from the cells. Further, membrane cholesterol has been shown to alter properties of the cell membrane such as fluidity and bending stiffness. Membrane cholesterol content is increased in some cardiovascular diseases, for example, in individuals with acute coronary syndromes and chronic stable angina, and therefore, because of the potential clinical relevance, we investigated the influence of altered RBC membrane cholesterol levels on ATP release. Because of the correlation between statins and reduced membrane cholesterol in vivo, we also investigated the effects of simvastatin on RBC deformation and ATP release. We found that reducing membrane cholesterol increases cell deformability and ATP release. We also found that simvastatin increases deformability by acting directly on the membrane in the absence of the liver, and that ATP release was increased for cells with enriched cholesterol after treatment with simvastatin. Copyright © 2012 Elsevier Inc. All rights reserved.

Otoferlin acts as a Ca2+ sensor for vesicle fusion and vesicle pool replenishment at auditory hair cell ribbon synapses

PubMed Central

Goutman, Juan D; Auclair, Sarah Marie; Boutet de Monvel, Jacques; Tertrais, Margot; Emptoz, Alice; Parrin, Alexandre; Nouaille, Sylvie; Guillon, Marc; Sachse, Martin; Ciric, Danica; Bahloul, Amel; Hardelin, Jean-Pierre; Sutton, Roger Bryan; Avan, Paul; Krishnakumar, Shyam S; Rothman, James E

2017-01-01

Hearing relies on rapid, temporally precise, and sustained neurotransmitter release at the ribbon synapses of sensory cells, the inner hair cells (IHCs). This process requires otoferlin, a six C2-domain, Ca2+-binding transmembrane protein of synaptic vesicles. To decipher the role of otoferlin in the synaptic vesicle cycle, we produced knock-in mice (Otof Ala515,Ala517/Ala515,Ala517) with lower Ca2+-binding affinity of the C2C domain. The IHC ribbon synapse structure, synaptic Ca2+ currents, and otoferlin distribution were unaffected in these mutant mice, but auditory brainstem response wave-I amplitude was reduced. Lower Ca2+ sensitivity and delay of the fast and sustained components of synaptic exocytosis were revealed by membrane capacitance measurement upon modulations of intracellular Ca2+ concentration, by varying Ca2+ influx through voltage-gated Ca2+-channels or Ca2+ uncaging. Otoferlin thus functions as a Ca2+ sensor, setting the rates of primed vesicle fusion with the presynaptic plasma membrane and synaptic vesicle pool replenishment in the IHC active zone. PMID:29111973

Otoferlin acts as a Ca2+ sensor for vesicle fusion and vesicle pool replenishment at auditory hair cell ribbon synapses.

PubMed

Michalski, Nicolas; Goutman, Juan D; Auclair, Sarah Marie; Boutet de Monvel, Jacques; Tertrais, Margot; Emptoz, Alice; Parrin, Alexandre; Nouaille, Sylvie; Guillon, Marc; Sachse, Martin; Ciric, Danica; Bahloul, Amel; Hardelin, Jean-Pierre; Sutton, Roger Bryan; Avan, Paul; Krishnakumar, Shyam S; Rothman, James E; Dulon, Didier; Safieddine, Saaid; Petit, Christine

2017-11-07

Hearing relies on rapid, temporally precise, and sustained neurotransmitter release at the ribbon synapses of sensory cells, the inner hair cells (IHCs). This process requires otoferlin, a six C 2 -domain, Ca 2+ -binding transmembrane protein of synaptic vesicles. To decipher the role of otoferlin in the synaptic vesicle cycle, we produced knock-in mice ( Otof Ala515,Ala517/Ala515,Ala517 ) with lower Ca 2+ -binding affinity of the C 2 C domain. The IHC ribbon synapse structure, synaptic Ca 2+ currents, and otoferlin distribution were unaffected in these mutant mice, but auditory brainstem response wave-I amplitude was reduced. Lower Ca 2+ sensitivity and delay of the fast and sustained components of synaptic exocytosis were revealed by membrane capacitance measurement upon modulations of intracellular Ca 2+ concentration, by varying Ca 2+ influx through voltage-gated Ca 2+ -channels or Ca 2+ uncaging. Otoferlin thus functions as a Ca 2+ sensor, setting the rates of primed vesicle fusion with the presynaptic plasma membrane and synaptic vesicle pool replenishment in the IHC active zone.

Planar Supported Membranes with Mobile SNARE Proteins and Quantitative Fluorescence Microscopy Assays to Study Synaptic Vesicle Fusion

PubMed Central

Kiessling, Volker; Liang, Binyong; Kreutzberger, Alex J. B.; Tamm, Lukas K.

2017-01-01

Synaptic vesicle membrane fusion, the process by which neurotransmitter gets released at the presynaptic membrane is mediated by a complex interplay between proteins and lipids. The realization that the lipid bilayer is not just a passive environment where other molecular players like SNARE proteins act, but is itself actively involved in the process, makes the development of biochemical and biophysical assays particularly challenging. We summarize in vitro assays that use planar supported membranes and fluorescence microscopy to address some of the open questions regarding the molecular mechanisms of SNARE-mediated membrane fusion. Most of the assays discussed in this mini-review were developed in our lab over the last 15 years. We emphasize the sample requirements that we found are important for the successful application of these methods. PMID:28360838

Chronic administration of docosahexaenoic acid or eicosapentaenoic acid, but not arachidonic acid, alone or in combination with uridine, increases brain phosphatide and synaptic protein levels in gerbils.

PubMed

Cansev, M; Wurtman, R J

2007-08-24

Synthesis of phosphatidylcholine, the most abundant brain membrane phosphatide, requires three circulating precursors: choline; a pyrimidine (e.g. uridine); and a polyunsaturated fatty acid. Supplementing a choline-containing diet with the uridine source uridine-5'-monophosphate (UMP) or, especially, with UMP plus the omega-3 fatty acid docosahexaenoic acid (given by gavage), produces substantial increases in membrane phosphatide and synaptic protein levels within gerbil brain. We now compare the effects of various polyunsaturated fatty acids, given alone or with UMP, on these synaptic membrane constituents. Gerbils received, daily for 4 weeks, a diet containing choline chloride with or without UMP and/or, by gavage, an omega-3 (docosahexaenoic or eicosapentaenoic acid) or omega-6 (arachidonic acid) fatty acid. Both of the omega-3 fatty acids elevated major brain phosphatide levels (by 18-28%, and 21-27%) and giving UMP along with them enhanced their effects significantly. Arachidonic acid, given alone or with UMP, was without effect. After UMP plus docosahexaenoic acid treatment, total brain phospholipid levels and those of each individual phosphatide increased significantly in all brain regions examined (cortex, striatum, hippocampus, brain stem, and cerebellum). The increases in brain phosphatides in gerbils receiving an omega-3 (but not omega-6) fatty acid, with or without UMP, were accompanied by parallel elevations in levels of pre- and post-synaptic proteins (syntaxin-3, PSD-95 and synapsin-1) but not in those of a ubiquitous structural protein, beta-tubulin. Hence administering omega-3 polyunsaturated fatty acids can enhance synaptic membrane levels in gerbils, and may do so in patients with neurodegenerative diseases, especially when given with a uridine source, while the omega-6 polyunsaturated fatty acid arachidonic acid is ineffective.

Chronic administration of docosahexaenoic acid or eicosapentaenoic acid, but not arachidonic acid, alone or in combination with uridine, increases brain phosphatide and synaptic protein levels in gerbils

PubMed Central

Cansev, M.; Wurtman, R. J.

2007-01-01

Synthesis of phosphatidylcholine, the most abundant brain membrane phosphatide, requires three circulating precursors: choline; a pyrimidine (e.g., uridine); and a polyunsaturated fatty acid. Supplementing a choline-containing diet with the uridine source uridine-5′-monophosphate (UMP) or, especially, with UMP plus the omega-3 fatty acid docosahexaenoic acid (given by gavage), produces substantial increases in membrane phosphatide and synaptic protein levels within gerbil brain. We now compare the effects of various polyunsaturated fatty acids, given alone or with UMP, on these synaptic membrane constituents. Gerbils received, daily for 4 weeks, a diet containing choline chloride with or without UMP and/or, by gavage, an omega-3 (docosahexaenoic or eicosapentaenoic acid) or omega-6 (arachidonic acid) fatty acid. Both of the omega-3 fatty acids elevated major brain phosphatide levels (by 18-28%, and 21-27%) and giving UMP along with them enhanced their effects significantly. Arachidonic acid, given alone or with UMP, was without effect. After UMP plus docosahexaenoic acid treatment, total brain phospholipids levels and those of each individual phosphatide increased significantly in all brain regions examined (cortex, striatum, hippocampus, brain stem, and cerebellum). The increases in brain phosphatides in gerbils receiving an omega-3 (but not omega-6) fatty acid, with or without UMP, were accompanied by parallel elevations in levels of pre- and post-synaptic proteins (syntaxin-3, PSD-95 and Synapsin-1) but not in those of a ubiquitous structural protein, β-tubulin. Hence administering omega-3 polyunsaturated fatty acids can enhance synaptic membrane levels in gerbils, and may do so in patients with neurodegenerative diseases, especially when given with a uridine source, while the omega-6 polyunsaturated fatty acid arachidonic acid is ineffective. PMID:17683870

Learning of Precise Spike Times with Homeostatic Membrane Potential Dependent Synaptic Plasticity.

PubMed

Albers, Christian; Westkott, Maren; Pawelzik, Klaus

2016-01-01

Precise spatio-temporal patterns of neuronal action potentials underly e.g. sensory representations and control of muscle activities. However, it is not known how the synaptic efficacies in the neuronal networks of the brain adapt such that they can reliably generate spikes at specific points in time. Existing activity-dependent plasticity rules like Spike-Timing-Dependent Plasticity are agnostic to the goal of learning spike times. On the other hand, the existing formal and supervised learning algorithms perform a temporally precise comparison of projected activity with the target, but there is no known biologically plausible implementation of this comparison. Here, we propose a simple and local unsupervised synaptic plasticity mechanism that is derived from the requirement of a balanced membrane potential. Since the relevant signal for synaptic change is the postsynaptic voltage rather than spike times, we call the plasticity rule Membrane Potential Dependent Plasticity (MPDP). Combining our plasticity mechanism with spike after-hyperpolarization causes a sensitivity of synaptic change to pre- and postsynaptic spike times which can reproduce Hebbian spike timing dependent plasticity for inhibitory synapses as was found in experiments. In addition, the sensitivity of MPDP to the time course of the voltage when generating a spike allows MPDP to distinguish between weak (spurious) and strong (teacher) spikes, which therefore provides a neuronal basis for the comparison of actual and target activity. For spatio-temporal input spike patterns our conceptually simple plasticity rule achieves a surprisingly high storage capacity for spike associations. The sensitivity of the MPDP to the subthreshold membrane potential during training allows robust memory retrieval after learning even in the presence of activity corrupted by noise. We propose that MPDP represents a biophysically plausible mechanism to learn temporal target activity patterns.

Learning of Precise Spike Times with Homeostatic Membrane Potential Dependent Synaptic Plasticity

PubMed Central

Albers, Christian; Westkott, Maren; Pawelzik, Klaus

2016-01-01

Precise spatio-temporal patterns of neuronal action potentials underly e.g. sensory representations and control of muscle activities. However, it is not known how the synaptic efficacies in the neuronal networks of the brain adapt such that they can reliably generate spikes at specific points in time. Existing activity-dependent plasticity rules like Spike-Timing-Dependent Plasticity are agnostic to the goal of learning spike times. On the other hand, the existing formal and supervised learning algorithms perform a temporally precise comparison of projected activity with the target, but there is no known biologically plausible implementation of this comparison. Here, we propose a simple and local unsupervised synaptic plasticity mechanism that is derived from the requirement of a balanced membrane potential. Since the relevant signal for synaptic change is the postsynaptic voltage rather than spike times, we call the plasticity rule Membrane Potential Dependent Plasticity (MPDP). Combining our plasticity mechanism with spike after-hyperpolarization causes a sensitivity of synaptic change to pre- and postsynaptic spike times which can reproduce Hebbian spike timing dependent plasticity for inhibitory synapses as was found in experiments. In addition, the sensitivity of MPDP to the time course of the voltage when generating a spike allows MPDP to distinguish between weak (spurious) and strong (teacher) spikes, which therefore provides a neuronal basis for the comparison of actual and target activity. For spatio-temporal input spike patterns our conceptually simple plasticity rule achieves a surprisingly high storage capacity for spike associations. The sensitivity of the MPDP to the subthreshold membrane potential during training allows robust memory retrieval after learning even in the presence of activity corrupted by noise. We propose that MPDP represents a biophysically plausible mechanism to learn temporal target activity patterns. PMID:26900845

Synaptic hyperpolarization and inhibition of turtle cochlear hair cells.

PubMed

Art, J J; Fettiplace, R; Fuchs, P A

1984-11-01

Intracellular recordings were made from turtle cochlear hair cells in order to examine the properties of the post-synaptic potentials evoked by electrical stimulation of the efferent axons. Single shocks to the efferents generated a hair cell membrane hyperpolarization with an average amplitude generally less than 1 mV and lasting for about 100 ms. With short trains of shocks, the size of the post-synaptic potential grew markedly to a maximum of 20-30 mV. The interaction between pairs of shocks separated by a varying interval was studied. For an interval of 4 ms, the response to the second shock was increased on average by a factor of 3 and the conditioning effect of the first shock decayed with a time constant of about 100 ms. We suggest the augmentation in response to trains of shocks may be partly due to facilitation of efferent transmitter release. The efferent post-synaptic potentials could be reversibly abolished by perfusion with perilymphs containing 3 microM-curare or atropine, and infusion of acetylcholine gave a transient membrane hyperpolarization. These observations are consistent with efferent action being mediated via a cholinergic synapse onto the hair cells. The post-synaptic potentials could be reversed in polarity by injection of hyperpolarizing currents through the recording electrode. The reversal potential was estimated as about -80 mV, 30 mV negative to the resting potential. Near reversal, a small brief depolarization was evident and may constitute a minor component of the synaptic response. The value of the reversal potential was unaffected by substitution of the perilymphatic chloride, but was altered in a predictable manner by changes in extracellular potassium concentration indicating that the post-synaptic potentials arise mainly by an increase in the permeability of the hair cell membrane to potassium ions. Throughout the post-synaptic hyperpolarization there was a reduction in the sensitivity of the hair cell to tones at its characteristic frequency. The desensitization, maximal for low sound pressures, varied in different cells from a factor of 1.6 to 28. At the peak of the largest synaptic potentials, the receptor potential remained negative to the resting potential with all but the loudest characteristic frequency tone s. We suggest that there are two factors in efferent inhibition; one a r duction in the receptor potential at the hair cell's characteristic frequency and the other a hyperpolarization of its membrane potential which should reduce the release of excitatory transmitter onto the afferent terminals.

Flow Cytometric Analysis of Presynaptic Nerve Terminals Isolated from Rats Subjected to Hypergravity

NASA Astrophysics Data System (ADS)

Borisova, Tatiana

2008-06-01

Flow cytometric studies revealed an insignificant decrease in cell size heterogeneity and cytoplasmic granularity of rat brain nerve terminals (synaptosomes) isolated from animals subjected to centrifuge-induced hypergravity as compared to control ones. The analysis of plasma membrane potential using the potentiometric optical dye rhodamine 6G showed a decrease in fluorescence intensity by 10 % at steady state level in hypergravity synaptosomes. To monitor synaptic vesicle acidification we used pH-sensitive fluorescent dye acridine orange and demonstrated a lower fluorescence intensity level at steady state (10%) after hypergravity as compared to controls. Thus, exposure to hypergravity resulted in depolarization of the synaptosomal plasma membrane and diminution in synaptic vesicle acidification that may be a cause leading to altered synaptic neurotransmission.

Contributions of Bcl-xL to acute and long term changes in bioenergetics during neuronal plasticity.

PubMed

Jonas, Elizabeth A

2014-08-01

Mitochondria manufacture and release metabolites and manage calcium during neuronal activity and synaptic transmission, but whether long term alterations in mitochondrial function contribute to the neuronal plasticity underlying changes in organism behavior patterns is still poorly understood. Although normal neuronal plasticity may determine learning, in contrast a persistent decline in synaptic strength or neuronal excitability may portend neurite retraction and eventual somatic death. Anti-death proteins such as Bcl-xL not only provide neuroprotection at the neuronal soma during cell death stimuli, but also appear to enhance neurotransmitter release and synaptic growth and development. It is proposed that Bcl-xL performs these functions through its ability to regulate mitochondrial release of bioenergetic metabolites and calcium, and through its ability to rapidly alter mitochondrial positioning and morphology. Bcl-xL also interacts with proteins that directly alter synaptic vesicle recycling. Bcl-xL translocates acutely to sub-cellular membranes during neuronal activity to achieve changes in synaptic efficacy. After stressful stimuli, pro-apoptotic cleaved delta N Bcl-xL (ΔN Bcl-xL) induces mitochondrial ion channel activity leading to synaptic depression and this is regulated by caspase activation. During physiological states of decreased synaptic stimulation, loss of mitochondrial Bcl-xL and low level caspase activation occur prior to the onset of long term decline in synaptic efficacy. The degree to which Bcl-xL changes mitochondrial membrane permeability may control the direction of change in synaptic strength. The small molecule Bcl-xL inhibitor ABT-737 has been useful in defining the role of Bcl-xL in synaptic processes. Bcl-xL is crucial to the normal health of neurons and synapses and its malfunction may contribute to neurodegenerative disease. Copyright © 2013. Published by Elsevier B.V.

Energetics and Kinetics of trans-SNARE Zippering

NASA Astrophysics Data System (ADS)

Rebane, Aleksander A.; Shu, Tong; Krishnakumar, Shyam; Rothman, James E.; Zhang, Yongli

Synaptic exocytosis relies on assembly of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins into a four-helix bundle to drive membrane fusion. Complementary SNAREs anchored to the synaptic vesicle (v-SNARE) and the plasma membrane (t-SNARE) associate from their N-termini, transiting a half-assembled intermediate (trans-SNARE), and ending at their C-termini with a rapid power stroke that leads to membrane fusion. Although cytosolic SNARE assembly has been characterized, it remains unknown how membranes modulate the energetics and kinetics of SNARE assembly. Here, we present optical tweezers measurements on folding of single membrane proteins in phospholipid bilayers. To our knowledge, this is the first such report. We measured the energetics, kinetics, and assembly intermediates of trans-SNAREs formed between a t-SNARE inserted into a bead-supported bilayer and a v-SNARE in a nanodisc. We found that the repulsive force of the apposed membranes increases the lifetime of the half-assembled intermediate. Our findings provide a single-molecule platform to study the regulation of trans-SNARE assembly by proteins that act on the half-assembled state, and thus reveal the mechanistic basis of the speed and high fidelity of synaptic transmission. This work was supported by US National Institutes of Health Grants F31 GM119312-01 (to A.A.R) and R01 GM093341 (to Y.Z.).

Cholesterol regulates multiple forms of vesicle endocytosis at a mammalian central synapse.

PubMed

Yue, Hai-Yuan; Xu, Jianhua

2015-07-01

Endocytosis in synapses sustains neurotransmission by recycling vesicle membrane and maintaining the homeostasis of synaptic membrane. A role of membrane cholesterol in synaptic endocytosis remains controversial because of conflicting observations, technical limitations in previous studies, and potential interference from non-specific effects after cholesterol manipulation. Furthermore, it remains unclear whether cholesterol participates in distinct forms of endocytosis that function under different activity levels. In this study, applying the whole-cell membrane capacitance measurement to monitor endocytosis in real time at the rat calyx of Held terminals, we found that disrupting cholesterol with dialysis of cholesterol oxidase or methyl-β-cyclodextrin impaired three different forms of endocytosis, including slow endocytosis, rapid endocytosis, and endocytosis of the retrievable membrane that exists at the surface before stimulation. The effects were observed when disruption of cholesterol was mild enough not to change Ca(2+) channel current or vesicle exocytosis, indicative of stringent cholesterol requirement in synaptic endocytosis. Extracting cholesterol with high concentrations of methyl-β-cyclodextrin reduced exocytosis, mainly by decreasing the readily releasable pool and the vesicle replenishment after readily releasable pool depletion. Our study suggests that cholesterol is an important, universal regulator in multiple forms of vesicle endocytosis at mammalian central synapses. © 2015 International Society for Neurochemistry.

Mixing, diffusion, and percolation in binary supported membranes containing mixtures of lipids and amphiphilic block copolymers.

PubMed

Gettel, Douglas L; Sanborn, Jeremy; Patel, Mira A; de Hoog, Hans-Peter; Liedberg, Bo; Nallani, Madhavan; Parikh, Atul N

2014-07-23

Substrate-mediated fusion of small polymersomes, derived from mixtures of lipids and amphiphilic block copolymers, produces hybrid, supported planar bilayers at hydrophilic surfaces, monolayers at hydrophobic surfaces, and binary monolayer/bilayer patterns at amphiphilic surfaces, directly responding to local measures of (and variations in) surface free energy. Despite the large thickness mismatch in their hydrophobic cores, the hybrid membranes do not exhibit microscopic phase separation, reflecting irreversible adsorption and limited lateral reorganization of the polymer component. With increasing fluid-phase lipid fraction, these hybrid, supported membranes undergo a fluidity transition, producing a fully percolating fluid lipid phase beyond a critical area fraction, which matches the percolation threshold for the immobile point obstacles. This then suggests that polymer-lipid hybrid membranes might be useful models for studying obstructed diffusion, such as occurs in lipid membranes containing proteins.

Effect of hyperthermia on the repair of sublethal radiation damage in normal and membrane fatty acid substituted fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wolters, H.; Kelholt, D.; Konings, A.W.

1987-02-01

The interaction of heat and X irradiation was studied in normal and polyunsaturated fatty acid (PUFA) substituted mouse fibroblast LM cells. As a result of the substitution the membranes of the PUFA cells were more fluid than the membranes of the normal cells. Three different heat doses were applied (60 min 42 degrees C, 20 min 43 degrees C, and 10 min 44 degrees C) in combination with single or split doses of X rays. Heat radiosensitization was the largest for the 60 min 42 degrees C treatment. Heat radiosensitization and the heat-induced inhibition of the rate of sublethal damagemore » repair were the same for the normal and the PUFA cells. It is concluded from the experiments reported that the processes of hyperthermic inhibition of SLD repair and hyperthermic radiosensitization are independent of membrane fluidity and membrane fatty acid composition.« less

The impact of humic and fulvic acids on the dynamic properties of liposome membranes: the ESR method.

PubMed

Man, Dariusz; Pisarek, Izabella; Braczkowski, Michał; Pytel, Barbara; Olchawa, Ryszard

2014-06-01

This paper presents the results of research on the influence of two fractions of humic substances (HS): fulvic acids (FA) and humic acids (HA), as a function of concentration, on the liposome membranes formed from egg yolk lecithin (EYL). The concentration of HS in relation to EYL changed from 0% to 10% by weight. The influence of HS on various areas of membranes: interphase water-lipid, in the lipid layer just below the polar part of the membrane and in the middle of the lipid bilayer, was investigated by different spin labels (TEMPO, DOXYL 5, DOXYL 16). The study showed that HA slightly decreased the fluidity of the analyzed membranes on the surface layer, while FA significantly liquidated the center of the lipid bilayer. The strong effect of both fractions of HS on the concentration of free radicals as a function of time was also described.

Interaction of Impulsive Pressures of Cavitation Bubbles with Cell Membranes during Sonoporation

NASA Astrophysics Data System (ADS)

Kodama, Tetsuya; Koshiyama, Ken-ichiro; Tomita, Yukio; Suzuki, Maiko; Yano, Takeru; Fujikawa, Shigeo

2006-05-01

Ultrasound contrast agents (UCAs), are capable of enhancing non-invasive cytoplasmic molecular delivery in the presence of ultrasound. Collapse of UCAs may generate nano-scale cavitation bubbles, resulting in the transient permeabilization of the cell membrane. In the present study, we investigated the interaction of a cavitation bubble-induced shock wave with a cell membrane using acoustic theory and molecular dynamics (MD) simulation. From the theory, we obtained the shock wave propagation distance from the center of a cavitation bubble that would induce membrane damage. The MD simulation determined the relationship between the uptake of water molecules into the lipid bilayer and the shock wave. The interaction of the shock wave induced a structural change of the bilayer and subsequently increased the fluidity of each molecule. These changes in the bilayer due to shock waves may be an important factor in the use of UCAs to produce the transient membrane permeability during sonoporation.

Differences in the Electrophysiological Properties of Mouse Somatosensory Layer 2/3 Neurons In Vivo and Slice Stem from Intrinsic Sources Rather than a Network-Generated High Conductance State

PubMed Central

2018-01-01

Abstract Synaptic activity in vivo can potentially alter the integration properties of neurons. Using recordings in awake mice, we targeted somatosensory layer 2/3 pyramidal neurons and compared neuronal properties with those from slices. Pyramidal cells in vivo had lower resistance and gain values, as well as broader spikes and increased spike frequency adaptation compared to the same cells in slices. Increasing conductance in neurons using dynamic clamp to levels observed in vivo, however, did not lessen the differences between in vivo and slice conditions. Further, local application of tetrodotoxin (TTX) in vivo blocked synaptic-mediated membrane voltage fluctuations but had little impact on pyramidal cell membrane input resistance and time constant values. Differences in electrophysiological properties of layer 2/3 neurons in mouse somatosensory cortex, therefore, stem from intrinsic sources separate from synaptic-mediated membrane voltage fluctuations. PMID:29662946

Synaptic activity-related classical protein kinase C isoform localization in the adult rat neuromuscular synapse.

PubMed

Besalduch, Núria; Tomàs, Marta; Santafé, Manel M; Garcia, Neus; Tomàs, Josep; Lanuza, Maria Angel

2010-01-10

Protein kinase C (PKC) is essential for signal transduction in a variety of cells, including neurons and myocytes, and is involved in both acetylcholine release and muscle fiber contraction. Here, we demonstrate that the increases in synaptic activity by nerve stimulation couple PKC to transmitter release in the rat neuromuscular junction and increase the level of alpha, betaI, and betaII isoforms in the membrane when muscle contraction follows the stimulation. The phosphorylation activity of these classical PKCs also increases. It seems that the muscle has to contract in order to maintain or increase classical PKCs in the membrane. We use immunohistochemistry to show that PKCalpha and PKCbetaI were located in the nerve terminals, whereas PKCalpha and PKCbetaII were located in the postsynaptic and the Schwann cells. Stimulation and contraction do not change these cellular distributions, but our results show that the localization of classical PKC isoforms in the membrane is affected by synaptic activity.

Detection of erythrocyte membrane structural abnormalities in lecithin: cholesterol acyltransferase deficiency using a spin label approach.

PubMed

Maraviglia, B; Herring, F G; Weeks, G; Godin, D V

1979-01-01

The membrane fluidity of erythrocytes from patients with Lecithin: cholesterol acyltransferase (LCAT) deficiency was studied by means of electron spin resonance. The temperature dependence of the separation of the outer extrema of the spectra of 2-(3-carboxy-propyl)-4,4-dimethyl, 2-tridecyl-3-oxazolidinyloxyl spin probe was monitored for normal, presumed carrier and clinically affected subjects. The temperature profile of controls was significantly different from that of the presumed carriers and the clinically affected individuals. The results show that the compositional abnormalities previously noted in erythrocyte membranes from patients with LCAT deficiency are associated with alterations in the physiocochemical state of the membrane. An investigation of the spectral lineshapes below 10 degrees C allowed a distinction to be made at the membrane level between clinically affected subjects and clinically normal heterozygous carriers. Alterations in the temperature dependence of elec-ron spin resonance parameters may provide a sensitive index of red cell membrane alterations in pathological states of generalized membrane involvement.

Synaptophysin regulates the kinetics of synaptic vesicle endocytosis in central neurons

PubMed Central

Kwon, Sung E.; Chapman, Edwin R.

2011-01-01

Summary Despite being the most abundant synaptic vesicle membrane protein, the function of synaptophysin remains enigmatic. For example, synaptic transmission was reported to be completely normal in synaptophysin knockout mice; however, direct experiments to monitor the synaptic vesicle cycle have not been carried out. Here, using optical imaging and electrophysiological experiments, we demonstrate that synaptophysin is required for kinetically efficient endocytosis of synaptic vesicles in cultured hippocampal neurons. Truncation analysis revealed that distinct structural elements of synaptophysin differentially regulate vesicle retrieval during and after stimulation. Thus, synaptophysin regulates at least two phases of endocytosis to ensure vesicle availability during and after sustained neuronal activity. PMID:21658579

G protein betagamma-subunits activated by serotonin mediate presynaptic inhibition by regulating vesicle fusion properties.

PubMed

Photowala, Huzefa; Blackmer, Trillium; Schwartz, Eric; Hamm, Heidi E; Alford, Simon

2006-03-14

Neurotransmitters are thought to be released as quanta, where synaptic vesicles deliver packets of neurotransmitter to the synaptic cleft by fusion with the plasma membrane. However, synaptic vesicles may undergo incomplete fusion. We provide evidence that G protein-coupled receptors inhibit release by causing such incomplete fusion. 5-hydroxytryptamine (5-HT) receptor signaling potently inhibits excitatory postsynaptic currents (EPSCs) between lamprey reticulospinal axons and their postsynaptic targets by a direct action on the vesicle fusion machinery. We show that 5-HT receptor-mediated presynaptic inhibition, at this synapse, involves a reduction in EPSC quantal size. Quantal size was measured directly by comparing unitary quantal amplitudes of paired EPSCs before and during 5-HT application and indirectly by determining the effect of 5-HT on the relationship between mean-evoked EPSC amplitude and variance. Results from FM dye-labeling experiments indicate that 5-HT prevents full fusion of vesicles. 5-HT reduces FM1-43 staining of vesicles with a similar efficacy to its effect on the EPSC. However, destaining of FM1-43-labeled vesicles is abolished by lower concentrations of 5-HT that leave a substantial EPSC. The use of a water-soluble membrane impermeant quenching agent in the extracellular space reduced FM1-43 fluorescence during stimulation in 5-HT. Thus vesicles contact the extracellular space during inhibition of synaptic transmission by 5-HT. We conclude that 5-HT, via free Gbetagamma, prevents the collapse of synaptic vesicles into the presynaptic membrane.

Stability, folding dynamics, and long-range conformational transition of the synaptic t-SNARE complex

PubMed Central

Zhang, Xinming; Rebane, Aleksander A.; Ma, Lu; Li, Feng; Jiao, Junyi; Qu, Hong; Pincet, Frederic; Rothman, James E.

2016-01-01

Synaptic soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) couple their stepwise folding to fusion of synaptic vesicles with plasma membranes. In this process, three SNAREs assemble into a stable four-helix bundle. Arguably, the first and rate-limiting step of SNARE assembly is the formation of an activated binary target (t)-SNARE complex on the target plasma membrane, which then zippers with the vesicle (v)-SNARE on the vesicle to drive membrane fusion. However, the t-SNARE complex readily misfolds, and its structure, stability, and dynamics are elusive. Using single-molecule force spectroscopy, we modeled the synaptic t-SNARE complex as a parallel three-helix bundle with a small frayed C terminus. The helical bundle sequentially folded in an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a central ionic layer, with total unfolding energy of ∼17 kBT, where kB is the Boltzmann constant and T is 300 K. Peptide binding to the CTD activated the t-SNARE complex to initiate NTD zippering with the v-SNARE, a mechanism likely shared by the mammalian uncoordinated-18-1 protein (Munc18-1). The NTD zippering then dramatically stabilized the CTD, facilitating further SNARE zippering. The subtle bidirectional t-SNARE conformational switch was mediated by the ionic layer. Thus, the t-SNARE complex acted as a switch to enable fast and controlled SNARE zippering required for synaptic vesicle fusion and neurotransmission. PMID:27911771

Theoretical foundations of the sound analog membrane potential that underlies coincidence detection in the barn owl

PubMed Central

Ashida, Go; Funabiki, Kazuo; Carr, Catherine E.

2013-01-01

A wide variety of neurons encode temporal information via phase-locked spikes. In the avian auditory brainstem, neurons in the cochlear nucleus magnocellularis (NM) send phase-locked synaptic inputs to coincidence detector neurons in the nucleus laminaris (NL) that mediate sound localization. Previous modeling studies suggested that converging phase-locked synaptic inputs may give rise to a periodic oscillation in the membrane potential of their target neuron. Recent physiological recordings in vivo revealed that owl NL neurons changed their spike rates almost linearly with the amplitude of this oscillatory potential. The oscillatory potential was termed the sound analog potential, because of its resemblance to the waveform of the stimulus tone. The amplitude of the sound analog potential recorded in NL varied systematically with the interaural time difference (ITD), which is one of the most important cues for sound localization. In order to investigate the mechanisms underlying ITD computation in the NM-NL circuit, we provide detailed theoretical descriptions of how phase-locked inputs form oscillating membrane potentials. We derive analytical expressions that relate presynaptic, synaptic, and postsynaptic factors to the signal and noise components of the oscillation in both the synaptic conductance and the membrane potential. Numerical simulations demonstrate the validity of the theoretical formulations for the entire frequency ranges tested (1–8 kHz) and potential effects of higher harmonics on NL neurons with low best frequencies (<2 kHz). PMID:24265616

Effects and Location of Coplanar and Noncoplanar PCB in a Lipid Bilayer: A Solid-State NMR Study.

PubMed

Totland, Christian; Nerdal, Willy; Steinkopf, Signe

2016-08-02

Coplanar and noncoplanar polychlorinated biphenyls (PCBs) are known to have different routes and degree of toxicity. Here, the effects of noncoplanar PCB 52 and coplanar PCB 77 present at 2 mol % in a model system consisting of POPC liposomes (50% hydrated) are investigated by solid-state (13)C and (31)P NMR at 298 K. Both PCBs intercalate horizontally in the outer part of the bilayer, near the segments of the acyl chain close to the glycerol group. Despite similar membrane locations, the coplanar PCB 77 shows little effect on the bilayer properties overall, except for the four nearest neighboring lipids, while the effect of PCB 52 is more dramatic. The first ∼2 layers of lipids around each PCB 52 in the bilayer form a high fluidity lamellar phase, whereas lipids beyond these layers form a lamellar phase with a slight increase in fluidity compared to a bilayer without PCB 52. Further, a third high mobility domain is observed. The explanation for this is the interference of several high fluidity lamellar phases caused by interactions of PCB 52 molecules in different leaflets of the model bilayer. This causes formation of high curvature toroidal region in the bilayer and might induce formation of channels.

Musa paradisica RCI complements AtRCI and confers Na+ tolerance and K+ sensitivity in Arabidopsis.

PubMed

Liu, Bing; Feng, Dongru; Zhang, Bipei; Mu, Peiqiang; Zhang, Yang; He, Yanming; Qi, Kangbiao; Wang, Jinfa; Wang, Hongbin

2012-03-01

The mechanisms involved in Na⁺/K⁺ uptake and extrusion are important in plant salt tolerance. In this study, we investigated the physiological role of a plasma membrane (PM)-localized protein, MpRCI, from plantain in transgenic Arabidopsis under NaCl and KCl stress and determined its effect on PM fluidity and H⁺-ATPase activity. The MpRCI gene exhibited high homology to the AtRCI2 gene family in Arabidopsis and was therefore able to complement for loss of the yeast AtRCI2-related PMP3 gene. Results of phenotypic espial and atomic emission spectrophotometer (AES) assays indicated that MpRCI overexpression in the AtRCI2A knockout mutant with reduced shoot Na⁺ and increased K⁺ exhibited increased Na⁺-tolerance and K⁺-sensitivity under NaCl or KCl treatments, respectively. Furthermore, comparisons of PM fluidity and H⁺-ATPase activity in shoots, with expression or absence of MpRCI/AtRCI2A expression under NaCl or KCl stress, showed MpRCI maintained PM fluidity and H⁺-ATPase activity under stress conditions. Results suggest that MpRCI plays an essential role in Na⁺/K⁺ flux in plant cells. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Quantification of Randomly-methylated-{beta}-cyclodextrin effect on liposome: An ESR study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grammenos, A., E-mail: A.Grammenos@ulg.ac.be; Bahri, M.A.; Guelluy, P.H.

2009-12-04

In the present work, the effect of Randomly-methylated-{beta}-cyclodextrin (Rameb) on the microviscosity of dimyristoyl-L-{alpha} phosphatidylcholine (DMPC) bilayer was investigated using the electron spin resonance (ESR) technique. The ability of Rameb to extract membrane cholesterol was demonstrated. For the first time, the percentage of cholesterol extracted by Rameb from cholesterol doped DMPC bilayer was monitored and quantified throughout a wide Rameb concentration range. The effect of cholesterol on the inner part of the membrane was also investigated using 16-doxyl stearic acid spin label (16-DSA). 16-DSA seems to explore two different membrane domains and report their respective microviscosities. ESR experiments also establishmore » that the presence of 30% of cholesterol in DMPC liposomes suppresses the jump in membrane fluidity at lipids phase-transition temperature (23.9 {sup o}C).« less

Protein Kinase Cϵ (PKCϵ) Promotes Synaptogenesis through Membrane Accumulation of the Postsynaptic Density Protein PSD-95*

PubMed Central

Sen, Abhik; Hongpaisan, Jarin; Wang, Desheng; Nelson, Thomas J.; Alkon, Daniel L.

2016-01-01

Protein kinase Cϵ (PKCϵ) promotes synaptic maturation and synaptogenesis via activation of synaptic growth factors such as BDNF, NGF, and IGF. However, many of the detailed mechanisms by which PKCϵ induces synaptogenesis are not fully understood. Accumulation of PSD-95 to the postsynaptic density (PSD) is known to lead to synaptic maturation and strengthening of excitatory synapses. Here we investigated the relationship between PKCϵ and PSD-95. We show that the PKCϵ activators dicyclopropanated linoleic acid methyl ester and bryostatin 1 induce phosphorylation of PSD-95 at the serine 295 residue, increase the levels of PSD-95, and enhance its membrane localization. Elimination of the serine 295 residue in PSD-95 abolished PKCϵ-induced membrane accumulation. Knockdown of either PKCϵ or JNK1 prevented PKCϵ activator-mediated membrane accumulation of PSD-95. PKCϵ directly phosphorylated PSD-95 and JNK1 in vitro. Inhibiting PKCϵ, JNK, or calcium/calmodulin-dependent kinase II activity prevented the effects of PKCϵ activators on PSD-95 phosphorylation. Increase in membrane accumulation of PKCϵ and phosphorylated PSD-95 (p-PSD-95S295) coincided with an increased number of synapses and increased amplitudes of excitatory post-synaptic potentials (EPSPs) in adult rat hippocampal slices. Knockdown of PKCϵ also reduced the synthesis of PSD-95 and the presynaptic protein synaptophysin by 30 and 44%, respectively. Prolonged activation of PKCϵ increased synapse number by 2-fold, increased presynaptic vesicle density, and greatly increased PSD-95 clustering. These results indicate that PKCϵ promotes synaptogenesis by activating PSD-95 phosphorylation directly through JNK1 and calcium/calmodulin-dependent kinase II and also by inducing expression of PSD-95 and synaptophysin. PMID:27330081

Protein Kinase Cϵ (PKCϵ) Promotes Synaptogenesis through Membrane Accumulation of the Postsynaptic Density Protein PSD-95.

PubMed

Sen, Abhik; Hongpaisan, Jarin; Wang, Desheng; Nelson, Thomas J; Alkon, Daniel L

2016-08-05

Protein kinase Cϵ (PKCϵ) promotes synaptic maturation and synaptogenesis via activation of synaptic growth factors such as BDNF, NGF, and IGF. However, many of the detailed mechanisms by which PKCϵ induces synaptogenesis are not fully understood. Accumulation of PSD-95 to the postsynaptic density (PSD) is known to lead to synaptic maturation and strengthening of excitatory synapses. Here we investigated the relationship between PKCϵ and PSD-95. We show that the PKCϵ activators dicyclopropanated linoleic acid methyl ester and bryostatin 1 induce phosphorylation of PSD-95 at the serine 295 residue, increase the levels of PSD-95, and enhance its membrane localization. Elimination of the serine 295 residue in PSD-95 abolished PKCϵ-induced membrane accumulation. Knockdown of either PKCϵ or JNK1 prevented PKCϵ activator-mediated membrane accumulation of PSD-95. PKCϵ directly phosphorylated PSD-95 and JNK1 in vitro Inhibiting PKCϵ, JNK, or calcium/calmodulin-dependent kinase II activity prevented the effects of PKCϵ activators on PSD-95 phosphorylation. Increase in membrane accumulation of PKCϵ and phosphorylated PSD-95 (p-PSD-95(S295)) coincided with an increased number of synapses and increased amplitudes of excitatory post-synaptic potentials (EPSPs) in adult rat hippocampal slices. Knockdown of PKCϵ also reduced the synthesis of PSD-95 and the presynaptic protein synaptophysin by 30 and 44%, respectively. Prolonged activation of PKCϵ increased synapse number by 2-fold, increased presynaptic vesicle density, and greatly increased PSD-95 clustering. These results indicate that PKCϵ promotes synaptogenesis by activating PSD-95 phosphorylation directly through JNK1 and calcium/calmodulin-dependent kinase II and also by inducing expression of PSD-95 and synaptophysin. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

A Stimulation Function of Synaptotagmin-1 in Ternary SNARE Complex Formation Dependent on Munc18 and Munc13

PubMed Central

Li, Yun; Wang, Shen; Li, Tianzhi; Zhu, Le; Xu, Yuanyuan; Ma, Cong

2017-01-01

The Ca2+ sensor synaptotagmin-1 (Syt1) plays an essential function in synaptic exocytosis. Recently, Syt1 has been implicated in synaptic vesicle priming, a maturation step prior to Ca2+-triggered membrane fusion that is believed to involve formation of the ternary SNARE complex and require priming proteins Munc18-1 and Munc13-1. However, the mechanisms of Syt1 in synaptic vesicle priming are still unclear. In this study, we found that Syt1 stimulates the transition from the Munc18-1/syntaxin-1 complex to the ternary SNARE complex catalyzed by Munc13-1. This stimulation can be further enhanced in a membrane-containing environment. Further, we showed that Syt1, together with Munc18-1 and Munc13-1, stimulates trans ternary SNARE complex formation on membranes in a manner resistant to disassembly factors NSF and α-SNAP. Disruption of a proposed Syt1/SNARE binding interface strongly abrogated the stimulation function of Syt1. Our results suggest that binding of Syt1 to an intermediate SNARE assembly with Munc18-1 and Munc13-1 is critical for the stimulation function of Syt1 in ternary SNARE complex formation, and this stimulation may underlie the priming function of Syt1 in synaptic exocytosis. PMID:28860966

Calsyntenin-3 molecular architecture and interaction with neurexin 1α.

PubMed

Lu, Zhuoyang; Wang, Yun; Chen, Fang; Tong, Huimin; Reddy, M V V V Sekhar; Luo, Lin; Seshadrinathan, Suchithra; Zhang, Lei; Holthauzen, Luis Marcelo F; Craig, Ann Marie; Ren, Gang; Rudenko, Gabby

2014-12-12

Calsyntenin 3 (Cstn3 or Clstn3), a recently identified synaptic organizer, promotes the development of synapses. Cstn3 localizes to the postsynaptic membrane and triggers presynaptic differentiation. Calsyntenin members play an evolutionarily conserved role in memory and learning. Cstn3 was recently shown in cell-based assays to interact with neurexin 1α (n1α), a synaptic organizer that is implicated in neuropsychiatric disease. Interaction would permit Cstn3 and n1α to form a trans-synaptic complex and promote synaptic differentiation. However, it is contentious whether Cstn3 binds n1α directly. To understand the structure and function of Cstn3, we determined its architecture by electron microscopy and delineated the interaction between Cstn3 and n1α biochemically and biophysically. We show that Cstn3 ectodomains form monomers as well as tetramers that are stabilized by disulfide bonds and Ca(2+), and both are probably flexible in solution. We show further that the extracellular domains of Cstn3 and n1α interact directly and that both Cstn3 monomers and tetramers bind n1α with nanomolar affinity. The interaction is promoted by Ca(2+) and requires minimally the LNS domain of Cstn3. Furthermore, Cstn3 uses a fundamentally different mechanism to bind n1α compared with other neurexin partners, such as the synaptic organizer neuroligin 2, because Cstn3 does not strictly require the sixth LNS domain of n1α. Our structural data suggest how Cstn3 as a synaptic organizer on the postsynaptic membrane, particularly in tetrameric form, may assemble radially symmetric trans-synaptic bridges with the presynaptic synaptic organizer n1α to recruit and spatially organize proteins into networks essential for synaptic function. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Entropic elasticity based coarse-grained model of lipid membranes

NASA Astrophysics Data System (ADS)

Feng, Shuo; Hu, Yucai; Liang, Haiyi

2018-04-01

Various models for lipid bilayer membranes have been presented to investigate their morphologies. Among them, the aggressive coarse-grained models, where the membrane is represented by a single layer of particles, are computationally efficient and of practical importance for simulating membrane dynamics at the microscopic scale. In these models, soft potentials between particle pairs are used to maintain the fluidity of membranes, but the underlying mechanism of the softening requires further clarification. We have analyzed the membrane area decrease due to thermal fluctuations, and the results demonstrate that the intraparticle part of entropic elasticity is responsible for the softening of the potential. Based on the stretching response of the membrane, a bottom-up model is developed with an entropic effect explicitly involved. The model reproduces several essential properties of the lipid membrane, including the fluid state and a plateau in the stretching curve. In addition, the area compressibility modulus, bending rigidity, and spontaneous curvature display linear dependence on model parameters. As a demonstration, we have investigated the closure and morphology evolution of membrane systems driven by spontaneous curvature, and vesicle shapes observed experimentally are faithfully reproduced.

Accumulation of K+ in the synaptic cleft modulates activity by influencing both vestibular hair cell and calyx afferent in the turtle

PubMed Central

Contini, Donatella; Price, Steven D.

2016-01-01

Key points In the synaptic cleft between type I hair cells and calyceal afferents, K+ ions accumulate as a function of activity, dynamically altering the driving force and permeation through ion channels facing the synaptic cleft.High‐fidelity synaptic transmission is possible due to large conductances that minimize hair cell and afferent time constants in the presence of significant membrane capacitance.Elevated potassium maintains hair cells near a potential where transduction currents are sufficient to depolarize them to voltages necessary for calcium influx and synaptic vesicle fusion.Elevated potassium depolarizes the postsynaptic afferent by altering ion permeation through hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels, and contributes to depolarizing the afferent to potentials where a single EPSP (quantum) can generate an action potential.With increased stimulation, hair cell depolarization increases the frequency of quanta released, elevates [K+]cleft and depolarizes the afferent to potentials at which smaller and smaller EPSPs would be sufficient to trigger APs. Abstract Fast neurotransmitters act in conjunction with slower modulatory effectors that accumulate in restricted synaptic spaces found at giant synapses such as the calyceal endings in the auditory and vestibular systems. Here, we used dual patch‐clamp recordings from turtle vestibular hair cells and their afferent neurons to show that potassium ions accumulating in the synaptic cleft modulated membrane potentials and extended the range of information transfer. High‐fidelity synaptic transmission was possible due to large conductances that minimized hair cell and afferent time constants in the presence of significant membrane capacitance. Increased potassium concentration in the cleft maintained the hair cell near potentials that promoted the influx of calcium necessary for synaptic vesicle fusion. The elevated potassium concentration also depolarized the postsynaptic neuron by altering ion permeation through hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels. This depolarization enabled the afferent to reliably generate action potentials evoked by single AMPA‐dependent EPSPs. Depolarization of the postsynaptic afferent could also elevate potassium in the synaptic cleft, and would depolarize other hair cells enveloped by the same neuritic process increasing the fidelity of neurotransmission at those synapses as well. Collectively, these data demonstrate that neuronal activity gives rise to potassium accumulation, and suggest that potassium ion action on HCN channels can modulate neurotransmission, preserving the fidelity of high‐speed synaptic transmission by dynamically shifting the resting potentials of both presynaptic and postsynaptic cells. PMID:27633787

Strychnine Binding Associated with Glycine Receptors of the Central Nervous System

PubMed Central

Young, Anne B.; Snyder, Solomon H.

1973-01-01

[3H]Strychnine binds to synaptic-membrane fractions of the spinal cord in a selective fashion, indicating an interaction with postsynaptic glycine receptors. Displacement of strychnine by glycine and other amino acids parallels their glycine-like neurophysiologic activity. The regional localization of strychnine binding in the central nervous system correlates closely with endogenous glycine concentrations. In subcellular fractionation experiments, strychnine binding is most enhanced in synaptic-membrane fractions. Strychnine binding is saturable, with affinity constants for glycine and strychnine of 10 and 0.03 μM, respectively. PMID:4200724

Lens lipids.

PubMed

Zelenka, P S

1984-11-01

Lens cells can synthesize, degrade, and remodel lipids. Endogenous lipid synthesis, in conjunction with uptake of exogenous cholesterol and certain fatty acids, leads to the formation of a plasma membrane that is especially rich in sphingomyelin, cholesterol, and long-chain saturated fatty acids. As a result of this unusual lipid composition, lens membranes have very low fluidity, which is restricted even further by lipid-protein interactions. The composition and metabolism of membrane lipids may affect the formation of various types of cataracts. Diets rich in vegetable oils offer some protection against the formation of osmotic cataracts and the hereditary cataract of the RCS rat, although the mechanism of this effect is not clear. Vitamin E also protects against the formation of several types of cataract in vivo and in vitro, suggesting that lipid peroxidation may play a role in cataractogenesis. Certain drugs which inhibit lipid synthesis or degradation are cataractogenic, and a deficiency in cataractogenic, and a deficiency in phosphatidylserine is associated with a loss of Na+/K+ ATPase activity in several types of cataract. Human senile cataracts show a marked loss of protein-lipid interactions, although the overall lipid composition is normal. This loss of protein-lipid interactions may be related to oxidative damage to membrane-associated proteins. Interestingly, the decrease in the fluidity of lens membranes with age would counteract the formation of aqueous pores in the membrane, which can result from the oxidative cross-linking of membrane-associated proteins. Certain pathways of lipid metabolism seem to have regulatory functions. Among these are phosphatidylinositol turnover, phosphatidylethanolamine methylation, and arachidonic acid metabolism. All of these pathways function in the lens. Phosphatidylinositol turnover is correlated with the rate of lens epithelial cell division, while phosphatidylethanolamine methylation seems to be related to the initiation of lens fiber cell formation. Both pathways are associated with the release and metabolism of arachidonic acid in other cell types. While it is not known whether phosphatidylinositol turnover or phosphatidylethanolamine methylation result in the release of arachidonic acid in the lens, recent work has shown that lens cells from a variety of species can metabolize arachidonic acid by both the cyclooxygenase and lipoxygenase pathways. The possible physiological significance of these metabolites to the lens is yet to be determined.

Acute Stress Suppresses Synaptic Inhibition and Increases Anxiety via Endocannabinoid Release in the Basolateral Amygdala

PubMed Central

Itoga, Christy A.; Fisher, Marc O.; Solomonow, Jonathan; Roltsch, Emily A.; Gilpin, Nicholas W.

2016-01-01

Stress and glucocorticoids stimulate the rapid mobilization of endocannabinoids in the basolateral amygdala (BLA). Cannabinoid receptors in the BLA contribute to anxiogenesis and fear-memory formation. We tested for rapid glucocorticoid-induced endocannabinoid regulation of synaptic inhibition in the rat BLA. Glucocorticoid application to amygdala slices elicited a rapid, nonreversible suppression of spontaneous, but not evoked, GABAergic synaptic currents in BLA principal neurons; the effect was also seen with a membrane-impermeant glucocorticoid, but not with intracellular glucocorticoid application, implicating a membrane-associated glucocorticoid receptor. The glucocorticoid suppression of GABA currents was not blocked by antagonists of nuclear corticosteroid receptors, or by inhibitors of gene transcription or protein synthesis, but was blocked by inhibiting postsynaptic G-protein activity, suggesting a postsynaptic nongenomic steroid signaling mechanism that stimulates the release of a retrograde messenger. The rapid glucocorticoid-induced suppression of inhibition was prevented by blocking CB1 receptors and 2-arachidonoylglycerol (2-AG) synthesis, and it was mimicked and occluded by CB1 receptor agonists, indicating it was mediated by the retrograde release of the endocannabinoid 2-AG. The rapid glucocorticoid effect in BLA neurons in vitro was occluded by prior in vivo acute stress-induced, or prior in vitro glucocorticoid-induced, release of endocannabinoid. Acute stress also caused an increase in anxiety-like behavior that was attenuated by blocking CB1 receptor activation and inhibiting 2-AG synthesis in the BLA. Together, these findings suggest that acute stress causes a long-lasting suppression of synaptic inhibition in BLA neurons via a membrane glucocorticoid receptor-induced release of 2-AG at GABA synapses, which contributes to stress-induced anxiogenesis. SIGNIFICANCE STATEMENT We provide a cellular mechanism in the basolateral amygdala (BLA) for the rapid stress regulation of anxiogenesis in rats. We demonstrate a nongenomic glucocorticoid induction of long-lasting suppression of synaptic inhibition that is mediated by retrograde endocannabinoid release at GABA synapses. The rapid glucocorticoid-induced endocannabinoid suppression of synaptic inhibition is initiated by a membrane-associated glucocorticoid receptor in BLA principal neurons. We show that acute stress increases anxiety-like behavior via an endocannabinoid-dependent mechanism centered in the BLA. The stress-induced endocannabinoid modulation of synaptic transmission in the BLA contributes, therefore, to the stress regulation of anxiety, and may play a role in anxiety disorders of the amygdala. PMID:27511017

Damage and protection of the photosynthetic apparatus from UV-B radiation. II. Effect of quercetin at different pH.

PubMed

Dobrikova, Anelia G; Apostolova, Emilia L

2015-07-20

The effect of the exogenously added quercetin against the UV-B inhibition of the photosystem II (PSII) functions in isolated pea thylakoid membranes suspended at different pH of the medium (6.5, 7.6 and 8.4) was investigated. The data revealed that the interaction of this flavonoid with the membranes depends on the pH and influences the initial S0-S1 state distribution of PSII in the dark, the energy transfer between pigment-protein complexes of the photosynthetic apparatus and the membrane fluidity. Quercetin also displays a different UV-protective effect depending on its location in the membranes, as the effect is more pronounced at pH 8.4 when it is located at the membrane surface. The results suggest that quercetin induces structural changes in thylakoid membranes, one of the possible reasons for its protection of the photosynthetic apparatus. Copyright © 2015 Elsevier GmbH. All rights reserved.

Cholesterol segregates into submicrometric domains at the living erythrocyte membrane: evidence and regulation.

PubMed

Carquin, Mélanie; Conrard, Louise; Pollet, Hélène; Van Der Smissen, Patrick; Cominelli, Antoine; Veiga-da-Cunha, Maria; Courtoy, Pierre J; Tyteca, Donatienne

2015-12-01

Although cholesterol is essential for membrane fluidity and deformability, the level of its lateral heterogeneity at the plasma membrane of living cells is poorly understood due to lack of appropriate probe. We here report on the usefulness of the D4 fragment of Clostridium perfringens toxin fused to mCherry (theta*), as specific, non-toxic, sensitive and quantitative cholesterol-labeling tool, using erythrocyte flat membrane. By confocal microscopy, theta* labels cholesterol-enriched submicrometric domains in coverslip-spread but also gel-suspended (non-stretched) fresh erythrocytes, suggesting in vivo relevance. Cholesterol domains on spread erythrocytes are stable in time and space, restricted by membrane:spectrin anchorage via 4.1R complexes, and depend on temperature and sphingomyelin, indicating combined regulation by extrinsic membrane:cytoskeleton interaction and by intrinsic lipid packing. Cholesterol domains partially co-localize with BODIPY-sphingomyelin-enriched domains. In conclusion, we show that theta* is a useful vital probe to study cholesterol organization and demonstrate that cholesterol forms submicrometric domains in living cells.

Cooperative binding of Annexin A5 to phosphatidylserine on apoptotic cell membranes

NASA Astrophysics Data System (ADS)

Janko, Christina; Jeremic, Ivica; Biermann, Mona; Chaurio, Ricardo; Schorn, Christine; Muñoz, Luis E.; Herrmann, Martin

2013-12-01

Healthy cells exhibit an asymmetric plasma membrane with phosphatidylserine (PS) located on the cytoplasmic leaflet of the plasma membrane bilayer. Annexin A5-FITC, a PS binding protein, is commonly used to evaluate apoptosis in flow cytometry. PS exposed by apoptotic cells serves as a major ‘eat-me’ signal for phagocytes. Although exposition of PS has been observed after alternative stimuli, no clearance of viable, PS exposing cells has been detected. Thus, besides PS exposure, membranes of viable and apoptotic cells might exhibit specific characteristics. Here, we show that Annexin A5 binds in a cooperative manner to different types of dead cells. Shrunken apoptotic cells thereby showed the highest Hill coefficient values. Contrarily, parafomaldehyde fixation of apoptotic cells completely abrogates the cooperativity effect seen with dead and dying cells. We tend to speculate that the cooperative binding of Annexin A5 to the membranes of apoptotic cells reflects higher fluidity of the exposed membranes facilitating PS clustering.

Aspirin Increases the Solubility of Cholesterol in Lipid Membranes

NASA Astrophysics Data System (ADS)

Alsop, Richard; Barrett, Matthew; Zheng, Sonbo; Dies, Hannah; Rheinstadter, Maikel

2014-03-01

Aspirin (ASA) is often prescribed for patients with high levels of cholesterol for the secondary prevention of myocardial events, a regimen known as the Low-Dose Aspirin Therapy. We have recently shown that Aspirin partitions in lipid bilayers. However, a direct interplay between ASA and cholesterol has not been investigated. Cholesterol is known to insert itself into the membrane in a dispersed state at moderate concentrations (under ~37.5%) and decrease fluidity of membranes. We prepared model lipid membranes containing varying amounts of both ASA and cholesterol molecules. The structure of the bilayers as a function of ASA and cholesterol concentration was determined using high-resolution X-ray diffraction. At cholesterol levels of more than 40mol%, immiscible cholesterol plaques formed. Adding ASA to the membranes was found to dissolve the cholesterol plaques, leading to a fluid lipid bilayer structure. We present first direct evidence for an interaction between ASA and cholesterol on the level of the cell membrane.

A Novel Form of Compensation in the Tg2576 Amyloid Mouse Model of Alzheimer’s Disease

PubMed Central

Somogyi, Attila; Katonai, Zoltán; Alpár, Alán; Wolf, Ervin

2016-01-01

One century after its first description, pathology of Alzheimer’s disease (AD) is still poorly understood. Amyloid-related dendritic atrophy and membrane alterations of susceptible brain neurons in AD, and in animal models of AD are widely recognized. However, little effort has been made to study the potential effects of combined morphological and membrane alterations on signal transfer and synaptic integration in neurons that build up affected neural networks in AD. In this study spatial reconstructions and electrophysiological measurements of layer II/III pyramidal neurons of the somatosensory cortex from wild-type (WT) and transgenic (TG) human amyloid precursor protein (hAPP) overexpressing Tg2576 mice were used to build faithful segmental cable models of these neurons. Local synaptic activities were simulated in various points of the dendritic arbors and properties of subthreshold dendritic impulse propagation and predictors of synaptic input pattern recognition ability were quantified and compared in modeled WT and TG neurons. Despite the widespread dendritic degeneration and membrane alterations in mutant mouse neurons, surprisingly little, or no change was detected in steady-state and 50 Hz sinusoidal voltage transfers, current transfers, and local and propagation delays of PSPs traveling along dendrites of TG neurons. Synaptic input pattern recognition ability was also predicted to be unaltered in TG neurons in two different soma-dendritic membrane models investigated. Our simulations predict the way how subthreshold dendritic signaling and pattern recognition are preserved in TG neurons: amyloid-related membrane alterations compensate for the pathological effects that dendritic atrophy has on subthreshold dendritic signal transfer and integration in layer II/III somatosensory neurons of this hAPP mouse model for AD. Since neither propagation of single PSPs nor integration of multiple PSPs (pattern recognition) changes in TG neurons, we conclude that AD-related neuronal hyperexcitability cannot be accounted for by altered subthreshold dendritic signaling in these neurons but hyperexcitability is related to changes in active membrane properties and network connectivity. PMID:27378850

Synaptic Activity and Muscle Contraction Increases PDK1 and PKCβI Phosphorylation in the Presynaptic Membrane of the Neuromuscular Junction.

PubMed

Hurtado, Erica; Cilleros, Víctor; Just, Laia; Simó, Anna; Nadal, Laura; Tomàs, Marta; Garcia, Neus; Lanuza, Maria A; Tomàs, Josep

2017-01-01

Conventional protein kinase C βI (cPKCβI) is a conventional protein kinase C (PKC) isoform directly involved in the regulation of neurotransmitter release in the neuromuscular junction (NMJ). It is located exclusively at the nerve terminal and both synaptic activity and muscle contraction modulate its protein levels and phosphorylation. cPKCβI molecular maturation includes a series of phosphorylation steps, the first of which is mediated by phosphoinositide-dependent kinase 1 (PDK1). Here, we sought to localize PDK1 in the NMJ and investigate the hypothesis that synaptic activity and muscle contraction regulate in parallel PDK1 and cPKCβI phosphorylation in the membrane fraction. To differentiate the presynaptic and postsynaptic activities, we abolished muscle contraction with μ-conotoxin GIIIB (μ-CgTx-GIIIB) in some experiments before stimulation of the phrenic nerve (1 Hz, 30 min). Then, we analyzed total and membrane/cytosol fractions of skeletal muscle by Western blotting. Results showed that PDK1 is located exclusively in the nerve terminal of the NMJ. After nerve stimulation with and without coincident muscle contraction, total PDK1 and phosphorylated PDK1 (pPDK1) protein levels remained unaltered. However, synaptic activity specifically enhanced phosphorylation of PDK1 in the membrane, an important subcellular location for PDK1 function. This increase in pPDK1 coincides with a significant increase in the phosphorylation of its substrate cPKCβI also in the membrane fraction. Moreover, muscle contraction maintains PDK1 and pPDK1 but increases cPKCβI protein levels and its phosphorylation. Thus, even though PDK1 activity is maintained, pcPKCβI levels increase in concordance with total cPKCβI. Together, these results indicate that neuromuscular activity could induce the membrane targeting of pPDK1 in the nerve terminal of the NMJ to promote the phosphorylation of the cPKCβI, which is involved in ACh release.

Synaptic Activity and Muscle Contraction Increases PDK1 and PKCβI Phosphorylation in the Presynaptic Membrane of the Neuromuscular Junction

PubMed Central

Hurtado, Erica; Cilleros, Víctor; Just, Laia; Simó, Anna; Nadal, Laura; Tomàs, Marta; Garcia, Neus; Lanuza, Maria A.; Tomàs, Josep

2017-01-01

Conventional protein kinase C βI (cPKCβI) is a conventional protein kinase C (PKC) isoform directly involved in the regulation of neurotransmitter release in the neuromuscular junction (NMJ). It is located exclusively at the nerve terminal and both synaptic activity and muscle contraction modulate its protein levels and phosphorylation. cPKCβI molecular maturation includes a series of phosphorylation steps, the first of which is mediated by phosphoinositide-dependent kinase 1 (PDK1). Here, we sought to localize PDK1 in the NMJ and investigate the hypothesis that synaptic activity and muscle contraction regulate in parallel PDK1 and cPKCβI phosphorylation in the membrane fraction. To differentiate the presynaptic and postsynaptic activities, we abolished muscle contraction with μ-conotoxin GIIIB (μ-CgTx-GIIIB) in some experiments before stimulation of the phrenic nerve (1 Hz, 30 min). Then, we analyzed total and membrane/cytosol fractions of skeletal muscle by Western blotting. Results showed that PDK1 is located exclusively in the nerve terminal of the NMJ. After nerve stimulation with and without coincident muscle contraction, total PDK1 and phosphorylated PDK1 (pPDK1) protein levels remained unaltered. However, synaptic activity specifically enhanced phosphorylation of PDK1 in the membrane, an important subcellular location for PDK1 function. This increase in pPDK1 coincides with a significant increase in the phosphorylation of its substrate cPKCβI also in the membrane fraction. Moreover, muscle contraction maintains PDK1 and pPDK1 but increases cPKCβI protein levels and its phosphorylation. Thus, even though PDK1 activity is maintained, pcPKCβI levels increase in concordance with total cPKCβI. Together, these results indicate that neuromuscular activity could induce the membrane targeting of pPDK1 in the nerve terminal of the NMJ to promote the phosphorylation of the cPKCβI, which is involved in ACh release. PMID:28890686

A Novel Form of Compensation in the Tg2576 Amyloid Mouse Model of Alzheimer's Disease.

PubMed

Somogyi, Attila; Katonai, Zoltán; Alpár, Alán; Wolf, Ervin

2016-01-01

One century after its first description, pathology of Alzheimer's disease (AD) is still poorly understood. Amyloid-related dendritic atrophy and membrane alterations of susceptible brain neurons in AD, and in animal models of AD are widely recognized. However, little effort has been made to study the potential effects of combined morphological and membrane alterations on signal transfer and synaptic integration in neurons that build up affected neural networks in AD. In this study spatial reconstructions and electrophysiological measurements of layer II/III pyramidal neurons of the somatosensory cortex from wild-type (WT) and transgenic (TG) human amyloid precursor protein (hAPP) overexpressing Tg2576 mice were used to build faithful segmental cable models of these neurons. Local synaptic activities were simulated in various points of the dendritic arbors and properties of subthreshold dendritic impulse propagation and predictors of synaptic input pattern recognition ability were quantified and compared in modeled WT and TG neurons. Despite the widespread dendritic degeneration and membrane alterations in mutant mouse neurons, surprisingly little, or no change was detected in steady-state and 50 Hz sinusoidal voltage transfers, current transfers, and local and propagation delays of PSPs traveling along dendrites of TG neurons. Synaptic input pattern recognition ability was also predicted to be unaltered in TG neurons in two different soma-dendritic membrane models investigated. Our simulations predict the way how subthreshold dendritic signaling and pattern recognition are preserved in TG neurons: amyloid-related membrane alterations compensate for the pathological effects that dendritic atrophy has on subthreshold dendritic signal transfer and integration in layer II/III somatosensory neurons of this hAPP mouse model for AD. Since neither propagation of single PSPs nor integration of multiple PSPs (pattern recognition) changes in TG neurons, we conclude that AD-related neuronal hyperexcitability cannot be accounted for by altered subthreshold dendritic signaling in these neurons but hyperexcitability is related to changes in active membrane properties and network connectivity.

Correlating Fluorescence and High-Resolution Scanning Electron Microscopy (HRSEM) for the study of GABAA receptor clustering induced by inhibitory synaptic plasticity.

PubMed

Orlando, Marta; Ravasenga, Tiziana; Petrini, Enrica Maria; Falqui, Andrea; Marotta, Roberto; Barberis, Andrea

2017-10-23

Both excitatory and inhibitory synaptic contacts display activity dependent dynamic changes in their efficacy that are globally termed synaptic plasticity. Although the molecular mechanisms underlying glutamatergic synaptic plasticity have been extensively investigated and described, those responsible for inhibitory synaptic plasticity are only beginning to be unveiled. In this framework, the ultrastructural changes of the inhibitory synapses during plasticity have been poorly investigated. Here we combined confocal fluorescence microscopy (CFM) with high resolution scanning electron microscopy (HRSEM) to characterize the fine structural rearrangements of post-synaptic GABA A Receptors (GABA A Rs) at the nanometric scale during the induction of inhibitory long-term potentiation (iLTP). Additional electron tomography (ET) experiments on immunolabelled hippocampal neurons allowed the visualization of synaptic contacts and confirmed the reorganization of post-synaptic GABA A R clusters in response to chemical iLTP inducing protocol. Altogether, these approaches revealed that, following the induction of inhibitory synaptic potentiation, GABA A R clusters increase in size and number at the post-synaptic membrane with no other major structural changes of the pre- and post-synaptic elements.

[Molecular organization of glutamate-sensitive chemoexcitatory membranes of nerve cells. Binding of L-[3H]glutamate to synaptic membranes of the rat cerebral cortex].

PubMed

Dambinova, S A; Gorodinskiĭ, A I

1984-01-01

The binding of L-[3H]glutamate to rat cerebral cortex synaptic membranes was investigated. Two types of binding sites, a Na+-independent (Kd = 140-160 nm; Bmax = 3.8-4.5 pmol-mg of protein) and a Na+-dependent (Kd = 2.0 microM; Bmax = 45-50 pmol/mg of protein) ones, were detected. The dependence of Na+-insensitive binding on time and temperature and membrane content in a sample was determined. Mono- and divalent cations (5-10 mM) potentiated specific binding by 2.1-3.3 times. The Na+-dependent binding is associated with active transport systems, while the Na+-independent one-with true receptor binding. The relationship between CNS glutamate receptors and Na+-independent binding sites is discussed.

Seminal plasma applied post-thawing affects boar sperm physiology: a flow cytometry study.

PubMed

Fernández-Gago, Rocío; Domínguez, Juan Carlos; Martínez-Pastor, Felipe

2013-09-01

Cryopreservation induces extensive biophysical and biochemical changes in the sperm. In the present study, we used flow cytometry to assess the capacitation-like status of frozen-thawed boar spermatozoa and its relationship with intracellular calcium, assessment of membrane fluidity, modification of thiol groups in plasma membrane proteins, reactive oxygen species (ROS) levels, viability, acrosomal status, and mitochondrial activity. This experiment was performed to verify the effect of adding seminal plasma on post-thaw sperm functions. To determine these effects after cryopreservation, frozen-thawed semen from seven boars was examined after supplementation with different concentrations of pooled seminal plasma (0%, 10%, and 50%) at various times of incubation from 0 to 4 hours. Incubation caused a decrease in membrane integrity and an increase in acrosomal damage, with small changes in other parameters (P > 0.05). Although 10% seminal plasma showed few differences with 0% (ROS increase at 4 hours, P < 0.05), 50% seminal plasma caused important changes. Membrane fluidity increased considerably from the beginning of the experiment, and ROS and free thiols in the cell surface increased by 2 hours of incubation. By the end of the experiment, viability decreased and acrosomal damage increased in the 50% seminal plasma samples. The addition of 50% of seminal plasma seems to modify the physiology of thawed boar spermatozoa, possibly through membrane changes and ROS increase. Although some effects were detrimental, the stimulatory effect of 50% seminal plasma could favor the performance of post-thawed boar semen, as showed in the field (García JC, Domínguez JC, Peña FJ, Alegre B, Gonzalez R, Castro MJ, Habing GG, Kirkwood RN. Thawing boar semen in the presence of seminal plasma: effects on sperm quality and fertility. Anim Reprod Sci 2010;119:160-5). Copyright © 2013 Elsevier Inc. All rights reserved.

Identification of spinal 5-HT sub 3 receptors and their role in the modulation of nociceptive responses in the rat

DOE Office of Scientific and Technical Information (OSTI.GOV)

Glaum, S.R.

1988-01-01

The project consisted of two related studies: (1) the characterization of serotonin binding sites in crude and purified synaptic membranes prepared from the rat spinal cord, and (2) the association of serotonin binding sites with functional 5-HT receptor responses in the modulation of nociceptive information at the level of the spinal cord. The first series of experiments involved the preparation of membranes from the dorsal and ventral halves of the rat spinal cord and the demonstration of specific ({sup 3}H)serotonin binding to these membranes. High affinity binding sites which conformed to the 5-HT{sub 3} subtype were identified in dorsal, butmore » not ventral spinal cord synaptic membranes. These experiments also confirmed the presence of high affinity ({sup 3}H)5-HT binding sites in dorsal spinal cord synaptic membranes of the 5-HT{sub 1} subtype. The second group of studies demonstrated the ability of selective 5-HT{sub 3} antagonists to inhibit the antinociceptive response to intrathecally administered 5-HT, as measured by a change in tail flick and hot plate latencies. Intrathecal pretreatment with the selective 5-HT{sub 3} antagonists ICS 205-930 or MDL 72222 abolished the antinociceptive effects of 5-HT. Furthermore, the selective 5-HT{sub 3} agonist 2-methyl-5-HT mimicked the antinociceptive effects of 5-HT.« less

Synaptic input correlations leading to membrane potential decorrelation of spontaneous activity in cortex.

PubMed

Graupner, Michael; Reyes, Alex D

2013-09-18

Correlations in the spiking activity of neurons have been found in many regions of the cortex under multiple experimental conditions and are postulated to have important consequences for neural population coding. While there is a large body of extracellular data reporting correlations of various strengths, the subthreshold events underlying the origin and magnitude of signal-independent correlations (called noise or spike count correlations) are unknown. Here we investigate, using intracellular recordings, how synaptic input correlations from shared presynaptic neurons translate into membrane potential and spike-output correlations. Using a pharmacologically activated thalamocortical slice preparation, we perform simultaneous recordings from pairs of layer IV neurons in the auditory cortex of mice and measure synaptic potentials/currents, membrane potentials, and spiking outputs. We calculate cross-correlations between excitatory and inhibitory inputs to investigate correlations emerging from the network. We furthermore evaluate membrane potential correlations near resting potential to study how excitation and inhibition combine and affect spike-output correlations. We demonstrate directly that excitation is correlated with inhibition thereby partially canceling each other and resulting in weak membrane potential and spiking correlations between neurons. Our data suggest that cortical networks are set up to partially cancel correlations emerging from the connections between neurons. This active decorrelation is achieved because excitation and inhibition closely track each other. Our results suggest that the numerous shared presynaptic inputs do not automatically lead to increased spiking correlations.

Molecular dynamics study of lipid bilayers modeling the plasma membranes of mouse hepatocytes and hepatomas.

PubMed

Andoh, Yoshimichi; Aoki, Noriyuki; Okazaki, Susumu

2016-02-28

Molecular dynamics (MD) calculations of lipid bilayers modeling the plasma membranes of normal mouse hepatocytes and hepatomas in water have been performed under physiological isothermal-isobaric conditions (310.15 K and 1 atm). The changes in the membrane properties induced by hepatic canceration were investigated and were compared with previous MD calculations included in our previous study of the changes in membrane properties induced by murine thymic canceration. The calculated model membranes for normal hepatocytes and hepatomas comprised 23 and 24 kinds of lipids, respectively. These included phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. We referred to previously published experimental values for the mole fraction of the lipids adopted in the present calculations. The calculated structural and dynamic properties of the membranes such as lateral structure, order parameters, lateral self-diffusion constants, and rotational correlation times all showed that hepatic canceration causes plasma membranes to become more ordered laterally and less fluid. Interestingly, this finding contrasts with the less ordered structure and increased fluidity of plasma membranes induced by thymic canceration observed in our previous MD study.

Molecular dynamics study of lipid bilayers modeling the plasma membranes of mouse hepatocytes and hepatomas

NASA Astrophysics Data System (ADS)

Andoh, Yoshimichi; Aoki, Noriyuki; Okazaki, Susumu

2016-02-01

Molecular dynamics (MD) calculations of lipid bilayers modeling the plasma membranes of normal mouse hepatocytes and hepatomas in water have been performed under physiological isothermal-isobaric conditions (310.15 K and 1 atm). The changes in the membrane properties induced by hepatic canceration were investigated and were compared with previous MD calculations included in our previous study of the changes in membrane properties induced by murine thymic canceration. The calculated model membranes for normal hepatocytes and hepatomas comprised 23 and 24 kinds of lipids, respectively. These included phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. We referred to previously published experimental values for the mole fraction of the lipids adopted in the present calculations. The calculated structural and dynamic properties of the membranes such as lateral structure, order parameters, lateral self-diffusion constants, and rotational correlation times all showed that hepatic canceration causes plasma membranes to become more ordered laterally and less fluid. Interestingly, this finding contrasts with the less ordered structure and increased fluidity of plasma membranes induced by thymic canceration observed in our previous MD study.

Orientation selectivity of synaptic input to neurons in mouse and cat primary visual cortex.

PubMed

Tan, Andrew Y Y; Brown, Brandon D; Scholl, Benjamin; Mohanty, Deepankar; Priebe, Nicholas J

2011-08-24

Primary visual cortex (V1) is the site at which orientation selectivity emerges in mammals: visual thalamus afferents to V1 respond equally to all stimulus orientations, whereas their target V1 neurons respond selectively to stimulus orientation. The emergence of orientation selectivity in V1 has long served as a model for investigating cortical computation. Recent evidence for orientation selectivity in mouse V1 opens cortical computation to dissection by genetic and imaging tools, but also raises two essential questions: (1) How does orientation selectivity in mouse V1 neurons compare with that in previously described species? (2) What is the synaptic basis for orientation selectivity in mouse V1? A comparison of orientation selectivity in mouse and in cat, where such measures have traditionally been made, reveals that orientation selectivity in mouse V1 is weaker than in cat V1, but that spike threshold plays a similar role in narrowing selectivity between membrane potential and spike rate. To uncover the synaptic basis for orientation selectivity, we made whole-cell recordings in vivo from mouse V1 neurons, comparing neuronal input selectivity-based on membrane potential, synaptic excitation, and synaptic inhibition-to output selectivity based on spiking. We found that a neuron's excitatory and inhibitory inputs are selective for the same stimulus orientations as is its membrane potential response, and that inhibitory selectivity is not broader than excitatory selectivity. Inhibition has different dynamics than excitation, adapting more rapidly. In neurons with temporally modulated responses, the timing of excitation and inhibition was different in mice and cats.

Orientation Selectivity of Synaptic Input to Neurons in Mouse and Cat Primary Visual Cortex

PubMed Central

Tan (陈勇毅), Andrew Y. Y.; Brown, Brandon D.; Scholl, Benjamin; Mohanty, Deepankar; Priebe, Nicholas J.

2011-01-01

Primary visual cortex (V1) is the site at which orientation selectivity emerges in mammals: visual thalamus afferents to V1 respond equally to all stimulus orientations whereas their target V1 neurons respond selectively to stimulus orientation. The emergence of orientation selectivity in V1 has long served as a model for investigating cortical computation. Recent evidence for orientation selectivity in mouse V1 opens cortical computation to dissection by genetic and imaging tools, but also raises two essential questions: 1) how does orientation selectivity in mouse V1 neurons compare with that in previously described species? 2) what is the synaptic basis for orientation selectivity in mouse V1? A comparison of orientation selectivity in mouse and in cat, where such measures have traditionally been made, reveals that orientation selectivity in mouse V1 is weaker than in cat V1, but that spike threshold plays a similar role in narrowing selectivity between membrane potential and spike rate. To uncover the synaptic basis for orientation selectivity, we made whole-cell recordings in vivo from mouse V1 neurons, comparing neuronal input selectivity - based on membrane potential, synaptic excitation, and synaptic inhibition - to output selectivity based on spiking. We found that a neuron's excitatory and inhibitory inputs are selective for the same stimulus orientations as is its membrane potential response, and that inhibitory selectivity is not broader than excitatory selectivity. Inhibition has different dynamics than excitation, adapting more rapidly. In neurons with temporally modulated responses, the timing of excitation and inhibition was different in mice and cats. PMID:21865476

STriatal-Enriched protein tyrosine Phosphatase (STEP) Regulates the PTPα/Fyn Signaling Pathway

PubMed Central

Xu, Jian; Kurup, Pradeep; Foscue, Ethan; Lombroso, Paul J.

2015-01-01

The tyrosine kinase Fyn has two regulatory tyrosine residues that when phosphorylated either activate (Tyr420) or inhibit (Tyr531) Fyn activity. Within the central nervous system, two protein tyrosine phosphatases (PTPs) target these regulatory tyrosines in Fyn. PTPα dephosphorylates Tyr531 and activates Fyn, while STEP (STriatal-Enriched protein tyrosine Phosphatase) dephosphorylates Tyr420 and inactivates Fyn. Thus, PTPα and STEP have opposing functions in the regulation of Fyn; however, whether there is cross talk between these two PTPs remains unclear. Here, we used molecular techniques in primary neuronal cultures and in vivo to demonstrate that STEP negatively regulates PTPα by directly dephosphorylating PTPα at its regulatory Tyr789. Dephosphorylation of Tyr789 prevents the translocation of PTPα to synaptic membranes, blocking its ability to interact with and activate Fyn. Genetic or pharmacologic reduction of STEP61 activity increased the phosphorylation of PTPα at Tyr789, as well as increased translocation of PTPα to synaptic membranes. Activation of PTPα and Fyn and trafficking of GluN2B to synaptic membranes are necessary for ethanol intake behaviors in rodents. We tested the functional significance of STEP61 in this signaling pathway by ethanol administration to primary cultures as well as in vivo, and demonstrated that the inactivation of STEP61 by ethanol leads to the activation of PTPα, its translocation to synaptic membranes, and the activation of Fyn. These findings indicate a novel mechanism by which STEP61 regulates PTPα and suggest that STEP and PTPα coordinate the regulation of Fyn. PMID:25951993

Specific multi-nutrient enriched diet enhances hippocampal cholinergic transmission in aged rats.

PubMed

Cansev, Mehmet; van Wijk, Nick; Turkyilmaz, Mesut; Orhan, Fulya; Sijben, John W C; Broersen, Laus M

2015-01-01

Fortasyn Connect (FC) is a specific nutrient combination designed to target synaptic dysfunction in Alzheimer's disease by providing neuronal membrane precursors and other supportive nutrients. The aim of the present study was to investigate the effects of FC on hippocampal cholinergic neurotransmission in association with its effects on synaptic membrane formation in aged rats. Eighteen-month-old male Wistar rats were randomized to receive a control diet for 4 weeks or an FC-enriched diet for 4 or 6 weeks. At the end of the dietary treatments, acetylcholine (ACh) release was investigated by in vivo microdialysis in the right hippocampi. On completion of microdialysis studies, the rats were sacrificed, and the left hippocampi were obtained to determine the levels of choline, ACh, membrane phospholipids, synaptic proteins, and choline acetyltransferase. Our results revealed that supplementation with FC diet for 4 or 6 weeks, significantly enhanced basal and stimulated hippocampal ACh release and ACh tissue levels, along with levels of phospholipids. Feeding rats the FC diet for 6 weeks significantly increased the levels of choline acetyltransferase, the presynaptic marker Synapsin-1, and the postsynaptic marker PSD-95, but decreased levels of Nogo-A, a neurite outgrowth inhibitor. These data show that the FC diet enhances hippocampal cholinergic neurotransmission in aged rats and suggest that this effect is mediated by enhanced synaptic membrane formation. These data provide further insight into cellular and molecular mechanisms by which FC may support memory processes in Alzheimer's disease. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Impact of Fast Sodium Channel Inactivation on Spike Threshold Dynamics and Synaptic Integration

PubMed Central

Platkiewicz, Jonathan; Brette, Romain

2011-01-01

Neurons spike when their membrane potential exceeds a threshold value. In central neurons, the spike threshold is not constant but depends on the stimulation. Thus, input-output properties of neurons depend both on the effect of presynaptic spikes on the membrane potential and on the dynamics of the spike threshold. Among the possible mechanisms that may modulate the threshold, one strong candidate is Na channel inactivation, because it specifically impacts spike initiation without affecting the membrane potential. We collected voltage-clamp data from the literature and we found, based on a theoretical criterion, that the properties of Na inactivation could indeed cause substantial threshold variability by itself. By analyzing simple neuron models with fast Na inactivation (one channel subtype), we found that the spike threshold is correlated with the mean membrane potential and negatively correlated with the preceding depolarization slope, consistent with experiments. We then analyzed the impact of threshold dynamics on synaptic integration. The difference between the postsynaptic potential (PSP) and the dynamic threshold in response to a presynaptic spike defines an effective PSP. When the neuron is sufficiently depolarized, this effective PSP is briefer than the PSP. This mechanism regulates the temporal window of synaptic integration in an adaptive way. Finally, we discuss the role of other potential mechanisms. Distal spike initiation, channel noise and Na activation dynamics cannot account for the observed negative slope-threshold relationship, while adaptive conductances (e.g. K+) and Na inactivation can. We conclude that Na inactivation is a metabolically efficient mechanism to control the temporal resolution of synaptic integration. PMID:21573200

Identification of the glucose transporter in mammalian cell membranes using an /sup 125/(I)-forskolin photoaffinity label

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ruoho, A.; Wadzinski, B.; Shanahan, M.

1987-05-01

The glucose transporter has been identified in a variety of mammlian cell membranes using a carrier-free photoactivatable radioiodinated derivative of forskolin, 3-iodo-4-azidophenethylamido-7-0-succinyldeacetyl-forskolin, (I-125)IAPS-Fsk, at 1-10 nM. The membranes which have been photolabeled with (I-125)IAPS-Fsk are: rat cardiac sarcolemmal membranes, rat cortex and cerebellum synaptic membranes, human placental membranes, and wild type S49 lymphoma cell membranes. The glucose transporter in rat cardiac sarcolemmal membranes and rat cortex and cerebellum synaptic membranes was determined to be 45 kDa by SDS-PAGE. Photolysis of human placental membranes and S49 lymphoma membranes with (I-125)IAPS-Fsk followed by SDS-PAGE indicated specific derivatization of a broad band (45-55more » kDa) in placental membranes and a narrower band (45 kDa) in the S49 lymphoma membranes. Digestion of the (I-125)IPAS-Fsk labelled placental and S49 lymphoma membranes with endo-B-galactosidase showed a reduction in the apparent molecular weight of the radiolabelled band to 40 kDa. Trypsinization of labelled placental and lymphoma membranes produced an 18 kDa radiolabelled proteolytic fragment. (I-125)IAPS-Fsk is a highly effective probe for identifying low levels of glucose transporters in mammalian tissues.« less

[Influence of mediator diffusion on trigger mode of a synapse].

PubMed

Vasilev, A N; Kulish, M V

2014-01-01

The model of postsynaptic membrane activation, is proposed in the paper. This model takes into account inhomogeneity of mediator's space distribution in the region of the synaptic cleft as well as nonlinear nature of interaction between the mediator and receptors on the postsynaptic membrane. Based on equations of this model stationary solutions are calculated for mediator distribution in the synaptic cleft and the number of activated receptors. Kinetics of reactions for activation and deactivation of receptors is analyzed within the concept of a trigger mode of the synapse. It is shown that activation-deactivation processes and redistribution of the mediator in the cleft can be interpreted as successive transitions between two stationary states of the system. Time of transitions between these states is found and its dependence on system parameters (in particular on the width of the synaptic cleft) is analyzed.

Self-organizing actin patterns shape membrane architecture but not cell mechanics

NASA Astrophysics Data System (ADS)

Fritzsche, M.; Li, D.; Colin-York, H.; Chang, V. T.; Moeendarbary, E.; Felce, J. H.; Sezgin, E.; Charras, G.; Betzig, E.; Eggeling, C.

2017-02-01

Cell-free studies have demonstrated how collective action of actin-associated proteins can organize actin filaments into dynamic patterns, such as vortices, asters and stars. Using complementary microscopic techniques, we here show evidence of such self-organization of the actin cortex in living HeLa cells. During cell adhesion, an active multistage process naturally leads to pattern transitions from actin vortices over stars into asters. This process is primarily driven by Arp2/3 complex nucleation, but not by myosin motors, which is in contrast to what has been theoretically predicted and observed in vitro. Concomitant measurements of mechanics and plasma membrane fluidity demonstrate that changes in actin patterning alter membrane architecture but occur functionally independent of macroscopic cortex elasticity. Consequently, tuning the activity of the Arp2/3 complex to alter filament assembly may thus be a mechanism allowing cells to adjust their membrane architecture without affecting their macroscopic mechanical properties.

Self-organizing actin patterns shape membrane architecture but not cell mechanics

PubMed Central

Fritzsche, M.; Li, D.; Colin-York, H.; Chang, V. T.; Moeendarbary, E.; Felce, J. H.; Sezgin, E.; Charras, G.; Betzig, E.; Eggeling, C.

2017-01-01

Cell-free studies have demonstrated how collective action of actin-associated proteins can organize actin filaments into dynamic patterns, such as vortices, asters and stars. Using complementary microscopic techniques, we here show evidence of such self-organization of the actin cortex in living HeLa cells. During cell adhesion, an active multistage process naturally leads to pattern transitions from actin vortices over stars into asters. This process is primarily driven by Arp2/3 complex nucleation, but not by myosin motors, which is in contrast to what has been theoretically predicted and observed in vitro. Concomitant measurements of mechanics and plasma membrane fluidity demonstrate that changes in actin patterning alter membrane architecture but occur functionally independent of macroscopic cortex elasticity. Consequently, tuning the activity of the Arp2/3 complex to alter filament assembly may thus be a mechanism allowing cells to adjust their membrane architecture without affecting their macroscopic mechanical properties. PMID:28194011

Observing polymersome dynamics in controlled microscale flows

NASA Astrophysics Data System (ADS)

Kumar, Subhalakshmi; Shenoy, Anish; Schroeder, Charles

2015-03-01

Achieving an understanding of single particle rheology for large yet deformable particles with controlled membrane viscoelasticity is major challenge in soft materials. In this work, we directly visualize the dynamics of single polymersomes (~ 10 μm in size) in an extensional flow using optical microscopy. We generate polymer vesicular structures composed of polybutadiene-block-polyethylene oxide (PB-b-PEO) copolymers. Single polymersomes are confined near the stagnation point of a planar extensional flow using an automated microfluidic trap, thereby enabling the direct observation of polymersome dynamics under fluid flows with controlled strains and strain rates. In a series of experiments, we investigate the effect of varying elasticity in vesicular membranes on polymersome deformation, along with the impact of decreasing membrane fluidity upon increasing diblock copolymer molecular weight. Overall, we believe that this approach will enable precise characterization of the role of membrane properties on single particle rheology for deformable polymersomes.

Mechanism of permeability-enhancing effect of EDTA and boric acid on the corneal penetration of 4-[1-hydroxy-1-methylethyl]-2-propyl-1-[4-[2-[tetrazole-5-yl]phenyl]phenyl] methylimidazole-5-carboxylic acid monohydrate (CS-088).

PubMed

Kikuchi, Takayuki; Suzuki, Masahiko; Kusai, Akira; Iseki, Ken; Sasaki, Hitoshi; Nakashima, Kenichiro

2005-08-11

This study was conducted to clarify the penetration properties of 4-[1-hydroxy-1-methylethyl]-2-propyl-1-[4-[2-[tetrazole-5-yl]phenyl]phenyl]methylimidazole-5-carboxylic acid monohydrate (CS-088), an ophthalmic agent, and the mechanism of the permeability-enhancing effect of EDTA and boric acid (EDTA/boric acid) on the corneal penetration of CS-088. In the absence of additives, corneal permeability decreased with increasing concentration of CS-088 as CS-088 monomers self-associate to form dimers. Presence of EDTA/boric acid caused no significant changes in the physicochemical properties of CS-088, the apparent partition coefficient or the mean particle size of CS-088. EDTA/boric acid induced only a slight change in the zeta potential of liposomes used as a model of the biological membrane. On the other hand, EDTA/boric acid significantly increased membrane fluidity of liposomes, whereas other buffering agents tested did not. This effect was synergistic and concentration-dependent for both EDTA and boric acid as was observed in in vitro corneal penetration of CS-088. In accordance with the result, the rate of CS-088 permeation into the liposomes significantly increased by the addition of EDTA/boric acid. Therefore, it was demonstrated that EDTA/boric acid promotes corneal penetration of CS-088 through the transcellular pathway by increasing membrane fluidity. Conversely, other buffering agents decreased corneal permeability of CS-088 by inducing further self-association of CS-088 aggregates.

Using a patterned grating structure to create lipid bilayer platforms insensitive to air bubbles.

PubMed

Han, Chung-Ta; Chao, Ling

2015-01-07

Supported lipid bilayers (SLBs) have been used for various biosensing applications. The bilayer structure enables embedded lipid membrane species to maintain their native orientation, and the two-dimensional fluidity is crucial for numerous biomolecular interactions to occur. The platform integrated with a microfluidic device for reagent transport and exchange has great potential to be applied with surface analytical tools. However, SLBs can easily be destroyed by air bubbles during assay reagent transport and exchange. Here, we created a patterned obstacle grating structured surface in a microfluidic channel to protect SLBs from being destroyed by air bubbles. Unlike all of the previous approaches using chemical modification or adding protection layers to strengthen lipid bilayers, the uniqueness of this approach is that it uses the patterned obstacles to physically trap water above the bilayers to prevent the air-water interface from directly coming into contact with and peeling the bilayers. We showed that our platform with certain grating geometry criteria can provide promising protection to SLBs from air bubbles. The required obstacle distance was found to decrease when we increased the air-bubble movement speed. In addition, the interaction assay results from streptavidin and biotinylated lipids in the confined SLBs suggested that receptors at the SLBs retained the interaction ability after air-bubble treatment. The results showed that the developed SLB platform can preserve both high membrane fluidity and high accessibility to the outside environment, which have never been simultaneously achieved before. Incorporating the built platforms with some surface analytical tools could open the bottleneck of building highly robust in vitro cell-membrane-related bioassays.

Membrane Electromechanics at Hair-Cell Synapses

NASA Astrophysics Data System (ADS)

Brownell, W. E.; Farrell, B.; Raphael, R. M.

2003-02-01

Both outer hair cell electromotility and neurotransmission at the inner hair cell synapse are rapid mechanical events that are synchronized to the hair-cell receptor potential. We analyze whether the forces and potentials resulting from membrane flexoelectricity could affect synaptic vesicle fusion. The results suggest that the coupling of membrane curvature with membrane potential is of sufficient magnitude to influence neurotransmitter release.

The Multifaceted Role of SNARE Proteins in Membrane Fusion

PubMed Central

Han, Jing; Pluhackova, Kristyna; Böckmann, Rainer A.

2017-01-01

Membrane fusion is a key process in all living organisms that contributes to a variety of biological processes including viral infection, cell fertilization, as well as intracellular transport, and neurotransmitter release. In particular, the various membrane-enclosed compartments in eukaryotic cells need to exchange their contents and communicate across membranes. Efficient and controllable fusion of biological membranes is known to be driven by cooperative action of SNARE proteins, which constitute the central components of the eukaryotic fusion machinery responsible for fusion of synaptic vesicles with the plasma membrane. During exocytosis, vesicle-associated v-SNARE (synaptobrevin) and target cell-associated t-SNAREs (syntaxin and SNAP-25) assemble into a core trans-SNARE complex. This complex plays a versatile role at various stages of exocytosis ranging from the priming to fusion pore formation and expansion, finally resulting in the release or exchange of the vesicle content. This review summarizes current knowledge on the intricate molecular mechanisms underlying exocytosis triggered and catalyzed by SNARE proteins. Particular attention is given to the function of the peptidic SNARE membrane anchors and the role of SNARE-lipid interactions in fusion. Moreover, the regulatory mechanisms by synaptic auxiliary proteins in SNARE-driven membrane fusion are briefly outlined. PMID:28163686

The Multifaceted Role of SNARE Proteins in Membrane Fusion.

PubMed

Han, Jing; Pluhackova, Kristyna; Böckmann, Rainer A

2017-01-01

Membrane fusion is a key process in all living organisms that contributes to a variety of biological processes including viral infection, cell fertilization, as well as intracellular transport, and neurotransmitter release. In particular, the various membrane-enclosed compartments in eukaryotic cells need to exchange their contents and communicate across membranes. Efficient and controllable fusion of biological membranes is known to be driven by cooperative action of SNARE proteins, which constitute the central components of the eukaryotic fusion machinery responsible for fusion of synaptic vesicles with the plasma membrane. During exocytosis, vesicle-associated v-SNARE (synaptobrevin) and target cell-associated t-SNAREs (syntaxin and SNAP-25) assemble into a core trans-SNARE complex. This complex plays a versatile role at various stages of exocytosis ranging from the priming to fusion pore formation and expansion, finally resulting in the release or exchange of the vesicle content. This review summarizes current knowledge on the intricate molecular mechanisms underlying exocytosis triggered and catalyzed by SNARE proteins. Particular attention is given to the function of the peptidic SNARE membrane anchors and the role of SNARE-lipid interactions in fusion. Moreover, the regulatory mechanisms by synaptic auxiliary proteins in SNARE-driven membrane fusion are briefly outlined.

Fast, temperature-sensitive and clathrin-independent endocytosis at central synapses

PubMed Central

Delvendahl, Igor; Vyleta, Nicholas P.; von Gersdorff, Henrique; Hallermann, Stefan

2016-01-01

The fusion of neurotransmitter-filled vesicles during synaptic transmission is balanced by endocytotic membrane retrieval. Despite extensive research, the speed and mechanisms of synaptic vesicle endocytosis have remained controversial. Here, we establish low-noise time-resolved membrane capacitance measurements that allow monitoring changes in surface membrane area elicited by single action potentials and stronger stimuli with high-temporal resolution at physiological temperature in individual bonafide mature central synapses. We show that single action potentials trigger very rapid endocytosis, retrieving presynaptic membrane with a time constant of 470 ms. This fast endocytosis is independent of clathrin, but mediated by dynamin and actin. In contrast, stronger stimuli evoke a slower mode of endocytosis that is clathrin-, dynamin-, and actin-dependent. Furthermore, the speed of endocytosis is highly temperature-dependent with a Q10 of ~3.5. These results demonstrate that distinct molecular modes of endocytosis with markedly different kinetics operate at central synapses. PMID:27146271

Neurolastin, a dynamin family GTPase, regulates excitatory synapses and spine density

PubMed Central

Madan Lomash, Richa; Gu, Xinglong; Youle, Richard J.; Lu, Wei; Roche, Katherine W.

2015-01-01

SUMMARY Membrane trafficking and spinogenesis contribute significantly to changes in synaptic strength during development and in various paradigms of synaptic plasticity. GTPases of the dynamin family are key players regulating membrane trafficking. Here, we identify a brain-specific dynamin family GTPase, neurolastin (RNF112/Znf179), with closest homology to atlastin. We demonstrate that neurolastin has functional GTPase and RING domains, making it a unique protein identified with this multi-enzymatic domain organization. We also show that neurolastin is a peripheral membrane protein, which localizes to endosomes and affects endosomal membrane dynamics via its RING domain. In addition, neurolastin knockout mice have fewer dendritic spines, and rescue of the wildtype phenotype requires both the GTPase and RING domains. Furthermore, we find fewer functional synapses and reduced paired pulse facilitation in neurolastin knockout mice. Thus, we identify neurolastin as a dynamin family GTPase that affects endosome size and spine density. PMID:26212327

Neuron Specific Rab4 Effector GRASP-1 Coordinates Membrane Specialization and Maturation of Recycling Endosomes

PubMed Central

Hoogenraad, Casper C.; Popa, Ioana; Futai, Kensuke; Sanchez-Martinez, Emma; Wulf, Phebe S.; van Vlijmen, Thijs; Dortland, Bjorn R.; Oorschot, Viola; Govers, Roland; Monti, Maria; Heck, Albert J. R.; Sheng, Morgan; Klumperman, Judith; Rehmann, Holger; Jaarsma, Dick; Kapitein, Lukas C.; van der Sluijs, Peter

2010-01-01

The endosomal pathway in neuronal dendrites is essential for membrane receptor trafficking and proper synaptic function and plasticity. However, the molecular mechanisms that organize specific endocytic trafficking routes are poorly understood. Here, we identify GRIP-associated protein-1 (GRASP-1) as a neuron-specific effector of Rab4 and key component of the molecular machinery that coordinates recycling endosome maturation in dendrites. We show that GRASP-1 is necessary for AMPA receptor recycling, maintenance of spine morphology, and synaptic plasticity. At the molecular level, GRASP-1 segregates Rab4 from EEA1/Neep21/Rab5-positive early endosomal membranes and coordinates the coupling to Rab11-labelled recycling endosomes by interacting with the endosomal SNARE syntaxin 13. We propose that GRASP-1 connects early and late recycling endosomal compartments by forming a molecular bridge between Rab-specific membrane domains and the endosomal SNARE machinery. The data uncover a new mechanism to achieve specificity and directionality in neuronal membrane receptor trafficking. PMID:20098723

Membrane Interactions of Phytochemicals as Their Molecular Mechanism Applicable to the Discovery of Drug Leads from Plants.

PubMed

Tsuchiya, Hironori

2015-10-16

In addition to interacting with functional proteins such as receptors, ion channels, and enzymes, a variety of drugs mechanistically act on membrane lipids to change the physicochemical properties of biomembranes as reported for anesthetic, adrenergic, cholinergic, non-steroidal anti-inflammatory, analgesic, antitumor, antiplatelet, antimicrobial, and antioxidant drugs. As well as these membrane-acting drugs, bioactive plant components, phytochemicals, with amphiphilic or hydrophobic structures, are presumed to interact with biological membranes and biomimetic membranes prepared with phospholipids and cholesterol, resulting in the modification of membrane fluidity, microviscosity, order, elasticity, and permeability with the potencies being consistent with their pharmacological effects. A novel mechanistic point of view of phytochemicals would lead to a better understanding of their bioactivities, an insight into their medicinal benefits, and a strategic implication for discovering drug leads from plants. This article reviews the membrane interactions of different classes of phytochemicals by highlighting their induced changes in membrane property. The phytochemicals to be reviewed include membrane-interactive flavonoids, terpenoids, stilbenoids, capsaicinoids, phloroglucinols, naphthodianthrones, organosulfur compounds, alkaloids, anthraquinonoids, ginsenosides, pentacyclic triterpene acids, and curcuminoids. The membrane interaction's applicability to the discovery of phytochemical drug leads is also discussed while referring to previous screening and isolating studies.

Acetylcholine Mediates a Slow Synaptic Potential in Hippocampal Pyramidal Cells

NASA Astrophysics Data System (ADS)

Cole, A. E.; Nicoll, R. A.

1983-09-01

The hippocampal slice preparation was used to study the role of acetylcholine as a synaptic transmitter. Bath-applied acetylcholine had three actions on pyramidal cells: (i) depolarization associated with increased input resistance, (ii) blockade of calcium-activated potassium responses, and (iii) blockade of accommodation of cell discharge. All these actions were reversed by the muscarinic antagonist atropine. Stimulation of sites in the slice known to contain cholinergic fibers mimicked all the actions. Furthermore, these evoked synaptic responses were enhanced by the cholinesterase inhibitor eserine and were blocked by atropine. These findings provide electrophysiological support for the role of acetylcholine as a synaptic transmitter in the brain and demonstrate that nonclassical synaptic responses involving the blockade of membrane conductances exist in the brain.

Characterization of the N-terminal segment used by the barley yellow dwarf virus movement protein to promote interaction with the nuclear membrane of host plant cells.

PubMed

Dennison, Sarah Rachel; Harris, Frederick; Brandenburg, Klaus; Phoenix, David Andrew

2007-11-01

The barley yellow dwarf virus movement protein (BYDV-MP) requires its N-terminal sequence to promote the transport of viral RNA into the nuclear compartment of host plant cells. Here, graphical analysis predicts that this sequence would form a membrane interactive amphiphilic alpha-helix. Confirming this prediction, NT1, a peptide homologue of the BYDV-MP N-terminal sequence, was found to be alpha-helical (65%) in the presence of vesicles mimics of the nuclear membrane. The peptide increased the fluidity of these nuclear membrane mimics (rise in wavenumber of circa 0.5-1.0 cm(-1)) and induced surface pressure changes of 2 mN m(-1) in lipid monolayers with corresponding compositions. Taken with isotherm analysis these results suggest that BYDV-MP forms an N-terminal amphiphilic alpha-helix, which partitions into the nuclear membrane primarily through thermodynamically stable associations with the membrane lipid headgroup region. We speculate that these associations may play a role in targeting of the nuclear membrane by BYDM-MP.

Disruption of gel phase lipid packing efficiency by sucralose studied with merocyanine 540.

PubMed

Barker, Morgan; Kennedy, Anthony

2017-04-01

Sucralose, an artificial sweetener, displays very different behavior towards membranes than its synthetic precursor sucrose. The impact of both sugars on model dipalmitoylphosphatidylcholine model membranes was investigated using absorbance and flourescence spectroscopy and the membrane probe merocyanine 540. This probe molecule is highly sensitive to changes in membrane packing, microviscosity and polarity. This work focuses on the impact of sugars on the outer leaflet of unilamellar dipalmitoyl phosphatidylcholine model membranes. The choice of lipid permits access to the gel phase at room temperature and incorporation of the dye after liposome formation allows us to examine the direct impact of the sugar on the outer leaflet while maximizing the response of the dye to changes in the bilayer. The results demonstrate that sucrose has no impact on the packing efficiency of lipids in unilamellar DPPC vesicles in the gel phase. Conversely sucralose decreases the packing efficiency of lipids in the gel phase and results in decreased microviscosity and increased membrane fluidity, which may be as a result of water disruption at the membrane water interface. Copyright © 2017 Elsevier B.V. All rights reserved.

Lipidomic adaptations of the Metarhizium robertsii strain in response to the presence of butyltin compounds.

PubMed

Stolarek, Paulina; Różalska, Sylwia; Bernat, Przemysław

2018-06-14

Metarhizium robertsii, a butyltin-resistant filamentous fungus, can rapid and complete biodegradation of di- (DBT) and tributyltin (TBT) under conditions of intensive aeration and ascorbic acid supplementation. In this paper, lipidomic investigations were performed to find the membrane adaptations necessary for effective butyltins degradation. HPLC-MS/MS analysis showed that the phospholipid profile was greatly modified during M. robertsii batch cultivation (pO 2  ≥ 20%), contributing to increased membrane fluidity and facilitated mass transfer, which could enhance butyltins biodegradation. Intensified biosynthesis of phospholipids, sphingolipids and ergosterol by the mycelia exposed to butyltins was noted. DIOC 6 (3) fluorescence intensity for TBT-treated mycelium increased 9-fold pointing to membrane hyperpolarization. Fluorescent studies showed improved membrane rigidity and integrity in response to butyltins presence. Vitamin C supplementation restored membrane composition and dynamic properties, followed by supposed acceleration of transport of monobutyltin and its biodegradation thus protecting the M. robertsii cells against oxidative and nitrosative stress. Copyright © 2018 Elsevier B.V. All rights reserved.

Chronic cigarette smoking alters erythrocyte membrane lipid composition and properties in male human volunteers.

PubMed

Padmavathi, Pannuru; Reddy, Vaddi Damodara; Kavitha, Godugu; Paramahamsa, Maturu; Varadacharyulu, Nallanchakravarthula

2010-11-01

Cigarette smoking is a major lifestyle factor influencing the health of human beings. The present study investigates smoking induced alterations on the erythrocyte membrane lipid composition, fluidity and the role of nitric oxide. Thirty experimental and control subjects (age 35+/-8) were selected for the study. Experimental subjects smoke 12+/-2 cigarettes per day for 7-10 years. In smokers elevated nitrite/nitrate levels in plasma and red cell lysates were observed. Smokers showed increased hemolysis, erythrocyte membrane lipid peroxidation, protein carbonyls, C/P ratio (cholesterol and phospholipid ratio), anisotropic (gamma) value with decreased Na(+)/K(+)-ATPase activity and sulfhydryl groups. Alterations in smokers erythrocyte membrane individual phospholipids were also evident from the study. Red cell lysate nitric oxide positively correlated with C/P ratio (r=0.565) and fluorescent anisotropic (gamma) value (r=0.386) in smokers. Smoking induced generation of reactive oxygen/nitrogen species might have altered erythrocyte membrane physico-chemical properties. Copyright 2010 Elsevier Inc. All rights reserved.

Neuronal activity-regulated gene transcription: how are distant synaptic signals conveyed to the nucleus?

PubMed Central

Matamales, Miriam

2012-01-01

Synaptic activity can trigger gene expression programs that are required for the stable change of neuronal properties, a process that is essential for learning and memory. Currently, it is still unclear how the stimulation of dendritic synapses can be coupled to transcription in the nucleus in a timely way given that large distances can separate these two cellular compartments. Although several mechanisms have been proposed to explain long distance communication between synapses and the nucleus, the possible co-existence of these models and their relevance in physiological conditions remain elusive. One model suggests that synaptic activation triggers the translocation to the nucleus of certain transcription regulators localised at postsynaptic sites that function as synapto-nuclear messengers. Alternatively, it has been hypothesised that synaptic activity initiates propagating regenerative intracellular calcium waves that spread through dendrites into the nucleus where nuclear transcription machinery is thereby regulated. It has also been postulated that membrane depolarisation of voltage-gated calcium channels on the somatic membrane is sufficient to increase intracellular calcium concentration and activate transcription without the need for transported signals from distant synapses. Here I provide a critical overview of the suggested mechanisms for coupling synaptic stimulation to transcription, the underlying assumptions behind them and their plausible physiological significance. PMID:24327840

Neuronal activity-regulated gene transcription: how are distant synaptic signals conveyed to the nucleus?

PubMed

Matamales, Miriam

2012-12-19

Synaptic activity can trigger gene expression programs that are required for the stable change of neuronal properties, a process that is essential for learning and memory. Currently, it is still unclear how the stimulation of dendritic synapses can be coupled to transcription in the nucleus in a timely way given that large distances can separate these two cellular compartments. Although several mechanisms have been proposed to explain long distance communication between synapses and the nucleus, the possible co-existence of these models and their relevance in physiological conditions remain elusive. One model suggests that synaptic activation triggers the translocation to the nucleus of certain transcription regulators localised at postsynaptic sites that function as synapto-nuclear messengers. Alternatively, it has been hypothesised that synaptic activity initiates propagating regenerative intracellular calcium waves that spread through dendrites into the nucleus where nuclear transcription machinery is thereby regulated. It has also been postulated that membrane depolarisation of voltage-gated calcium channels on the somatic membrane is sufficient to increase intracellular calcium concentration and activate transcription without the need for transported signals from distant synapses. Here I provide a critical overview of the suggested mechanisms for coupling synaptic stimulation to transcription, the underlying assumptions behind them and their plausible physiological significance.

Neuronal activity-regulated gene transcription: how are distant synaptic signals conveyed to the nucleus?

PubMed

Matamales, Miriam

2012-01-01

Synaptic activity can trigger gene expression programs that are required for the stable change of neuronal properties, a process that is essential for learning and memory. Currently, it is still unclear how the stimulation of dendritic synapses can be coupled to transcription in the nucleus in a timely way given that large distances can separate these two cellular compartments. Although several mechanisms have been proposed to explain long distance communication between synapses and the nucleus, the possible co-existence of these models and their relevance in physiological conditions remain elusive. One model suggests that synaptic activation triggers the translocation to the nucleus of certain transcription regulators localised at postsynaptic sites that function as synapto-nuclear messengers. Alternatively, it has been hypothesised that synaptic activity initiates propagating regenerative intracellular calcium waves that spread through dendrites into the nucleus where nuclear transcription machinery is thereby regulated. It has also been postulated that membrane depolarisation of voltage-gated calcium channels on the somatic membrane is sufficient to increase intracellular calcium concentration and activate transcription without the need for transported signals from distant synapses. Here I provide a critical overview of the suggested mechanisms for coupling synaptic stimulation to transcription, the underlying assumptions behind them and their plausible physiological significance.

Energetics, kinetics, and pathway of SNARE folding and assembly revealed by optical tweezers.

PubMed

Zhang, Yongli

2017-07-01

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are universal molecular engines that drive membrane fusion. Particularly, synaptic SNAREs mediate fast calcium-triggered fusion of neurotransmitter-containing vesicles with plasma membranes for synaptic transmission, the basis of all thought and action. During membrane fusion, complementary SNAREs located on two apposed membranes (often called t- and v-SNAREs) join together to assemble into a parallel four-helix bundle, releasing the energy to overcome the energy barrier for fusion. A long-standing hypothesis suggests that SNAREs act like a zipper to draw the two membranes into proximity and thereby force them to fuse. However, a quantitative test of this SNARE zippering hypothesis was hindered by difficulties to determine the energetics and kinetics of SNARE assembly and to identify the relevant folding intermediates. Here, we first review different approaches that have been applied to study SNARE assembly and then focus on high-resolution optical tweezers. We summarize the folding energies, kinetics, and pathways of both wild-type and mutant SNARE complexes derived from this new approach. These results show that synaptic SNAREs assemble in four distinct stages with different functions: slow N-terminal domain association initiates SNARE assembly; a middle domain suspends and controls SNARE assembly; and rapid sequential zippering of the C-terminal domain and the linker domain directly drive membrane fusion. In addition, the kinetics and pathway of the stagewise assembly are shared by other SNARE complexes. These measurements prove the SNARE zippering hypothesis and suggest new mechanisms for SNARE assembly regulated by other proteins. © 2017 The Protein Society.

Synaptic proteins associate with a sub-set of lipid rafts when isolated from nerve endings at physiological temperature.

PubMed

Gil, Carles; Cubí, Roger; Blasi, Juan; Aguilera, José

2006-10-06

Although the high presence of cholesterol in nerve terminals is well documented, specific roles of this lipid in transmitter release have remained elusive. Since cholesterol is a highly enriched component in the membrane microdomains known as lipid rafts, it is probable that these domains are very important in synaptic function. The extraction of lipid rafts using Brij 98 at 37 degrees C avoids the formation of nonspecific membrane aggregates at low temperature, allowing the isolation of more physiologically relevant lipid rafts. In the present work, we examine, by means of buoyancy analysis in sucrose gradients after solubilization of the membranes with Brij 98 or with Lubrol WX, the presence of proteins involved in exocytosis in detergent-resistant membranes (DRM) using rat brain synaptosomes as a neurological model. Significant proportions of the proteins tested in the present work, which are involved in neurotransmitter release, are found in Brij 98 raft fractions, demonstrating that significant pools of synaptic proteins are segregated in specific parts of the membrane at physiological temperature. On the other hand, Lubrol WX is unable to solubilize the major fraction of the proteins tested. Treatment of synaptosomes with methyl-beta-cyclodextrin (mbetaCD) causes alteration in the buoyancy properties of proteins initially present in Brij- as well as in Lubrol-resistant membranes, indicating the cholesterol-dependency of both kinds of microdomains. Finally, we detect the depolarization-induced enhancement of the cholesterol-dependent association of syntaxin 1 with Brij 98-rafts, under the same conditions in which prolonged neurotransmitter release is stimulated.

Formation and stability of synaptic receptor domains.

PubMed

Haselwandter, Christoph A; Calamai, Martino; Kardar, Mehran; Triller, Antoine; da Silveira, Rava Azeredo

2011-06-10

Neurotransmitter receptor molecules, concentrated in postsynaptic domains along with scaffold and a number of other molecules, are key regulators of signal transmission across synapses. Combining experiment and theory, we develop a quantitative description of synaptic receptor domains in terms of a reaction-diffusion model. We show that interactions between only receptors and scaffolds, together with the rapid diffusion of receptors on the cell membrane, are sufficient for the formation and stable characteristic size of synaptic receptor domains. Our work reconciles long-term stability of synaptic receptor domains with rapid turnover and diffusion of individual receptors, and suggests novel mechanisms for a form of short-term, postsynaptic plasticity.

Hyperforin inhibits vesicular uptake of monoamines by dissipating pH gradient across synaptic vesicle membrane.

PubMed

Roz, Netta; Rehavi, Moshe

2003-06-13

Extracts of Hypericum perforatum (St. John's wort) have antidepressant properties in depressed patients and exert antidepressant-like action in laboratory animals. The phloroglucinol derivative hyperforin has become a topic of interest, as this Hypericum component is a potent inhibitor of monoamines reuptake. The molecular mechanism by which hyperforin inhibits monoamines uptake is yet unclear. In the present study we try to clarify the mechanism by which hyperforin inhibits the synaptic vesicle transport of monoamines. The pH gradient across the synaptic vesicle membrane, induced by vacuolar type H(+)-ATPase, is the major driving force for vesicular monoamines uptake and storage. We suggest that hyperforin, like the protonophore FCCP, dissipates an existing Delta pH generated by an efflux of inwardly pumped protons. Proton transport was measured by acridine orange fluorescence quenching. Adding Mg-ATP to a medium containing 130 mM KCl and synaptic vesicles caused an immediate decrease in fluorescence of acridine orange and the addition of 1 microM FCCP abolished this effect. H(+)-ATPase dependent proton pumping was inhibited by hyperforin in a dose dependent manner (IC(50) = 1.9 x 10(-7) M). Hyperforin acted similarly to the protonophore FCCP, abolishing the ATP induced fluorescence quenching (IC(50) = 4.3 x 10(-7) M). Hyperforin and FCCP had similar potencies for inhibiting rat brain synaptosomal uptake of [3H]monoamines as well as vesicular monoamine uptake. The efflux of [3H]5HT from synaptic vesicles was sensitive to both drugs, thus 50% of preloaded [3H]5HT was released in the presence of 2.1 x 10(-7) M FCCP and 4 x 10(-7) M hyperforin. The effect of hyperforin on the pH gradient in synaptic vesicle membrane may explain its inhibitory effect on monoamines uptake, but could only partially explain its antidepressant properties.

A model for studying the energetics of sustained high frequency firing

PubMed Central

Morris, Catherine E.

2018-01-01

Regulating membrane potential and synaptic function contributes significantly to the energetic costs of brain signaling, but the relative costs of action potentials (APs) and synaptic transmission during high-frequency firing are unknown. The continuous high-frequency (200-600Hz) electric organ discharge (EOD) of Eigenmannia, a weakly electric fish, underlies its electrosensing and communication. EODs reflect APs fired by the muscle-derived electrocytes of the electric organ (EO). Cholinergic synapses at the excitable posterior membranes of the elongated electrocytes control AP frequency. Based on whole-fish O2 consumption, ATP demand per EOD-linked AP increases exponentially with AP frequency. Continual EOD-AP generation implies first, that ion homeostatic processes reliably counteract any dissipation of posterior membrane ENa and EK and second that high frequency synaptic activation is reliably supported. Both of these processes require energy. To facilitate an exploration of the expected energy demands of each, we modify a previous excitability model and include synaptic currents able to drive APs at frequencies as high as 600 Hz. Synaptic stimuli are modeled as pulsatile cation conductance changes, with or without a small (sustained) background conductance. Over the full species range of EOD frequencies (200–600 Hz) we calculate frequency-dependent “Na+-entry budgets” for an electrocyte AP as a surrogate for required 3Na+/2K+-ATPase activity. We find that the cost per AP of maintaining constant-amplitude APs increases nonlinearly with frequency, whereas the cost per AP for synaptic input current is essentially constant. This predicts that Na+ channel density should correlate positively with EOD frequency, whereas AChR density should be the same across fish. Importantly, calculated costs (inferred from Na+-entry through Nav and ACh channels) for electrocyte APs as frequencies rise are much less than expected from published whole-fish EOD-linked O2 consumption. For APs at increasingly high frequencies, we suggest that EOD-related costs external to electrocytes (including packaging of synaptic transmitter) substantially exceed the direct cost of electrocyte ion homeostasis. PMID:29708986

Cocaine sensitization increases subthreshold activity in dopamine neurons from the ventral tegmental area.

PubMed

Arencibia-Albite, Francisco; Vázquez-Torres, Rafael; Jiménez-Rivera, Carlos A

2017-02-01

The progressive escalation of psychomotor responses that results from repeated cocaine administration is termed sensitization. This phenomenon alters the intrinsic properties of dopamine (DA) neurons from the ventral tegmental area (VTA), leading to enhanced dopaminergic transmission in the mesocorticolimbic network. The mechanisms underlying this augmented excitation are nonetheless poorly understood. DA neurons display the hyperpolarization-activated, nonselective cation current, dubbed I h We recently demonstrated that I h and membrane capacitance are substantially reduced in VTA DA cells from cocaine-sensitized rats. The present study shows that 7 days of cocaine withdrawal did not normalize I h and capacitance. In cells from cocaine-sensitized animals, the amplitude of excitatory synaptic potentials, at -70 mV, was ∼39% larger in contrast to controls. Raise and decay phases of the synaptic signal were faster under cocaine, a result associated with a reduced membrane time constant. Synaptic summation was paradoxically elevated by cocaine exposure, as it consisted of a significantly reduced summation indexed but a considerably increased depolarization. These effects are at least a consequence of the reduced capacitance. I h attenuation is unlikely to explain such observations, since at -70 mV, no statistical differences exist in I h or input resistance. The neuronal shrinkage associated with a diminished capacitance may help to understand two fundamental elements of drug addiction: incentive sensitization and negative emotional states. A reduced cell size may lead to substantial enhancement of cue-triggered bursting, which underlies drug craving and reward anticipation, whereas it could also result in DA depletion, as smaller neurons might express low levels of tyrosine hydroxylase. This work uses a new approach that directly extracts important biophysical parameters from alpha function-evoked synaptic potentials. Two of these parameters are the cell membrane capacitance (C m ) and rate at any time point of the synaptic waveform. The use of such methodology shows that cocaine sensitization reduces C m and increases the speed of synaptic signaling. Paradoxically, although synaptic potentials show a faster decay under cocaine their temporal summation is substantially elevated. Copyright © 2017 the American Physiological Society.

Ex vivo effects of ibogaine on the activity of antioxidative enzymes in human erythrocytes.

PubMed

Nikolić-Kokić, Aleksandra; Oreščanin-Dušić, Zorana; Spasojević, Ivan; Slavić, Marija; Mijušković, Ana; Paškulin, Roman; Miljević, Čedo; Spasić, Mihajlo B; Blagojević, Duško P

2015-04-22

Ibogaine is a naturally occurring alkaloid with psychotropic and metabotropic effects, derived from the bark of the root of the West African Tabernanthe iboga plant. The tribes of Kongo basin have been using iboga as a stimulant, for medicinal purposes, and in rite of passage ceremonies, for centuries. Besides, it has been found that this drug has anti-addictive effects. Previous studies have demonstrated that ibogaine changed the quantity of ATP and energy related enzymes as well as the activity of antioxidant enzymes in cells thus altering redox equilibrium in a time manner. In this work, the mechanism of its action was further studied by measuring the effects of ibogaine in human erythrocytes in vitro on ATP liberation, membrane fluidity and antioxidant enzymes activity. Heparinized human blood samples were incubated with ibogaine (10 and 20 μM) at 37°C for 1h. Blood plasma was separated by centrifugation and the levels of ATP and uric acid were measured 10 min after the addition of ibogaine using standard kits. The activity of copper-zinc superoxide dismutase (SOD1), catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione reductase (GR) were measured in erythrocytes after incubation period. The stability of SOD1 activity was further tested through in vitro incubation with H2O2 and scanning of its electrophoretic profiles. Membrane fluidity was determined using an electron paramagnetic resonance spin-labelling method. Results showed that ibogaine treatment of erythrocytes in vitro increased ATP concentration in the blood plasma without changes in neither erythrocytes membrane fluidity nor uric acid concentration. Ibogaine also increased SOD1 activity in erythrocytes at both doses applied here. Treatment with 20 μM also elevated GR activity after in vitro incubation at 37°C. Electrophoretic profiles revealed that incubation with ibogaine mitigates H2O2 mediated suppression of SOD1 activity. Some of the effects of ibogaine seem to be mediated through its influence on energy metabolism, redox active processes and the effects of discrete fluctuations of individual reactive oxygen species on different levels of enzyme activities. Overall, ibogaine acts as a pro-antioxidant by increasing activity of antioxidative enzymes and as an adaptagene in oxidative distress. Copyright © 2015. Published by Elsevier Ireland Ltd.

Regulation of neuronal communication by G protein-coupled receptors.

PubMed

Huang, Yunhong; Thathiah, Amantha

2015-06-22

Neuronal communication plays an essential role in the propagation of information in the brain and requires a precisely orchestrated connectivity between neurons. Synaptic transmission is the mechanism through which neurons communicate with each other. It is a strictly regulated process which involves membrane depolarization, the cellular exocytosis machinery, neurotransmitter release from synaptic vesicles into the synaptic cleft, and the interaction between ion channels, G protein-coupled receptors (GPCRs), and downstream effector molecules. The focus of this review is to explore the role of GPCRs and G protein-signaling in neurotransmission, to highlight the function of GPCRs, which are localized in both presynaptic and postsynaptic membrane terminals, in regulation of intrasynaptic and intersynaptic communication, and to discuss the involvement of astrocytic GPCRs in the regulation of neuronal communication. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Mediterranean-style diet effect on the structural properties of the erythrocyte cell membrane of hypertensive patients: the Prevencion con Dieta Mediterranea Study.

PubMed

Barceló, Francisca; Perona, Javier S; Prades, Jesús; Funari, Sérgio S; Gomez-Gracia, Enrique; Conde, Manuel; Estruch, Ramon; Ruiz-Gutiérrez, Valentina

2009-11-01

A currently ongoing randomized trial has revealed that the Mediterranean diet, rich in virgin olive oil or nuts, reduces systolic blood pressure in high-risk cardiovascular patients. Here, we present a structural substudy to assess the effect of a Mediterranean-style diet supplemented with nuts or virgin olive oil on erythrocyte membrane properties in 36 hypertensive participants after 1 year of intervention. Erythrocyte membrane lipid composition, structural properties of reconstituted erythrocyte membranes, and serum concentrations of inflammatory markers are reported. After the intervention, the membrane cholesterol content decreased, whereas that of phospholipids increased in all of the dietary groups; the diminishing cholesterol:phospholipid ratio could be associated with an increase in the membrane fluidity. Moreover, reconstituted membranes from the nuts and virgin olive oil groups showed a higher propensity to form a nonlamellar inverted hexagonal phase structure that was related to an increase in phosphatidylethanolamine lipid class. These data suggest that the Mediterranean-style diet affects the lipid metabolism that is altered in hypertensive patients, influencing the structural membrane properties. The erythrocyte membrane modulation described provides insight in the structural bases underlying the beneficial effect of a Mediterranean-style diet in hypertensive subjects.

Biomimetic interfaces based on S-layer proteins, lipid membranes and functional biomolecules

PubMed Central

Schuster, Bernhard; Sleytr, Uwe B.

2014-01-01

Designing and utilization of biomimetic membrane systems generated by bottom-up processes is a rapidly growing scientific and engineering field. Elucidation of the supramolecular construction principle of archaeal cell envelopes composed of S-layer stabilized lipid membranes led to new strategies for generating highly stable functional lipid membranes at meso- and macroscopic scale. In this review, we provide a state-of-the-art survey of how S-layer proteins, lipids and polymers may be used as basic building blocks for the assembly of S-layer-supported lipid membranes. These biomimetic membrane systems are distinguished by a nanopatterned fluidity, enhanced stability and longevity and, thus, provide a dedicated reconstitution matrix for membrane-active peptides and transmembrane proteins. Exciting areas in the (lab-on-a-) biochip technology are combining composite S-layer membrane systems involving specific membrane functions with the silicon world. Thus, it might become possible to create artificial noses or tongues, where many receptor proteins have to be exposed and read out simultaneously. Moreover, S-layer-coated liposomes and emulsomes copying virus envelopes constitute promising nanoformulations for the production of novel targeting, delivery, encapsulation and imaging systems. PMID:24812051

Rationale and clinical data supporting nutritional intervention in Alzheimer's disease.

PubMed

Engelborghs, S; Gilles, C; Ivanoiu, A; Vandewoude, M

2014-01-01

Adequate nutrition plays an important role in the maintenance of cognitive function, particularly during aging. Malnutrition is amongst the risk factors for developing mild cognitive impairment (MCI) and Alzheimer's disease (AD). Epidemiological studies have associated deficiencies in some nutrients with a higher risk of cognitive dysfunction and/or AD. Cognitive decline in AD is correlated with synaptic loss and many of the components required to maintain optimal synaptic function are derived from dietary sources. As synapses are part of the neuronal membrane and are continuously being remodelled, the availability of sufficient levels of nutritional precursors (mainly uridine monophosphate, choline and omega-3 fatty acids) to make the phospholipids required to build neuronal membranes may have beneficial effects on synaptic degeneration in AD. In addition, B-vitamins, phospholipids and other micronutrients act as cofactors to enhance the supply of precursors required to make neuronal membranes and synapses. Despite this, no randomized controlled trial has hitherto provided evidence that any single nutrient has a beneficial effect on cognition or lowers the risk for AD. However, a multi-target approach using combinations of (micro)nutrients might have beneficial effects on cognitive function in neurodegenerative brain disorders like AD leading to synaptic degeneration. Here we review the clinical evidence for supplementation, based on a multi-target approach with a focus on key nutrients with a proposed role in synaptic dysfunction. Based on preclinical evidence, a nutrient mixture, Souvenaid(®) (Nutricia N.V., Zoetermeer, The Netherlands) was developed. Clinical trials with Souvenaid(®) have shown improved memory performance in patients with mild AD. Further clinical trials to evaluate the effects of nutritional intervention in MCI and early dementia due to AD are on-going.

Differential Regulation of Synaptic Vesicle Tethering and Docking by UNC-18 and TOM-1.

PubMed

Gracheva, Elena O; Maryon, Ed B; Berthelot-Grosjean, Martine; Richmond, Janet E

2010-01-01

The assembly of SNARE complexes between syntaxin, SNAP-25 and synaptobrevin is required to prime synaptic vesicles for fusion. Since Munc18 and tomosyn compete for syntaxin interactions, the interplay between these proteins is predicted to be important in regulating synaptic transmission. We explored this possibility, by examining genetic interactions between C. elegans unc-18(Munc18), unc-64(syntaxin) and tom-1(tomosyn). We have previously demonstrated that unc-18 mutants have reduced synaptic transmission, whereas tom-1 mutants exhibit enhanced release. Here we show that the unc-18 mutant release defect is associated with loss of two morphologically distinct vesicle pools; those tethered within 25 nm of the plasma membrane and those docked with the plasma membrane. In contrast, priming defective unc-13 mutants accumulate tethered vesicles, while docked vesicles are greatly reduced, indicating tethering is UNC-18-dependent and occurs in the absence of priming. C. elegans unc-64 mutants phenocopy unc-18 mutants, losing both tethered and docked vesicles, whereas overexpression of open syntaxin preferentially increases vesicle docking, suggesting UNC-18/closed syntaxin interactions are responsible for vesicle tethering. Given the competition between vertebrate tomosyn and Munc18, for syntaxin binding, we hypothesized that C. elegans TOM-1 may inhibit both UNC-18-dependent vesicle targeting steps. Consistent with this hypothesis, tom-1 mutants exhibit enhanced UNC-18 plasma membrane localization and a concomitant increase in both tethered and docked synaptic vesicles. Furthermore, in tom-1;unc-18 double mutants the docked, primed vesicle pool is preferentially rescued relative to unc-18 single mutants. Together these data provide evidence for the differential regulation of two vesicle targeting steps by UNC-18 and TOM-1 through competitive interactions with syntaxin.

Effect of presynaptic membrane potential on electrical vs. chemical synaptic transmission

PubMed Central

Evans, Colin G.; Ludwar, Bjoern Ch.; Kang, Timothy

2011-01-01

The growing realization that electrical coupling is present in the mammalian brain has sparked renewed interest in determining its functional significance and contrasting it with chemical transmission. One question of interest is whether the two types of transmission can be selectively regulated, e.g., if a cell makes both types of connections can electrical transmission occur in the absence of chemical transmission? We explore this issue in an experimentally advantageous preparation. B21, the neuron we study, is an Aplysia sensory neuron involved in feeding that makes electrical and chemical connections with other identified cells. Previously we demonstrated that chemical synaptic transmission is membrane potential dependent. It occurs when B21 is centrally depolarized prior to and during peripheral activation, but does not occur if B21 is peripherally activated at its resting membrane potential. In this article we study effects of membrane potential on electrical transmission. We demonstrate that maximal potentiation occurs in different voltage ranges for the two types of transmission, with potentiation of electrical transmission occurring at more hyperpolarized potentials (i.e., requiring less central depolarization). Furthermore, we describe a physiologically relevant type of stimulus that induces both spiking and an envelope of depolarization in the somatic region of B21. This depolarization does not induce functional chemical synaptic transmission but is comparable to the depolarization needed to maximally potentiate electrical transmission. In this study we therefore characterize a situation in which electrical and chemical transmission can be selectively controlled by membrane potential. PMID:21593394

The formation of ordered nanoclusters controls cadherin anchoring to actin and cell–cell contact fluidity

PubMed Central

Strale, Pierre-Olivier; Duchesne, Laurence; Peyret, Grégoire; Montel, Lorraine; Nguyen, Thao; Png, Evelyn; Tampé, Robert; Troyanovsky, Sergey; Hénon, Sylvie; Ladoux, Benoit

2015-01-01

Oligomerization of cadherins could provide the stability to ensure tissue cohesion. Cadherins mediate cell–cell adhesion by forming trans-interactions. They form cis-interactions whose role could be essential to stabilize intercellular junctions by shifting cadherin clusters from a fluid to an ordered phase. However, no evidence has been provided so far for cadherin oligomerization in cellulo and for its impact on cell–cell contact stability. Visualizing single cadherins within cell membrane at a nanometric resolution, we show that E-cadherins arrange in ordered clusters, providing the first demonstration of the existence of oligomeric cadherins at cell–cell contacts. Studying the consequences of the disruption of the cis-interface, we show that it is not essential for adherens junction formation. Its disruption, however, increased the mobility of junctional E-cadherin. This destabilization strongly affected E-cadherin anchoring to actin and cell–cell rearrangement during collective cell migration, indicating that the formation of oligomeric clusters controls the anchoring of cadherin to actin and cell–cell contact fluidity. PMID:26195669

In vivo collective cell migration requires an LPAR2-dependent increase in tissue fluidity

PubMed Central

Kuriyama, Sei; Theveneau, Eric; Benedetto, Alexandre; Parsons, Maddy; Tanaka, Masamitsu; Charras, Guillaume; Kabla, Alexandre

2014-01-01

Collective cell migration (CCM) and epithelial–mesenchymal transition (EMT) are common to cancer and morphogenesis, and are often considered to be mutually exclusive in spite of the fact that many cancer and embryonic cells that have gone through EMT still cooperate to migrate collectively. Here we use neural crest (NC) cells to address the question of how cells that have down-regulated cell–cell adhesions can migrate collectively. NC cell dissociation relies on a qualitative and quantitative change of the cadherin repertoire. We found that the level of cell–cell adhesion is precisely regulated by internalization of N-cadherin downstream of lysophosphatidic acid (LPA) receptor 2. Rather than promoting the generation of single, fully mesenchymal cells, this reduction of membrane N-cadherin only triggers a partial mesenchymal phenotype. This intermediate phenotype is characterized by an increase in tissue fluidity akin to a solid-like–to–fluid-like transition. This change of plasticity allows cells to migrate under physical constraints without abolishing cell cooperation required for collectiveness. PMID:25002680

Increased Training Intensity Induces Proper Membrane Localization of Actin Remodeling Proteins in the Hippocampus Preventing Cognitive Deficits: Implications for Fragile X Syndrome.

PubMed

Martinez, L A; Tejada-Simon, Maria Victoria

2018-06-01

Behavioral intervention therapy has proven beneficial in the treatment of autism and intellectual disabilities (ID), raising the possibility of certain changes in molecular mechanisms activated by these interventions that may promote learning. Fragile X syndrome (FXS) is a neurodevelopmental disorder characterized by autistic features and intellectual disability and can serve as a model to examine mechanisms that promote learning. FXS results from mutations in the fragile X mental retardation 1 gene (Fmr1) that prevents expression of the Fmr1 protein (FMRP), a messenger RNA (mRNA) translation regulator at synapses. Among many other functions, FMRP organizes a complex with the actin cytoskeleton-regulating small Rho GTPase Rac1. As in humans, Fmr1 KO mice lacking FMRP display autistic-like behaviors and deformities of actin-rich synaptic structures in addition to impaired hippocampal learning and synaptic plasticity. These features have been previously linked to proper function of actin remodeling proteins that includes Rac1. An important step in Rac1 activation and function is its translocation to the membrane, where it can influence synaptic actin cytoskeleton remodeling during hippocampus-dependent learning. Herein, we report that Fmr1 KO mouse hippocampus exhibits increased levels of membrane-bound Rac1, which may prevent proper learning-induced synaptic changes. We also determine that increasing training intensity during fear conditioning (FC) training restores contextual memory in Fmr1 KO mice and reduces membrane-bound Rac1 in Fmr1 KO hippocampus. Increased training intensity also results in normalized long-term potentiation in hippocampal slices taken from Fmr1 KO mice. These results point to interventional treatments providing new therapeutic options for FXS-related cognitive dysfunction.

Synaptic calcium regulation in hair cells of the chicken basilar papilla.

PubMed

Im, Gi Jung; Moskowitz, Howard S; Lehar, Mohammed; Hiel, Hakim; Fuchs, Paul Albert

2014-12-10

Cholinergic inhibition of hair cells occurs by activation of calcium-dependent potassium channels. A near-membrane postsynaptic cistern has been proposed to serve as a store from which calcium is released to supplement influx through the ionotropic ACh receptor. However, the time and voltage dependence of acetylcholine (ACh)-evoked potassium currents reveal a more complex relationship between calcium entry and release from stores. The present work uses voltage steps to regulate calcium influx during the application of ACh to hair cells in the chicken basilar papilla. When calcium influx was terminated at positive membrane potential, the ACh-evoked potassium current decayed exponentially over ∼100 ms. However, at negative membrane potentials, this current exhibited a secondary rise in amplitude that could be eliminated by dihydropyridine block of the voltage-gated calcium channels of the hair cell. Calcium entering through voltage-gated channels may transit through the postsynaptic cistern, since ryanodine and sarcoendoplasmic reticulum calcium-ATPase blockers altered the time course and magnitude of this secondary, voltage-dependent contribution to ACh-evoked potassium current. Serial section electron microscopy showed that efferent and afferent synaptic structures are juxtaposed, supporting the possibility that voltage-gated influx at afferent ribbon synapses influences calcium homeostasis during long-lasting cholinergic inhibition. In contrast, spontaneous postsynaptic currents ("minis") resulting from stochastic efferent release of ACh were made briefer by ryanodine, supporting the hypothesis that the synaptic cistern serves primarily as a calcium barrier and sink during low-level synaptic activity. Hypolemmal cisterns such as that at the efferent synapse of the hair cell can play a dynamic role in segregating near-membrane calcium for short-term and long-term signaling. Copyright © 2014 the authors 0270-6474/14/3416688-10$15.00/0.

Synaptic Calcium Regulation in Hair Cells of the Chicken Basilar Papilla

PubMed Central

Im, Gi Jung; Moskowitz, Howard S.; Lehar, Mohammed; Hiel, Hakim

2014-01-01

Cholinergic inhibition of hair cells occurs by activation of calcium-dependent potassium channels. A near-membrane postsynaptic cistern has been proposed to serve as a store from which calcium is released to supplement influx through the ionotropic ACh receptor. However, the time and voltage dependence of acetylcholine (ACh)-evoked potassium currents reveal a more complex relationship between calcium entry and release from stores. The present work uses voltage steps to regulate calcium influx during the application of ACh to hair cells in the chicken basilar papilla. When calcium influx was terminated at positive membrane potential, the ACh-evoked potassium current decayed exponentially over ∼100 ms. However, at negative membrane potentials, this current exhibited a secondary rise in amplitude that could be eliminated by dihydropyridine block of the voltage-gated calcium channels of the hair cell. Calcium entering through voltage-gated channels may transit through the postsynaptic cistern, since ryanodine and sarcoendoplasmic reticulum calcium-ATPase blockers altered the time course and magnitude of this secondary, voltage-dependent contribution to ACh-evoked potassium current. Serial section electron microscopy showed that efferent and afferent synaptic structures are juxtaposed, supporting the possibility that voltage-gated influx at afferent ribbon synapses influences calcium homeostasis during long-lasting cholinergic inhibition. In contrast, spontaneous postsynaptic currents (“minis”) resulting from stochastic efferent release of ACh were made briefer by ryanodine, supporting the hypothesis that the synaptic cistern serves primarily as a calcium barrier and sink during low-level synaptic activity. Hypolemmal cisterns such as that at the efferent synapse of the hair cell can play a dynamic role in segregating near-membrane calcium for short-term and long-term signaling. PMID:25505321

Association of Membrane Rafts and Postsynaptic Density: Proteomics, Biochemical, and Ultrastructural Analyses

PubMed Central

Suzuki, Tatsuo; Zhang, Jingping; Miyazawa, Shoko; Liu, Qian; Farzan, Michael R.; Yao, Wei-Dong

2011-01-01

Postsynaptic membrane rafts are believed to play important roles in synaptic signaling, plasticity, and maintenance. However, their molecular identities remain elusive. Further, how they interact with the well-established signaling specialization, the postsynaptic density (PSD), is poorly understood. We previously detected a number of conventional PSD proteins in detergent-resistant membranes (DRMs). Here, we have performed LC-MS/MS (liquid chromatography coupled with tandem mass spectrometry) analyses on postsynaptic membrane rafts and PSDs. Our comparative analysis identified an extensive overlap of protein components in the two structures. This overlapping could be explained, at least partly, by a physical association of the two structures. Meanwhile, a significant number of proteins displayed biased distributions to either rafts or PSDs, suggesting distinct roles for the two postsynaptic specializations. Using biochemical and electron microscopic methods, we directly detected membrane raft-PSD complexes. In vitro reconstitution experiments indicated that the formation of raft-PSD complexes was not due to the artificial reconstruction of once-solubilized membrane components and PSD structures, supporting that these complexes occurred in vivo. Taking together, our results provide evidence that postsynaptic membrane rafts and PSDs may be physically associated. Such association could be important in postsynaptic signal integration, synaptic function, and maintenance. PMID:21797867

Model lipid bilayers mimic non-specific interactions of gold nanoparticles with macrophage plasma membranes.

PubMed

Montis, Costanza; Generini, Viola; Boccalini, Giulia; Bergese, Paolo; Bani, Daniele; Berti, Debora

2018-04-15

Understanding the interaction between nanomaterials and biological interfaces is a key unmet goal that still hampers clinical translation of nanomedicine. Here we investigate and compare non-specific interaction of gold nanoparticles (AuNPs) with synthetic lipid and wild type macrophage membranes. A comprehensive data set was generated by systematically varying the structural and physicochemical properties of the AuNPs (size, shape, charge, surface functionalization) and of the synthetic membranes (composition, fluidity, bending properties and surface charge), which allowed to unveil the matching conditions for the interaction of the AuNPs with macrophage plasma membranes in vitro. This effort directly proved for the first time that synthetic bilayers can be set to mimic and predict with high fidelity key aspects of nanoparticle interaction with macrophage eukaryotic plasma membranes. It then allowed to model the experimental observations according to classical interface thermodynamics and in turn determine the paramount role played by non-specific contributions, primarily electrostatic, Van der Waals and bending energy, in driving nanoparticle-plasma membrane interactions. Copyright © 2018 Elsevier Inc. All rights reserved.

Complementary probes reveal that phosphatidylserine is required for the proper transbilayer distribution of cholesterol.

PubMed

Maekawa, Masashi; Fairn, Gregory D

2015-04-01

Cholesterol is an essential component of metazoan cellular membranes and it helps to maintain the structural integrity and fluidity of the plasma membrane. Here, we developed a cholesterol biosensor, termed D4H, based on the fourth domain of Clostridium perfringens theta-toxin, which recognizes cholesterol in the cytosolic leaflet of the plasma membrane and organelles. The D4H probe disassociates from the plasma membrane upon cholesterol extraction and after perturbations in cellular cholesterol trafficking. When used in combination with a recombinant version of the biosensor, we show that plasmalemmal phosphatidylserine is essential for retaining cholesterol in the cytosolic leaflet of the plasma membrane. In vitro experiments reveal that 1-stearoy-2-oleoyl phosphatidylserine can induce phase separation in cholesterol-containing lipid bilayers and shield cholesterol from cholesterol oxidase. Finally, the altered transbilayer distribution of cholesterol causes flotillin-1 to relocalize to endocytic organelles. This probe should be useful in the future to study pools of cholesterol in the cytosolic leaflet of the plasma membrane and organelles. © 2015. Published by The Company of Biologists Ltd.

Comparison Actin- and Glass-Supported Phospholipid Bilayer Diffusion Coefficients

PubMed Central

Sterling, Sarah M.; Dawes, Ryan; Allgeyer, Edward S.; Ashworth, Sharon L.; Neivandt, David J.

2015-01-01

The formation of biomimetic lipid membranes has the potential to provide insights into cellular lipid membrane dynamics. The construction of such membranes necessitates not only the utilization of appropriate lipids, but also physiologically relevant substrate/support materials. The substrate materials employed have been shown to have demonstrable effects on the behavior of the overlying lipid membrane, and thus must be studied before use as a model cushion support. To our knowledge, we report the formation and investigation of a novel actin protein-supported lipid membrane. Specifically, inner leaflet lateral mobility of globular actin-supported DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) bilayers, deposited via the Langmuir-Blodgett/Langmuir Schaefer methodology, was investigated by z-scan fluorescence correlation spectroscopy across a temperature range of 20–44°C. The actin substrate was found to decrease the diffusion coefficient when compared to an identical membrane supported on glass. The depression of the diffusion coefficient occurred across all measured temperatures. These results indicated that the actin substrate exerted a direct effect on the fluidity of the lipid membrane and highlighted the fact that the choice of substrate/support is critical in studies of model lipid membranes. PMID:25902434

Vesicle Fusion Observed by Content Transfer across a Tethered Lipid Bilayer

PubMed Central

Rawle, Robert J.; van Lengerich, Bettina; Chung, Minsub; Bendix, Poul Martin; Boxer, Steven G.

2011-01-01

Synaptic transmission is achieved by exocytosis of small, synaptic vesicles containing neurotransmitters across the plasma membrane. Here, we use a DNA-tethered freestanding bilayer as a target architecture that allows observation of content transfer of individual vesicles across the tethered planar bilayer. Tethering and fusion are mediated by hybridization of complementary DNA-lipid conjugates inserted into the two membranes, and content transfer is monitored by the dequenching of an aqueous content dye. By analyzing the diffusion profile of the aqueous dye after vesicle fusion, we are able to distinguish content transfer across the tethered bilayer patch from vesicle leakage above the patch. PMID:22004762

Synaptic transmission in the superior cervical ganglion of the cat after reinnervation by vagus fibres

PubMed Central

Ceccarelli, B.; Clementi, F.; Mantegazza, P.

1971-01-01

1. A vagus-sympathetic anastomosis was performed in the cat by connecting end to end the cranial trunk of the vagus to the cranial end of the cervical sympathetic trunk, both severed under the ganglia. 2. Forty to sixty days after the anastomosis, the ocular signs of sympathetic paralysis (such as myosis and prolapse of the nictitating membrane) which had developed shortly after the operation, had completely disappeared, thus suggesting the recovery of synaptic transmission in the ganglion. In case of plain preganglionic denervation after the same period the ocular signs of cervical sympathetic paralysis were still present. 3. Contraction of the nictitating membrane could be induced by electrical stimulation of both the vagus preanastomotic and the sympathetic postanastomotic—preganglionic trunks. Ganglionic blocking agents induced the blockade of the `new' ganglionic synaptic function, while nicotine and pilocarpine provoked a marked contraction of the nictitating membrane. 4. Electron microscopy showed that the preganglionic regeneration of vagus fibers resulted in the formation of new synapses, mainly of axodendritic type, identical to normal ganglionic synapses. Moreover, after cutting the preanastomotic trunk of the vagus, these new ganglionic presynaptic profiles degenerated, thus proving their vagal origin. 5. During restoration of the synaptic contacts readjustment of dendritic tips occurred. ImagesText-fig. 2Fig. 9Fig. 10Fig. 11Fig. 12Fig. 13Fig. 16Fig. 17Fig. 14Fig. 15Fig. 1Fig. 2Fig. 3Fig. 4Fig. 5Fig. 7Fig. 8 PMID:4326851

Proteomic Analysis of Rat Hippocampus under Simulated Microgravity

NASA Astrophysics Data System (ADS)

Wang, Yun; Li, Yujuan; Zhang, Yongqian; Liu, Yahui; Deng, Yulin

It has been found that microgravity may lead to impairments in cognitive functions performed by CNS. However, the exact mechanism of effects of microgravity on the learning and memory function in animal nervous system is not elucidated yet. Brain function is mainly mediated by membrane proteins and their dysfunction causes degeneration of the learning and memory. To induce simulated microgravity, the rat tail suspension model was established. Comparative O (18) labeling quantitative proteomic strategy was applied to detect the differentially expressed proteins in rat brain hippocampus. The proteins in membrane fraction from rat hippocampus were digested by trypsin and then the peptides were separated by off-gel for the first dimension with 24 wells device encompassing the pH range of 3 - 10. An off-gel fraction was subjected into LC-ESI-QTOF in triplicate. Preliminary results showed that nearly 77% of the peptides identified were specific to one fraction. 676 proteins were identified among which 108 proteins were found differentially expressed under simulated microgravity. Using the KOBAS server, many enriched pathways, such as metabolic pathway, synaptic vesicle cycle, endocytosis, calcium signaling pathway, and SNAREs pathway were identified. Furthermore, it has been found that neurotransmitter released by Ca (2+) -triggered synaptic vesicles fusion may play key role in neural function. Rab 3A might inhibit the membrane fusion and neurotransmitter release. The protein alteration of the synaptic vesicle cycle may further explain the effects of microgravity on learning and memory function in rats. Key words: Microgravity; proteomics; synaptic vesicle; O (18) ({}) -labeling

Cardiolipin effects on membrane structure and dynamics.

PubMed

Unsay, Joseph D; Cosentino, Katia; Subburaj, Yamunadevi; García-Sáez, Ana J

2013-12-23

Cardiolipin (CL) is a lipid with unique properties solely found in membranes generating electrochemical potential. It contains four acyl chains and tends to form nonlamellar structures, which are believed to play a key role in membrane structure and function. Indeed, CL alterations have been linked to disorders such as Barth syndrome and Parkinson's disease. However, the molecular effects of CL on membrane organization remain poorly understood. Here, we investigated the structure and physical properties of CL-containing membranes using confocal microscopy, fluorescence correlation spectroscopy, and atomic force microscopy. We found that the fluidity of the lipid bilayer increased and its mechanical stability decreased with CL concentration, indicating that CL decreases the packing of the membrane. Although the presence of up to 20% CL gave rise to flat, stable bilayers, the inclusion of 5% CL promoted the formation of flowerlike domains that grew with time. Surprisingly, we often observed two membrane-piercing events in atomic force spectroscopy experiments with CL-containing membranes. Similar behavior was observed with a lipid mixture mimicking the mitochondrial outer membrane composition. This suggests that CL promotes the formation of membrane areas with apposed double bilayers or nonlamellar structures, similar to those proposed for mitochondrial contact sites. All together, we show that CL induces membrane alterations that support the role of CL in facilitating bilayer structure remodeling, deformation, and permeabilization.

Electric Dipole Theory of Chemical Synaptic Transmission

PubMed Central

Wei, Ling Y.

1968-01-01

In this paper we propose that chemicals such as acetylcholine are electric dipoles which when oriented and arranged in a large array could produce an electric field strong enough to drive positive ions over the junction barrier of the post-synaptic membrane and thus initiate excitation or produce depolarization. This theory is able to explain a great number of facts such as cleft size, synaptic delay, nonregeneration, subthreshold integration, facilitation with repetition, and the calcium and magnesium effects. It also shows why and how acetylcholine could act as excitatory or inhibitory transmitters under different circumstances. Our conclusion is that the nature of synaptic transmission is essentially electrical, be it mediated by electrical or chemical transmitters. PMID:4296121

Degradation of neurotensin by rat brain synaptic membranes: involvement of a thermolysin-like metalloendopeptidase (enkephalinase), angiotensin-converting enzyme, and other unidentified peptidases.

PubMed

Checler, F; Vincent, J P; Kitabgi, P

1983-08-01

Neurotensin was inactivated by membrane-bound and soluble degrading activities present in purified preparations of rat brain synaptic membranes. Degradation products were identified by HPLC and amino acid analysis. The major points of cleavage of neurotensin were the Arg8-Arg9, Pro10-Tyr11, and Tyr11-Ile12 peptide bonds with the membrane-bound activity and the Arg8-Arg9 and Pro10-Tyr11 bonds with the soluble activity. Several lines of evidence indicated that the cleavage of the Arg8-Arg9 bond by the membrane-bound activity resulted mainly from the conversion of neurotensin1-10 to neurotensin1-8 by a dipeptidyl carboxypeptidase. In particular, captopril inhibited this cleavage with an IC50 (5.7 nM) close to its K1 (7 nM) for angiotensin-converting enzyme. Thiorphan inhibited the cleavage at the Tyr11-Ile12 bond by the membrane-bound activity with an IC50 (17 nM) similar to its K1 (4.7 nM) for enkephalinase. Both cleavages were inhibited by 1,10-phenanthroline. These and other data suggested that angiotensin-converting enzyme and a thermolysin-like metalloendopeptidase (enkephalinase) were the membrane-bound peptidases responsible for cleavages at the Arg8-Arg9 and Tyr11-Ile12 bonds, respectively. In contrast, captopril had no effect on the cleavage at the Arg8-Arg9 bond by the soluble activity, indicating that the enzyme responsible for this cleavage was different from angiotensin-converting enzyme. The cleavage at the Pro10-Tyr11 bond by both the membrane-bound and the soluble activities appeared to be catalyzed by an endopeptidase different from known brain proline endopeptidases. The possibility is discussed that the enzymes described here participate in physiological mechanisms of neurotensin inactivation at the synaptic level.

Silybum marianum oil attenuates oxidative stress and ameliorates mitochondrial dysfunction in mice treated with D-galactose

PubMed Central

Zhu, Shu Yun; Dong, Ying; Tu, Jie; Zhou, Yue; Zhou, Xing Hua; Xu, Bin

2014-01-01

Background: Silybum marianum has been used as herbal medicine for the treatment of liver disease, liver cirrhosis, and to prevent liver cancer in Europe and Asia since ancient times. Silybum marianum oil (SMO), a by-product of silymarin production, is rich in essential fatty acids, phospholipids, sterols, and vitamin E. However, it has not been very good development and use. Objective: In the present study, we used olive oil as a control to investigate the antioxidant and anti-aging effect of SMO in D-galactose (D-gal)-induced aging mice. Materials and Methods: D-gal was injected intraperitoneally (500 mg/kg body weight daily) for 7 weeks while SMO was simultaneously administered orally. The triglycerides (TRIG) and cholesterol (CHOL) levels were estimated in the serum. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), monoamine oxidase (MAO), malondialdehyde (MDA), caspase-3, and Bcl-2 were determined in the liver and brain. The activities of Na+-K+-adenosine triphosphatase (ATPase), Ca2+-Mg2+-ATPase, membrane potential (ΔΨm), and membrane fluidity of the liver mitochondrial were estimated. Results: SMO decreased levels of TRIG and CHOL in aging mice. SMO administration elevated the activities of SOD, GSH-Px, and T-AOC, which are suppressed by aging. The levels of MAO and MDA in the liver and brain were reduced by SMO administration in aging mice. Enzyme linked immunosorbent assay showed that SMO significantly decreased the concentration of caspase-3 and improved the activity of Bcl-2 in the liver and brain of aging mice. Furthermore, SMO significantly attenuated the D-gal induced liver mitochondrial dysfunction by improving the activities of Na+-K+-ATPase, Ca2+-Mg2+-ATPase, membrane potential (ΔΨm), and membrane fluidity. Conclusion: These results indicate that SMO effectively attenuated oxidative damage and improved apoptosis related factors as well as liver mitochondrial dysfunction in aging mice. PMID:24914315

Calcein release behavior from liposomal bilayer; influence of physicochemical/mechanical/structural properties of lipids.

PubMed

Maherani, Behnoush; Arab-Tehrany, Elmira; Kheirolomoom, Azadeh; Geny, David; Linder, Michel

2013-11-01

The design of the drug delivery depends upon different parameters. One of the most noticeable factors in design of the drug delivery is drug-release profile which determines the site of action, the concentration of the drug at the time of administration, the period of time that the drug must remain at a therapeutic concentration. To get a better understanding of drug release, large unilamellar liposomes containing calcein were prepared using 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1,2-palmitoyl-sn-glycero-3-phosphocholine, and a mixture of them; calcein was chosen as a model of hydrophilic drug. The calcein permeability across liposomal membrane (with different compositions) was evaluated on the basis of the first-order kinetic by spectrofluorometer. Also, the effects of liposome composition/fluidity as well as the incubation temperature/pH were investigated. Furthermore, we simulated the digestion condition in the gastrointestinal tract in humans, to mimic human gastro-duodenal digestion to monitor calcein release during the course of the digestion process. In vitro digestion model ''pH stat'' was used to systematically examine the influence of pH/enzyme on phospholipid liposomes digestion under simulated gastro-duodenal digestion. The results revealed that calcein permeates across liposomal membrane without membrane disruption. The release rate of calcein from the liposomes depends on the number and fluidity of bilayers and its mechanical/physical properties such as permeability, bending elasticity. Chemo-structural properties of drugs like as partition coefficient (Log P), H-bonding, polar surface area (PSA) are also determinative parameter in release behavior. Finally, stimulated emission depletion (STED) microscopy was used to study calcein translocation through liposomal bilayers. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Detrimental Effects of Non-Functional Spermatozoa on the Freezability of Functional Spermatozoa from Boar Ejaculate

PubMed Central

Martinez-Alborcia, Maria J.; Valverde, Anthony; Parrilla, Inmaculada; Vazquez, Juan M.; Martinez, Emilio A.; Roca, Jordi

2012-01-01

In the present study, the impact of non-functional spermatozoa on the cryopreservation success of functional boar spermatozoa was evaluated. Fifteen sperm-rich ejaculate fractions collected from five fertile boars were frozen with different proportions of induced non-functional sperm (0 –native semen sample-, 25, 50 and 75% non-functional spermatozoa). After thawing, the recovery of motile and viable spermatozoa was assessed, and the functional of the spermatozoa was evaluated from plasma membrane fluidity and intracellular reactive oxygen species (ROS) generation upon exposure to capacitation conditions. In addition, the lipid peroxidation of the plasma membrane was assessed by the indirect measurement of malondialdehyde (MDA) generation. The normalized (with respect to a native semen sample) sperm motility (assessed by CASA) and viability (cytometrically assessed after staining with Hoechst 33342, propidium iodide and fluorescein-conjugated peanut agglutinin) decreased (p<0.01) as the proportion of functional spermatozoa in the semen samples before freezing decreased, irrespective of the semen donor. However, the magnitude of the effect differed (p<0.01) among boars. Moreover, semen samples with the largest non-functional sperm subpopulation before freezing showed the highest (p<0.01) levels of MDA after thawing. The thawed viable spermatozoa of semen samples with a high proportion of non-functional spermatozoa before freezing were also functionally different from those of samples with a low proportion of non-functional spermatozoa. These differences consisted of higher (p<0.01) levels of intracellular ROS generation (assessed with 5-(and-6) chloromethyl-20,70-dichlorodihydrofluorescein diacetate acetyl ester; CM-H2DCFDA) and increased (p<0.01) membrane fluidity (assessed with Merocyanine 540). These findings indicate that non-functional spermatozoa in the semen samples before freezing negatively influence the freezability of functional spermatozoa. PMID:22567165

Temperature-dependent changes in the swimming behaviour of Tetrahymena pyriformis-NT1 and their interrelationships with electrophysiology and the state of membrane lipids.

PubMed

Connolly, J G; Brown, I D; Lee, A G; Kerkut, G A

1985-01-01

The swimming velocity and the amplitude of the helical swimming path of T. pyriformis-NT1 cells grown at 20 degrees C (Tg 20 degrees C) and 38 degrees C (Tg 38 degrees C) were monitored between 0 and 40 degrees C in the presence and absence of electric fields. Within physiological limits the swimming velocity increased and the amplitude decreased as temperature was raised. The temperature profiles of these properties were not linear, and showed discontinuities at different temperatures for the different cultures. The break points in Arrhenius plots of the resting potential, regenerative spike magnitude, repolarization time, swimming velocity and swimming amplitude are tabulated and compared. The initial breakpoints upon cooling were clustered about the breakpoints in fluorescence polarization of D.P.H. in extracted phospholipids, and around the transition temperatures estimated from the literature for the pellicular membrane of these cells. The average of the initial breakpoints on cooling was 22.9 degrees C for Tg 38 degrees C cells and 13.7 degrees C for Tg 20 degrees C cells, a shift of 9.2 degrees C. Unlike Paramecium there is no depolarizing receptor potential in Tetrahymena upon warming. It is suggested that this may be the basis of a behavioural difference between Tetrahymena and Paramecium--namely that in Tetrahymena maximum swimming velocity occurs above growth temperature whereas in Paramecium the two points coincide. Swimming velocity and resting potential were correlated with membrane fluidity within physiological limits, but for other parameters the relationship with fluidity was more complex.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of pentoxifylline on spontaneous and experimental metastasis of the mouse Neuro2a neuroblastoma.

PubMed

Amirkhosravi, A; Warnes, G; Biggerstaff, J; Malik, Z; May, K; Francis, J L

1997-07-01

Pentoxifylline (PTX) has been reported to have both direct and indirect anti-tumor effects in experimental tumor models. We studied the effect of PTX on (1) the proliferation of Neuro2a mouse neuroblastoma cells in vitro and in vivo, (2) spontaneous and experimental metastasis, (3) tumor cell membrane fluidity and (4) adhesion to a fibronectin-coated surface. PTX significantly reduced the proliferation of Neuro2a cells in vitro as determined by DNA measurement (P < 0.01) and total cell count (P < 0.02). In vivo, PTX reduced the growth of subcutaneously transplanted primary tumors in syngeneic A/J mice (P < 0.01; n = 15). All seven animals (100%) receiving intravenous tumor cells developed extensive liver metastasis. In contrast, only 1/11 (9%) of animals pre-treated with oral PTX and injected with PTX-treated cells developed liver metastases. Of five mice receiving PTX-treated cells without oral pretreatment of PTX, two out of five (40%) developed liver metastases. There was a slight, but not significant (P = 0.08) increase in both experimental and spontaneous lung metastases formation in PTX-treated animals. However, tumor nodule formation on the lung surface was inefficient. PTX also increased membrane fluidity of the Neuro2a cells and significantly decreased tumor cell adhesion to fibronectin-coated microtiter wells (P < 0.01). We conclude that PTX has a cytostatic effect on the Neuro2a mouse neuroblastoma and exerts an anti-tumor effect on liver metastases following intravenous administration of neuroblastoma cells. Whether these results are directly related to the changes in membrane properties caused by pentoxifylline remains to be established.

Cholecystokinin octa- and tetrapeptide degradation by synaptic membranes. II. Solubilization and separation of membrane-bound CCK-8 cleaving enzymes.

PubMed

Deschodt-Lanckman, M; Bui, N D; Koulischer, D; Paroutaud, P; Strosberg, A D

1983-01-01

Solubilization of rat synaptic membranes by Triton X-100, followed by DEAE-cellulose chromatography allowed the identification of different CCK-8 cleaving enzymes. The first one (in the order of elution) removed the N-terminal aspartic acid residue of CCK-8 and was active on L-aspartic acid beta naphtylamide, suggesting that a corresponded to an aminopeptidase A. Two aminopeptidases of broad specificity hydrolyzed sequentially all the peptide bonds of CCK-8 as far as the release of free tryptophan. The removal of the sulfated tyrosine residue of CCK-8 occurred at a slower rate than that of the unsulfated residue. Another peptidase converted CCK-8 into its C-terminal heptapeptide. This enzyme had a lower affinity for the sulfated octapeptide in comparison with the unsulfated form (app Km of respectively 180 and 40 muM). The CCK-7 generating proteases displayed a moderate regional variation in five rat brain areas, with the highest activity in olfactory bulbs membranes and the lowest in cerebellar membranes. This distribution followed (with a lower amplitude) that of the CCK receptors.

Neurotensin-metabolizing peptidases in rat fundus plasma membranes.

PubMed

Checler, F; Barelli, H; Kwan, C Y; Kitabgi, P; Vincent, J P

1987-08-01

The mechanisms by which neurotensin (NT) was inactivated by rat fundus plasma membranes were characterized. Primary inactivating cleavages occurred at the Arg8-Arg9, Pro10-Tyr11, and Ile12-Leu13 peptidyl bonds. Hydrolysis at the Arg8-Arg9 bond was fully abolished by the use of N-[1(R,S)-carboxy-2-phenylethyl]-alanyl-alanyl-phenylalanine-p- aminobenzoate, a result indicating the involvement at this site of a recently purified soluble metallopeptidase. Hydrolysis of the Pro10-Tyr11 bond was totally resistant to N-benzyloxycarbonyl-prolyl-prolinal and thiorphan, an observation suggesting that the peptidase responsible for this cleavage was different from proline endopeptidase and endopeptidase 24.11 and might correspond to a NT-degrading neutral metallopeptidase recently isolated from rat brain synaptic membranes. The enzyme acting at the Ile12-Leu13 bond has not yet been identified. Secondary cleavages occurring on NT degradation products were mainly generated by bestatin-sensitive aminopeptidases and post-proline dipeptidyl aminopeptidase. The content in NT-metabolizing peptidases present in rat fundus plasma membranes is compared with that previously established for purified rat brain synaptic membranes.

A review of the integrate-and-fire neuron model: II. Inhomogeneous synaptic input and network properties.

PubMed

Burkitt, A N

2006-08-01

The integrate-and-fire neuron model describes the state of a neuron in terms of its membrane potential, which is determined by the synaptic inputs and the injected current that the neuron receives. When the membrane potential reaches a threshold, an action potential (spike) is generated. This review considers the model in which the synaptic input varies periodically and is described by an inhomogeneous Poisson process, with both current and conductance synapses. The focus is on the mathematical methods that allow the output spike distribution to be analyzed, including first passage time methods and the Fokker-Planck equation. Recent interest in the response of neurons to periodic input has in part arisen from the study of stochastic resonance, which is the noise-induced enhancement of the signal-to-noise ratio. Networks of integrate-and-fire neurons behave in a wide variety of ways and have been used to model a variety of neural, physiological, and psychological phenomena. The properties of the integrate-and-fire neuron model with synaptic input described as a temporally homogeneous Poisson process are reviewed in an accompanying paper (Burkitt in Biol Cybern, 2006).

Brain cortex mitochondrial bioenergetics in synaptosomes and non-synaptic mitochondria during aging.

PubMed

Lores-Arnaiz, Silvia; Lombardi, Paulina; Karadayian, Analía G; Orgambide, Federico; Cicerchia, Daniela; Bustamante, Juanita

2016-02-01

Alterations in mitochondrial bioenergetics have been associated with brain aging. In order to evaluate the susceptibility of brain cortex synaptosomes and non-synaptic mitochondria to aging-dependent dysfunction, male Swiss mice of 3 or 17 months old were used. Mitochondrial function was evaluated by oxygen consumption, mitochondrial membrane potential and respiratory complexes activity, together with UCP-2 protein expression. Basal respiration and respiration driving proton leak were decreased by 26 and 33 % in synaptosomes from 17-months old mice, but spare respiratory capacity was not modified by aging. Succinate supported state 3 respiratory rate was decreased by 45 % in brain cortex non-synaptic mitochondria from 17-month-old mice, as compared with young animals, but respiratory control was not affected. Synaptosomal mitochondria would be susceptible to undergo calcium-induced depolarization in 17 months-old mice, while non-synaptic mitochondria would not be affected by calcium overload. UCP-2 was significantly up-regulated in both synaptosomal and submitochondrial membranes from 17-months old mice, compared to young animals. UCP-2 upregulation seems to be a possible mechanism by which mitochondria would be resistant to suffer oxidative damage during aging.

Cellular Effects of Perfluorinated Fatty Acids (PFDA).

DTIC Science & Technology

This is a proposal to investigate the effects of perfluorinated decanoic acid ( PFDA ) on the cell surface of liver cells and tissue. The major method...summarized as follows: (a) differentiated liver tissue culture cells in vitro do have the membrane fluidity affected by PFDA whereas undifferentiated, non...d) the effect on mobility occurs within 24 hours of exposure without further increase with time of exposure; (e) scanning EM demonstrates no gross structural abnormality of the surface as a result of the non-toxic levels of PFDA .

Fast, Temperature-Sensitive and Clathrin-Independent Endocytosis at Central Synapses.

PubMed

Delvendahl, Igor; Vyleta, Nicholas P; von Gersdorff, Henrique; Hallermann, Stefan

2016-05-04

The fusion of neurotransmitter-filled vesicles during synaptic transmission is balanced by endocytotic membrane retrieval. Despite extensive research, the speed and mechanisms of synaptic vesicle endocytosis have remained controversial. Here, we establish low-noise time-resolved membrane capacitance measurements that allow monitoring changes in surface membrane area elicited by single action potentials and stronger stimuli with high-temporal resolution at physiological temperature in individual bona-fide mature central synapses. We show that single action potentials trigger very rapid endocytosis, retrieving presynaptic membrane with a time constant of 470 ms. This fast endocytosis is independent of clathrin but mediated by dynamin and actin. In contrast, stronger stimuli evoke a slower mode of endocytosis that is clathrin, dynamin, and actin dependent. Furthermore, the speed of endocytosis is highly temperature dependent with a Q10 of ∼3.5. These results demonstrate that distinct molecular modes of endocytosis with markedly different kinetics operate at central synapses. Copyright © 2016 Elsevier Inc. All rights reserved.

Secretagogue-triggered Transfer of Membrane Proteins from Neuroendocrine Secretory Granules to Synaptic-like Microvesicles

PubMed Central

Strasser, Jane E.; Arribas, Monica; Blagoveshchenskaya, Anastasia D.; Cutler, Daniel F.

1999-01-01

The membrane proteins of all regulated secretory organelles (RSOs) recycle after exocytosis. However, the recycling of those membrane proteins that are targeted to both dense core granules (DCGs) and synaptic-like microvesicles (SLMVs) has not been addressed. Since neuroendocrine cells contain both RSOs, and the recycling routes that lead to either organelle overlap, transfer between the two pools of membrane proteins could occur during recycling. We have previously demonstrated that a chimeric protein containing the cytosolic and transmembrane domains of P-selectin coupled to horseradish peroxidase is targeted to both the DCG and the SLMV in PC12 cells. Using this chimera, we have characterized secretagogue-induced traffic in PC12 cells. After stimulation, this chimeric protein traffics from DCGs to the cell surface, internalizes into transferrin receptor (TFnR)-positive endosomes and thence to a population of secretagogue-responsive SLMVs. We therefore find a secretagogue-dependent rise in levels of HRP within SLMVs. In addition, the levels within SLMVs of the endogenous membrane protein, synaptotagmin, as well as a green fluorescent protein-tagged version of vesicle-associated membrane protein (VAMP)/synaptobrevin, also show a secretagogue-dependent increase. PMID:10436017

Silver nanoparticles induced alterations in multiple cellular targets, which are critical for drug susceptibilities and pathogenicity in fungal pathogen (Candida albicans)

PubMed Central

Radhakrishnan, Venkatraman Srinivasan; Reddy Mudiam, Mohana Krishna; Kumar, Manish; Dwivedi, Surya Prakash; Singh, Surinder Pal; Prasad, Tulika

2018-01-01

Purpose A significant increase in the incidence of fungal infections and drug resistance has been observed in the past decades due to limited availability of broad-spectrum antifungal drugs. Nanomedicines have shown significant antimicrobial potential against various drug-resistant microbes. Silver nanoparticles (AgNps) are known for their antimicrobial properties and lower host toxicity; however, for clinical applications, evaluation of their impact at cellular and molecular levels is essential. The present study aims to understand the cellular and molecular mechanisms of AgNp-induced toxicity in a common fungal pathogen, Candida albicans. Methods AgNps were synthesized by chemical reduction method and characterized using UV–visible spectroscopy, X-ray powder diffraction, transmission electron microscopy, scanning electron microscopy–energy dispersive X-ray spectroscopy, energy dispersive X-ray fluorescence, and zeta potential. The anti-Candida activity of AgNps was assessed by broth microdilution and spot assays. Effects of AgNps on cellular and molecular targets were assessed by monitoring the intracellular reactive oxygen species (ROS) production in the absence and presence of natural antioxidant, changes in surface morphology, cellular ultrastructure, membrane microenvironment, membrane fluidity, membrane ergosterol, and fatty acids. Results Spherical AgNps (10–30 nm) showed minimum inhibitory concentration (minimum concentration required to inhibit the growth of 90% of organisms) at 40 μg/mL. Our results demonstrated that AgNps induced dose-dependent intracellular ROS which exerted antifungal effects; however, even scavenging ROS by antioxidant could not offer protection from AgNp mediated killing. Treatment with AgNps altered surface morphology, cellular ultrastructure, membrane microenvironment, membrane fluidity, ergosterol content, and fatty acid composition, especially oleic acid. Conclusion To summarize, AgNps affected multiple cellular targets crucial for drug resistance and pathogenicity in the fungal cells. The study revealed new cellular targets of AgNps which include fatty acids like oleic acid, vital for hyphal morphogenesis (a pathogenic trait of Candida). Yeast to hypha transition being pivotal for virulence and biofilm formation, targeting virulence might emerge as a new paradigm for developing nano silver-based therapy for clinical applications in fungal therapeutics. PMID:29760548

Preparation of pH-sensitive anionic liposomes designed for drug delivery system (DDS) application.

PubMed

Aoki, Asami; Akaboshi, Hikaru; Ogura, Taku; Aikawa, Tatsuo; Kondo, Takeshi; Tobori, Norio; Yuasa, Makoto

2015-01-01

We prepared pH-sensitive anionic liposomes composed solely of anionic bilayer membrane components that were designed to promote efficient release of entrapped agents in response to acidic pH. The pH-sensitive anionic liposomes showed high dispersion stability at neutral pH, but the fluidity of the bilayer membrane was enhanced in an acidic environment. These liposomes were rather simple and were composed of dimyristoylphosphatidylcholine (DMPC), an anionic bilayer membrane component, and polyoxyethylene sorbitan monostearate (Tween 80). In particular, the present pH-sensitive anionic liposomes showed higher temporal stability than those of conventional DMPC/DPPC liposomes. We found that pHsensitive properties strongly depended on the molecular structure, pKa value, and amount of an incorporated anionic bilayer membrane component, such as sodium oleate (SO), dimyristoylphosphatidylserine (DMPS), or sodium β-sitosterol sulfate (SS). These results provide an opportunity to manipulate liposomal stability in a pH-dependent manner, which could lead to the formulation of a high performance drug delivery system (DDS).

Lab on a Biomembrane: Rapid prototyping and manipulation of 2D fluidic lipid bilayers circuits

PubMed Central

Ainla, Alar; Gözen, Irep; Hakonen, Bodil; Jesorka, Aldo

2013-01-01

Lipid bilayer membranes are among the most ubiquitous structures in the living world, with intricate structural features and a multitude of biological functions. It is attractive to recreate these structures in the laboratory, as this allows mimicking and studying the properties of biomembranes and their constituents, and to specifically exploit the intrinsic two-dimensional fluidity. Even though diverse strategies for membrane fabrication have been reported, the development of related applications and technologies has been hindered by the unavailability of both versatile and simple methods. Here we report a rapid prototyping technology for two-dimensional fluidic devices, based on in-situ generated circuits of phospholipid films. In this “lab on a molecularly thin membrane”, various chemical and physical operations, such as writing, erasing, functionalization, and molecular transport, can be applied to user-defined regions of a membrane circuit. This concept is an enabling technology for research on molecular membranes and their technological use. PMID:24067786

Functionalized Vesicles by Microfluidic Device.

PubMed

Vallejo, Derek; Lee, Shih-Hui; Lee, Abraham

2017-01-01

In recent years, lipid vesicles have become popular vehicles for the creation of biosensors. Vesicles can hold reaction components within a selective permeable membrane that provides an ideal environment for membrane protein biosensing elements. The lipid bilayer allows a protein to retain its native structure and function, and the membrane fluidity can allow for conformational changes and physiological interactions with target analytes. Here, we present two methods for the production of giant unilamellar vesicles (GUVs) within a microfluidic device that can be used as the basis for a biosensor. The vesicles are produced from water-in-oil-in-water (W/O/W) double emulsion templates using a nonvolatile oil phase. To create the GUVs, the oil can be removed via extraction with ethanol, or by altering the interfacial tension between the oil and carrier solution causing the oil to retract into a cap on one side of the structure, leaving behind an exposed lipid bilayer. Methods to integrate sensing elements and membrane protein pores onto the vesicles are also introduced in this work.

Changes in the Fatty Acid Profile and Phospholipid Molecular Species Composition of Human Erythrocyte Membranes after Hybrid Palm and Extra Virgin Olive Oil Supplementation.

PubMed

Pacetti, D; Gagliardi, R; Balzano, M; Frega, N G; Ojeda, M L; Borrero, M; Ruiz, A; Lucci, P

2016-07-13

This work aims to evaluate and compare, for the first time, the effects of extra virgin olive oil (EVOO) and hybrid palm oil (HPO) supplementation on the fatty acid profile and phospholipid (PL) molecular species composition of human erythrocyte membranes. Results supported the effectiveness of both HPO and EVOO supplementation (3 months, 25 mL/day) in decreasing the lipophilic index of erythrocytes with no significant differences between HPO and EVOO groups at month 3. On the other hand, the novel and rapid ultraperformance liquid chromatography-tandem mass spectrometry method used for PL analysis reveals an increase in the levels of phosphatidylcholine and phosphatidylethanolamine species esterified with polyunsaturated fatty acids. This work demonstrates the ability of both EVOO and HPO to increase the degree of unsaturation of erythrocyte membrane lipids with an improvement in membrane fluidity that could be associated with a lower risk of developing cardiovascular diseases.

Effect of Dialkyl Ammonium Cationic Surfactants on the Microfluidity of Membranes Containing Raft Domains.

PubMed

Uyama, Makoto; Inoue, Kaori; Kinoshita, Koichi; Miyahara, Reiji; Yokoyama, Hirokazu; Nakano, Minoru

2018-01-01

It has been reported that a lot of receptors localize in lipid raft domains and that the microfluidity of these domains regulates the activation of these receptors. In this study, we focused on the lipid raft and in order to evaluate the physicochemical effects of surfactants on microfluidity of lipid membranes, we used liposomes comprising of egg-yolk L-α-phosphatidylcholine, egg-yolk sphingomyelin, and cholesterol as a model of cell membranes containing raft domains. The microfluidity of the domains was characterized by fluorescence spectrometry using 1,6-diphenyl-1,3,5-hexatriene and 2-dimethylamino-6-lauroylnaphthalene. Among several surfactants, dialkylammonium-type cationic surfactants most efficiently increased the microfluidity. It is therefore concluded that (1) the electrostatic interaction between the cationic surfactant and eggPC/eggSM/cholesterol liposome could be important, (2) surfactants with alkyl chains more effectively inserted into membranes than those with acyl chains, and (3) cationic surfactants with lower T m values have a greater ability to increase the fluidity.

Modification of the erythrocyte membrane dielectric constant by alcohols.

PubMed

Orme, F W; Moronne, M M; Macey, R I

1988-08-01

Aliphatic alcohols are found to stimulate the transmembrane fluxes of a hydrophobic cation (tetraphenylarsonium, TPA) and anion (AN-12) 5-20 times in red blood cells. The results are analyzed using the Born-Parsegian equation (Parsegian, A., 1969, Nature (London) 221:844-846), together with the Clausius-Mossotti equation to calculate membrane dielectric energy barriers. Using established literature values of membrane thickness, native membrane dielectric constant, TPA ionic radius, and alcohol properties (partition coefficient, molar volume, dielectric constant), the TPA permeability data is predicted remarkably well by theory. If the radius of AN-12 is taken as 1.9 A, its permeability in the presence of butanol is also described by our analysis. Further, the theory quantitatively accounts for the data of Gutknecht and Tosteson (Gutknecht, J., Tosteson, D.C., 1970, J. Gen. Physiol. 55:359-374) covering alcohol-induced conductivity changes of 3 orders of magnitude in artificial bilayers. Other explanations including perturbations of membrane fluidity, surface charge, membrane thickness, and dipole potential are discussed. However, the large magnitude of the stimulation, the more pronounced effect on smaller ions, and the acceleration of both anions and cations suggest membrane dielectric constant change as the primary basis of alcohol effects.

Kinetic analysis of the interaction between poly(amidoamine) dendrimers and model lipid membranes.

PubMed

Tiriveedhi, Venkataswarup; Kitchens, Kelly M; Nevels, Kerrick J; Ghandehari, Hamidreza; Butko, Peter

2011-01-01

We used fluorescence spectroscopy and surface tensiometry to study the interaction between low-generation (G1 and G4) poly(amidoamine) (PAMAM) dendrimers, potential vehicles for intracellular drug delivery, and model lipid bilayers. Membrane association of fluorescently labeled dendrimers, measured by fluorescence anisotropy, increased with increasing size of the dendrimer and with increasing negative charge density in the membrane, indicating the electrostatic nature of the interaction. When the membrane was doped with pyrene-labeled phosphatidyl glycerol (pyrene-PG), pyrene excimer fluorescence demonstrated a dendrimer-induced selective aggregation of negatively charged lipids when the membrane was in the liquid crystalline state. A nonlinear Stern-Volmer quenching of dendrimer fluorescence with cobalt bromide suggested a dendrimer-induced aggregation of lipid vesicles, which increased with the dendrimer's generation number. Surface tensiometry measurements showed that dendrimers penetrated into the lipid monolayer only at subphysiologic surface pressures (<30mN/m). We conclude that the low-generation PAMAM dendrimers associate with lipid membranes predominantly electrostatically, without significantly compromising the bilayer integrity. They bind stronger to membranes with higher fluidity and lower surface pressure, which are characteristic of rapidly dividing cells. Copyright © 2010 Elsevier B.V. All rights reserved.

Effect of Melatonin and Cholesterol on the Structure of DOPC and DPPC Membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Drolle, E; Kucerka, Norbert; Hoopes, M I

The cell membrane plays an important role in the molecular mechanism of amyloid toxicity associated with Alzheimer's disease. The membrane's chemical composition and the incorporation of small molecules, such as melatonin and cholesterol, can alter its structure and physical properties, thereby affecting its interaction with amyloid peptides. Both melatonin and cholesterol have been recently linked to amyloid toxicity. Melatonin has been shown to have a protective role against amyloid toxicity. However, the underlying molecular mechanism of this protection is still not well understood, and cholesterol's role remains controversial. We used small-angle neutron diffraction (SAND) from oriented lipid multi-layers, small-angle neutronmore » scattering (SANS) from unilamellar vesicles experiments andMolecular Dynamics (MD) simulations to elucidate non-specific interactions of melatonin and cholesterol with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dipalmitoyl-snglycero-3-phosphocholine (DPPC) model membranes. We conclude that melatonin decreases the thickness of both model membranes by disordering the lipid hydrocarbon chains, thus increasing membrane fluidity. This result is in stark contrast to the much accepted ordering effect induced by cholesterol, which causes membranes to thicken.« less

Two-photon fluorescence anisotropy imaging

NASA Astrophysics Data System (ADS)

Li, Wei; Wang, Yi; Shao, Hanrong; He, Yonghong; Ma, Hui

2006-09-01

We have developed a novel method for imaging the fluorescence intensity and anisotropy by two-photon fluorescence microscopy and tested its capability in biological application. This method is applied to model sample including FITC and FITC-CD44 antibody solution and also FITC-CD44 stained cells. The fluorescence anisotropy (FA) of FITC-CD44ab solution is higher than the FITC solution with the same concentration. The fluorescence in cell sample has even higher FA than in solution because the rotation diffusion is restrained in membrane. The method is employed to study the effect of berberine a kind of Chinese medicine, on tumor metastasis. The results indicated that tumor cell membrane fluidity is decreasing with increasing the concentration of berberine in culture medium.

Study of the Peripheral Nerve Fibers Myelin Structure Changes during Activation of Schwann Cell Acetylcholine Receptors

PubMed Central

Verdiyan, Ekaterina E.; Allakhverdiev, Elvin S.; Maksimov, Georgy V.

2016-01-01

In the present paper we consider a new type of mechanism by which neurotransmitter acetylcholine (ACh) regulates the properties of peripheral nerve fibers myelin. Our data show the importance of the relationship between the changes in the number of Schwann cell (SC) acetylcholine receptors (AChRs) and the axon excitation (different intervals between action potentials (APs)). Using Raman spectroscopy, an effect of activation of SC AChRs on the myelin membrane fluidity was investigated. It was found, that ACh stimulates an increase in lipid ordering degree of the myelin lipids, thus providing evidence for specific role of the “axon-SC” interactions at the axon excitation. It was proposed, that during the axon excitation, the SC membrane K+- depolarization and the Ca2+—influx led to phospholipase activation or exocytosis of intracellular membrane vesicles and myelin structure reorganization. PMID:27455410

Biomolecular Interactions of Tannin Isolated from Oenothera gigas with Liposomes.

PubMed

Sekowski, Szymon; Ionov, Maksim; Dubis, Alina; Mavlyanov, Saidmukhtar; Bryszewska, Maria; Zamaraeva, Maria

2016-04-01

We have examined the interaction between hydrolysable tannin 1-O-galloyl-4,6-hexahydroxydiphenoyl-β-D-glucose (OGβDG) with neutral liposomes as a model of cell membranes composed of three lipids: lecithin, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) at different mass ratios. OGβDG in the concentration range 0.5-15 µg/ml (0.4-12 µM) strongly interacts with liposomal membranes by changing their structure, surface charge and fluidity. Used OGβDG molecules decrease and increase the rigidity of hydrophilic surface and hydrophobic parts of liposomes, respectively. At higher concentrations of tannin (>15 µM), liposomes are aggregated. Fourier Transform Infra-Red (FTIR) analysis showed that mainly -OH groups from OGβDG and also PO(2-) groups from phospholipids are responsible for the interaction. Obtained data indicate the importance of membrane lipid composition in interactions between tannins and cells.

Polyunsaturated Fatty Acids in Lipid Bilayers and Tubules

NASA Astrophysics Data System (ADS)

Hirst, Linda S.; Yuan, Jing; Pramudya, Yohannes; Nguyen, Lam T.

2007-03-01

Omega-3 polyunsaturated fatty acids (PUFAs) are found in a variety of biological membranes and have been implicated with lipid raft formation and possible function, typical molecules include DHA (Docosahexanoic Acid) and AA (Alphalinoleic Acid) which have been the focus of considerable attention in recent years. We are interested in the phase behavior of these molecules in the lipid bilayer. The addition of lipid molecules with polyunsaturated chains has a clear effect on the fluidity and curvature of the membrane and we investigate the effects the addition of polyunsaturated lipids on bilayer structure and tubule formation. Self-assembled cylindrical lipid tubules have attracted considerable attention because of their interesting structures and potential technological applications. Using x-ray diffraction techniques, Atomic Force Microscopy and confocal fluorescence imaging, both symmetric and mixed chain lipids were incorporated into model membranes and the effects on bilayer structure and tubule formation investigated.

Overturning dogma: tolerance of insects to mixed-sterol diets is not universal.

PubMed

Behmer, Spencer T

2017-10-01

Insects cannot synthesize sterols de novo, but like all eukaryotes they use them as cell membrane inserts where they influence membrane fluidity and rigidity. They also use a small amount for metabolic purposes, most notably as essential precursors for steroid hormones. It has been a long-held view that most insects require a small amount of specific sterol (often cholesterol) for metabolic purposes, but for membrane purposes (where the bulk of sterols are used) specificity in sterol structure was less important. Under this model, it was assumed that insects could tolerate mixed-sterol diets as long as a small amount of cholesterol was available. In the current paper this dogma is overturned, using data from plant-feeding insects that were fed mixed-sterol diets with different amounts and ratios of dietary sterols. Copyright © 2017 Elsevier Inc. All rights reserved.

Influence of doxorubicin on model cell membrane properties: insights from in vitro and in silico studies.

PubMed

Alves, Ana Catarina; Magarkar, Aniket; Horta, Miguel; Lima, Jose L F C; Bunker, Alex; Nunes, Cláudia; Reis, Salette

2017-07-24

Despite doxorubicin being commonly used in chemotherapy there still remain significant holes in our knowledge regarding its delivery efficacy and an observed resistance mechanism that is postulated to involve the cell membrane. One possible mechanism is the efflux by protein P-gp, which is found predominantly in cholesterol enriched domains. Thereby, a hypothesis for the vulnerability of doxorubicin to efflux through P-gp is its enhanced affinity for the ordered cholesterol rich regions of the plasma membrane. Thus, we have studied doxorubicin's interaction with model membranes in a cholesterol rich, ordered environment and in liquid-disordered cholesterol poor environment. We have combined three separate experimental protocols: UV-Vis spectrophotometry, fluorescence quenching and steady-state anisotropy and computational molecular dynamics modeling. Our results show that the presence of cholesterol induces a change in membrane structure and doesn't impair doxorubicin's membrane partitioning, but reduces drug's influence on membrane fluidity without directly interacting with it. It is thus possible that the resistance mechanism that lowers the efficacy of doxorubicin, results from an increased density in membrane regions where the efflux proteins are present. This work represents a successful approach, combining experimental and computational studies of membrane based systems to unveil the behavior of drugs and candidate drug molecules.

Deuterium Labeling Strategies for Creating Contrast in Structure-Function Studies of Model Bacterial Outer Membranes Using Neutron Reflectometry.

PubMed

Le Brun, Anton P; Clifton, Luke A; Holt, Stephen A; Holden, Peter J; Lakey, Jeremy H

2016-01-01

Studying the outer membrane of Gram-negative bacteria is challenging due to the complex nature of its structure. Therefore, simplified models are required to undertake structure-function studies of processes that occur at the outer membrane/fluid interface. Model membranes can be created by immobilizing bilayers to solid supports such as gold or silicon surfaces, or as monolayers on a liquid support where the surface pressure and fluidity of the lipids can be controlled. Both model systems are amenable to having their structure probed by neutron reflectometry, a technique that provides a one-dimensional depth profile through a membrane detailing its thickness and composition. One of the strengths of neutron scattering is the ability to use contrast matching, allowing molecules containing hydrogen and those enriched with deuterium to be highlighted or matched out against the bulk isotopic composition of the solvent. Lipopolysaccharides, a major component of the outer membrane, can be isolated for incorporation into model membranes. Here, we describe the deuteration of lipopolysaccharides from rough strains of Escherichia coli for incorporation into model outer membranes, and how the use of deuterated materials enhances structural analysis of model membranes by neutron reflectometry. © 2016 Elsevier Inc. All rights reserved.

A high affinity RIM-binding protein/Aplip1 interaction prevents the formation of ectopic axonal active zones.

PubMed

Siebert, Matthias; Böhme, Mathias A; Driller, Jan H; Babikir, Husam; Mampell, Malou M; Rey, Ulises; Ramesh, Niraja; Matkovic, Tanja; Holton, Nicole; Reddy-Alla, Suneel; Göttfert, Fabian; Kamin, Dirk; Quentin, Christine; Klinedinst, Susan; Andlauer, Till Fm; Hell, Stefan W; Collins, Catherine A; Wahl, Markus C; Loll, Bernhard; Sigrist, Stephan J

2015-08-14

Synaptic vesicles (SVs) fuse at active zones (AZs) covered by a protein scaffold, at Drosophila synapses comprised of ELKS family member Bruchpilot (BRP) and RIM-binding protein (RBP). We here demonstrate axonal co-transport of BRP and RBP using intravital live imaging, with both proteins co-accumulating in axonal aggregates of several transport mutants. RBP, via its C-terminal Src-homology 3 (SH3) domains, binds Aplip1/JIP1, a transport adaptor involved in kinesin-dependent SV transport. We show in atomic detail that RBP C-terminal SH3 domains bind a proline-rich (PxxP) motif of Aplip1/JIP1 with submicromolar affinity. Pointmutating this PxxP motif provoked formation of ectopic AZ-like structures at axonal membranes. Direct interactions between AZ proteins and transport adaptors seem to provide complex avidity and shield synaptic interaction surfaces of pre-assembled scaffold protein transport complexes, thus, favouring physiological synaptic AZ assembly over premature assembly at axonal membranes.

Ultrafast endocytosis at mouse hippocampal synapses

NASA Astrophysics Data System (ADS)

Watanabe, Shigeki; Rost, Benjamin R.; Camacho-Pérez, Marcial; Davis, M. Wayne; Söhl-Kielczynski, Berit; Rosenmund, Christian; Jorgensen, Erik M.

2013-12-01

To sustain neurotransmission, synaptic vesicles and their associated proteins must be recycled locally at synapses. Synaptic vesicles are thought to be regenerated approximately 20s after fusion by the assembly of clathrin scaffolds or in approximately 1s by the reversal of fusion pores via `kiss-and-run' endocytosis. Here we use optogenetics to stimulate cultured hippocampal neurons with a single stimulus, rapidly freeze them after fixed intervals and examine the ultrastructure using electron microscopy--`flash-and-freeze' electron microscopy. Docked vesicles fuse and collapse into the membrane within 30ms of the stimulus. Compensatory endocytosis occurs within 50 to 100ms at sites flanking the active zone. Invagination is blocked by inhibition of actin polymerization, and scission is blocked by inhibiting dynamin. Because intact synaptic vesicles are not recovered, this form of recycling is not compatible with kiss-and-run endocytosis; moreover, it is 200-fold faster than clathrin-mediated endocytosis. It is likely that `ultrafast endocytosis' is specialized to restore the surface area of the membrane rapidly.

Active Mechanisms of Vibration Encoding and Frequency Filtering in Central Mechanosensory Neurons.

PubMed

Azevedo, Anthony W; Wilson, Rachel I

2017-10-11

To better understand biophysical mechanisms of mechanosensory processing, we investigated two cell types in the Drosophila brain (A2 and B1 cells) that are postsynaptic to antennal vibration receptors. A2 cells receive excitatory synaptic currents in response to both directions of movement: thus, twice per vibration cycle. The membrane acts as a low-pass filter, so that voltage and spiking mainly track the vibration envelope rather than individual cycles. By contrast, B1 cells are excited by only forward or backward movement, meaning they are sensitive to vibration phase. They receive oscillatory synaptic currents at the stimulus frequency, and they bandpass filter these inputs to favor specific frequencies. Different cells prefer different frequencies, due to differences in their voltage-gated conductances. Both Na + and K + conductances suppress low-frequency synaptic inputs, so cells with larger voltage-gated conductances prefer higher frequencies. These results illustrate how membrane properties and voltage-gated conductances can extract distinct stimulus features into parallel channels. Copyright © 2017 Elsevier Inc. All rights reserved.

Activity-dependent trafficking of lysosomes in dendrites and dendritic spines.

PubMed

Goo, Marisa S; Sancho, Laura; Slepak, Natalia; Boassa, Daniela; Deerinck, Thomas J; Ellisman, Mark H; Bloodgood, Brenda L; Patrick, Gentry N

2017-08-07

In neurons, lysosomes, which degrade membrane and cytoplasmic components, are thought to primarily reside in somatic and axonal compartments, but there is little understanding of their distribution and function in dendrites. Here, we used conventional and two-photon imaging and electron microscopy to show that lysosomes traffic bidirectionally in dendrites and are present in dendritic spines. We find that lysosome inhibition alters their mobility and also decreases dendritic spine number. Furthermore, perturbing microtubule and actin cytoskeletal dynamics has an inverse relationship on the distribution and motility of lysosomes in dendrites. We also find trafficking of lysosomes is correlated with synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors. Strikingly, lysosomes traffic to dendritic spines in an activity-dependent manner and can be recruited to individual spines in response to local activation. These data indicate the position of lysosomes is regulated by synaptic activity and thus plays an instructive role in the turnover of synaptic membrane proteins. © 2017 Goo et al.

Activity-dependent trafficking of lysosomes in dendrites and dendritic spines

PubMed Central

Sancho, Laura; Slepak, Natalia; Boassa, Daniela; Deerinck, Thomas J.; Ellisman, Mark H.

2017-01-01

In neurons, lysosomes, which degrade membrane and cytoplasmic components, are thought to primarily reside in somatic and axonal compartments, but there is little understanding of their distribution and function in dendrites. Here, we used conventional and two-photon imaging and electron microscopy to show that lysosomes traffic bidirectionally in dendrites and are present in dendritic spines. We find that lysosome inhibition alters their mobility and also decreases dendritic spine number. Furthermore, perturbing microtubule and actin cytoskeletal dynamics has an inverse relationship on the distribution and motility of lysosomes in dendrites. We also find trafficking of lysosomes is correlated with synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid–type glutamate receptors. Strikingly, lysosomes traffic to dendritic spines in an activity-dependent manner and can be recruited to individual spines in response to local activation. These data indicate the position of lysosomes is regulated by synaptic activity and thus plays an instructive role in the turnover of synaptic membrane proteins. PMID:28630145

Vesicle fusion observed by content transfer across a tethered lipid bilayer.

PubMed

Rawle, Robert J; van Lengerich, Bettina; Chung, Minsub; Bendix, Poul Martin; Boxer, Steven G

2011-10-19

Synaptic transmission is achieved by exocytosis of small, synaptic vesicles containing neurotransmitters across the plasma membrane. Here, we use a DNA-tethered freestanding bilayer as a target architecture that allows observation of content transfer of individual vesicles across the tethered planar bilayer. Tethering and fusion are mediated by hybridization of complementary DNA-lipid conjugates inserted into the two membranes, and content transfer is monitored by the dequenching of an aqueous content dye. By analyzing the diffusion profile of the aqueous dye after vesicle fusion, we are able to distinguish content transfer across the tethered bilayer patch from vesicle leakage above the patch. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Proteomic analysis of synaptoneurosomes highlights the relevant role of local translation in the hippocampus.

PubMed

Benito, Itziar; Casañas, Juan José; Montesinos, María Luz

2018-06-19

Several proteomic analyses have been performed on synaptic fractions isolated from cortex or even total brain, resulting in preparations with a high synaptic heterogeneity and complexity. Synaptoneurosomes (SNs) are subcellular membranous elements that contain sealed pre- and post-synaptic components. They are obtained by subcellular fractionation of brain homogenates and serve as a suitable model to study many aspects of the synapse physiology. Here we report the proteomic content of SNs isolated from hippocampus of adult mice, a brain region involved in memory that presents lower synaptic heterogeneity than cortex. Interestingly, in addition to pre- and post-synaptic proteins, we found that proteins involved in RNA binding and translation were overrepresented in our preparation. These results validate the protocol we previously reported for SNs isolation, and, as reported by other authors, highlight the relevance of local synaptic translation for hippocampal physiology. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Wnt signaling pathway improves central inhibitory synaptic transmission in a mouse model of Duchenne muscular dystrophy.

PubMed

Fuenzalida, Marco; Espinoza, Claudia; Pérez, Miguel Ángel; Tapia-Rojas, Cheril; Cuitino, Loreto; Brandan, Enrique; Inestrosa, Nibaldo C

2016-02-01

The dystrophin-associated glycoprotein complex (DGC) that connects the cytoskeleton, plasma membrane and the extracellular matrix has been related to the maintenance and stabilization of channels and synaptic receptors, which are both essential for synaptogenesis and synaptic transmission. The dystrophin-deficient (mdx) mouse model of Duchenne muscular dystrophy (DMD) exhibits a significant reduction in hippocampal GABA efficacy, which may underlie the altered synaptic function and abnormal hippocampal long-term plasticity exhibited by mdx mice. Emerging studies have implicated Wnt signaling in the modulation of synaptic efficacy, neuronal plasticity and cognitive function. We report here that the activation of the non-canonical Wnt-5a pathway and Andrographolide, improves hippocampal mdx GABAergic efficacy by increasing the number of inhibitory synapses and GABA(A) receptors or GABA release. These results indicate that Wnt signaling modulates GABA synaptic efficacy and could be a promising novel target for DMD cognitive therapy. Copyright © 2015 Elsevier Inc. All rights reserved.

Heterotopic synaptic bodies in the auditory hair cells of adult lizards.

PubMed

Miller, M R; Beck, J

1987-07-01

The auditory hair cells of adults of eight species of lizards (three gekkonids: Coleonyx variegatus, Gekko gecko, and Cosymbotus platyurus; two teiids: Ameiva ameiva and Cnemidophorus tigris; one anguid: Celestus costatus; one lacertid: Podarcis (Lacerta) sicula; and one iguanid: Crotaphytus wislizeni) were studied by transmission electron microscopy. Heterotopic synaptic bodies were found in some of the auditory hair cells of all of the above species, occurring frequently in the gekkonids but infrequently in other species. The groups of heterotopic synaptic bodies occurred mainly in the infranuclear cytoplasm between the hair cell nucleus and the hair cell plasma membrane. The groups of synaptic bodies that were close to the hair cell nucleus were usually associated with specialized arrays of rough and smooth endoplasmic reticulum. The numbers of heterotopic synaptic bodies were greatest in the gekkonid species and were especially large in Coleonyx variegatus, where an average of 36.8 synaptic bodies occur in one group. The functional significance of the presence of heterotopic synaptic bodies in the auditory hair cells of adults animals is not known.

The structure and function of presynaptic endosomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jähne, Sebastian, E-mail: sebastian.jaehne1@stud.uni-goettingen.de; International Max Planck Research School for Neurosciences, 37077 Göttingen; Rizzoli, Silvio O.

The function of endosomes and of endosome-like structures in the presynaptic compartment is still controversial. This is in part due to the absence of a consensus on definitions and markers for these compartments. Synaptic endosomes are sometimes seen as stable organelles, permanently present in the synapse. Alternatively, they are seen as short-lived intermediates in synaptic vesicle recycling, arising from the endocytosis of large vesicles from the plasma membrane, or from homotypic fusion of small vesicles. In addition, the potential function of the endosome is largely unknown in the synapse. Some groups have proposed that the endosome is involved in themore » sorting of synaptic vesicle proteins, albeit others have produced data that deny this possibility. In this review, we present the existing evidence for synaptic endosomes, we discuss their potential functions, and we highlight frequent technical pitfalls in the analysis of this elusive compartment. We also sketch a roadmap to definitely determine the role of synaptic endosomes for the synaptic vesicle cycle. Finally, we propose a common definition of synaptic endosome-like structures.« less

Ba2+- and bupivacaine-sensitive background K+ conductances mediate rapid EPSP attenuation in oligodendrocyte precursor cells

PubMed Central

Chan, Chu-Fang; Kuo, Tzu-Wei; Weng, Ju-Yun; Lin, Yen-Chu; Chen, Ting-Yu; Cheng, Jen-Kun; Lien, Cheng-Chang

2013-01-01

Glutamatergic transmission onto oligodendrocyte precursor cells (OPCs) may regulate OPC proliferation, migration and differentiation. Dendritic integration of excitatory postsynaptic potentials (EPSPs) is critical for neuronal functions, and mechanisms regulating dendritic propagation and summation of EPSPs are well understood. However, little is known about EPSP attenuation and integration in OPCs. We developed realistic OPC models for synaptic integration, based on passive membrane responses of OPCs obtained by simultaneous dual whole-cell patch-pipette recordings. Compared with neurons, OPCs have a very low value of membrane resistivity, which is largely mediated by Ba2+- and bupivacaine-sensitive background K+ conductances. The very low membrane resistivity not only leads to rapid EPSP attenuation along OPC processes but also sharpens EPSPs and narrows the temporal window for EPSP summation. Thus, background K+ conductances regulate synaptic responses and integration in OPCs, thereby affecting activity-dependent neuronal control of OPC development and function. PMID:23940377

The Crossroads of Synaptic Growth Signaling, Membrane Traffic and Neurological Disease: Insights from Drosophila.

PubMed

Deshpande, Mugdha; Rodal, Avital A

2016-02-01

Neurons require target-derived autocrine and paracrine growth factors to maintain proper identity, innervation, homeostasis and survival. Neuronal growth factor signaling is highly dependent on membrane traffic, both for the packaging and release of the growth factors themselves, and for regulation of intracellular signaling by their transmembrane receptors. Here, we review recent findings from the Drosophila larval neuromuscular junction (NMJ) that illustrate how specific steps of intracellular traffic and inter-organelle interactions impinge on signaling, particularly in the bone morphogenic protein, Wingless and c-Jun-activated kinase pathways, regulating elaboration and stability of NMJ arbors, construction of synapses and synaptic transmission and homeostasis. These membrane trafficking and signaling pathways have been implicated in human motor neuron diseases including amyotrophic lateral sclerosis and hereditary spastic paraplegia, highlighting their importance for neuronal health and survival. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Neuron membrane trafficking and protein kinases involved in autism and ADHD.

PubMed

Kitagishi, Yasuko; Minami, Akari; Nakanishi, Atsuko; Ogura, Yasunori; Matsuda, Satoru

2015-01-30

A brain-enriched multi-domain scaffolding protein, neurobeachin has been identified as a candidate gene for autism patients. Mutations in the synaptic adhesion protein cell adhesion molecule 1 (CADM1) are also associated with autism spectrum disorder, a neurodevelopmental disorder of uncertain molecular origin. Potential roles of neurobeachin and CADM1 have been suggested to a function of vesicle transport in endosomal trafficking. It seems that protein kinase B (AKT) and cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) have key roles in the neuron membrane trafficking involved in the pathogenesis of autism. Attention deficit hyperactivity disorder (ADHD) is documented to dopaminergic insufficiencies, which is attributed to synaptic dysfunction of dopamine transporter (DAT). AKT is also essential for the DAT cell-surface redistribution. In the present paper, we summarize and discuss the importance of several protein kinases that regulate the membrane trafficking involved in autism and ADHD, suggesting new targets for therapeutic intervention.

Alterations of hepatocyte function with free radical generators and reparation or prevention with coffee polyphenols.

PubMed

Saidi Merzouk, Amel; Hafida, Merzouk; Medjdoub, Amel; Loukidi, Bouchra; Cherrak, Sabri; Merzouk, Sid Ahmed; Elhabiri, Mourad

2017-03-01

Liver diseases are linked in the majority of cases to oxidative stress that antioxidants could neutralize with reducing liver injury. Chlorogenic acid, a coffee polyphenol, possesses antioxidant prosperities. The aim of this study was to evaluate in vitro preventive and corrective effects of cholorogenic acid in hepatocyte toxicity induced by free radicals. Hepatocytes were isolated from adult male Wistar rats. To determine corrective effects and reparation, cells were first exposed to two free radical generators (hydrogen peroxide/iron sulfate for hydroxyl radical formation, and phenazine methosulfate/nicotinamide adenine dinucleotide for superoxide anion formation) for 12H and thereafter treated by chlorogenic acid (1 and 10 μM final concentration) for another 12H. To show preventive effects, cells were pretreated by chlorogenic acid and thereafter exposed to free radical generators. Hepatocyte proliferation, glucose uptake, ATP contents, membrane fluidity and integrity, and intracellular redox status were investigated after 24H culture. The results showed that chlorogenic acid reversed the decrease in cell proliferation, glucose uptake and ATP levels, the increased LDH release and the reduced membrane fluidity and restored the oxidant/antioxidant status under oxidative stress. When pre-treated with chlorogenic acid, hepatocytes became very resistant to oxidative conditions and cellular homeostasis was maintained. In conclusion, chlorogenic acid displayed not only corrective but also preventive effects in hepatocytes exposed to oxidative stress and could be beneficial in patients with or at risk of liver diseases.

Pro-inflammatory and anti-inflammatory compounds exert similar effects on P-glycoprotein in blood-brain barrier endothelial cells.

PubMed

Torres-Vergara, Pablo; Penny, Jeffrey

2018-06-01

The effects of anti-inflammatory glucocorticoids dexamethasone (DX) and hydrocortisone (HC), pro-inflammatory cytokine interleukin-1β (IL-1β) and dietary long-chain polyunsaturated fatty acids (PUFAs) on expression and activity of the ATP-binding cassette transporter P-glycoprotein (P-GP) were studied in porcine brain endothelial cells (PBECs). Primary PBECs were treated for 24 h with glucocorticoids, IL-1β and long-chain PUFAs. P-GP activity was determined by measuring intracellular calcein accumulation and P-GP expression by Western blotting. The effect of PUFAs on membrane fluidity was assessed by fluorescence recovery after photobleaching (FRAP). Dexamethasone, HC and IL-1β significantly increased P-GP expression and activity. The effect of IL-1β was attenuated by the IL-1 receptor antagonist (IL-1RA). This is the first report of the combined actions of IL-1β and IL-1RA on P-GP expression and the first evidence of glucocorticoid-mediated P-GP up-regulation in PBECs. Arachidonic acid (AA), docosahexaenoic acid (DHA) and eicosapentenoic acid (EPA) significantly decreased P-GP activity without affecting expression or membrane fluidity. AA, DHA and EPA counteracted IL-1β-mediated increases in P-GP activity, while AA and EPA, but not DHA, counteracted glucocorticoid-mediated increase in P-GP activity. While glucocorticoids and IL-1β possess opposing actions in inflammation, they demonstrate functional consistency by increasing P-GP expression and activity in PBECs. © 2018 Royal Pharmaceutical Society.

Receptosecretory nature of type III cells in the taste bud.

PubMed

Yoshie, Sumio

2009-01-01

Type III cells in taste buds form chemical synapses with intragemmal afferent nerve fibers and are characterized by the presence of membrane-bound vesicles in the cytoplasm. Although the vesicles differ in shape and size among species, they are primarily categorized into small clear (40 nm in diameter) and large dense-cored (90-200 nm) types. As such vesicles tend to be closely juxtaposed to the synaptic membrane of the cells, it is reasonable to consider that the vesicles include transmitter(s) towards the gustatory nerve. In the guinea-pig taste bud, stimulation with various taste substances (sucrose, sodium chloride, quinine hydrochloride, or monosodium L-glutamate) causes ultrastructural alterations of the type III cells. At the synapse, the presynaptic plasma membrane often displays invaginations of 90 nm in a mean diameter towards the cytoplasm, which indicates the dense-cored vesicles opening into the synaptic cleft by means of exocytosis. The vesicles are also exocytosed at the non-synaptic region into the intercellular space. These findings strongly suggest that the transmitters presumably contained in the vesicles are released to conduct the excitement of the type III cells to the nerves and also to exert their paracrine effects upon the surroundings, such as the Ebner's salivary gland, acting as local hormones.

Comparison of [corrected] actin- and glass-supported phospholipid bilayer diffusion coefficients.

PubMed

Sterling, Sarah M; Dawes, Ryan; Allgeyer, Edward S; Ashworth, Sharon L; Neivandt, David J

2015-04-21

The formation of biomimetic lipid membranes has the potential to provide insights into cellular lipid membrane dynamics. The construction of such membranes necessitates not only the utilization of appropriate lipids, but also physiologically relevant substrate/support materials. The substrate materials employed have been shown to have demonstrable effects on the behavior of the overlying lipid membrane, and thus must be studied before use as a model cushion support. To our knowledge, we report the formation and investigation of a novel actin protein-supported lipid membrane. Specifically, inner leaflet lateral mobility of globular actin-supported DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) bilayers, deposited via the Langmuir-Blodgett/Langmuir Schaefer methodology, was investigated by z-scan fluorescence correlation spectroscopy across a temperature range of 20-44°C. The actin substrate was found to decrease the diffusion coefficient when compared to an identical membrane supported on glass. The depression of the diffusion coefficient occurred across all measured temperatures. These results indicated that the actin substrate exerted a direct effect on the fluidity of the lipid membrane and highlighted the fact that the choice of substrate/support is critical in studies of model lipid membranes. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

In vivo collective cell migration requires an LPAR2-dependent increase in tissue fluidity.

PubMed

Kuriyama, Sei; Theveneau, Eric; Benedetto, Alexandre; Parsons, Maddy; Tanaka, Masamitsu; Charras, Guillaume; Kabla, Alexandre; Mayor, Roberto

2014-07-07

Collective cell migration (CCM) and epithelial-mesenchymal transition (EMT) are common to cancer and morphogenesis, and are often considered to be mutually exclusive in spite of the fact that many cancer and embryonic cells that have gone through EMT still cooperate to migrate collectively. Here we use neural crest (NC) cells to address the question of how cells that have down-regulated cell-cell adhesions can migrate collectively. NC cell dissociation relies on a qualitative and quantitative change of the cadherin repertoire. We found that the level of cell-cell adhesion is precisely regulated by internalization of N-cadherin downstream of lysophosphatidic acid (LPA) receptor 2. Rather than promoting the generation of single, fully mesenchymal cells, this reduction of membrane N-cadherin only triggers a partial mesenchymal phenotype. This intermediate phenotype is characterized by an increase in tissue fluidity akin to a solid-like-to-fluid-like transition. This change of plasticity allows cells to migrate under physical constraints without abolishing cell cooperation required for collectiveness. © 2014 Kuriyama et al.

Effect of primycin on growth-arrested cultures and cell integrity of Staphylococcus aureus.

PubMed

Feiszt, Péter; Schneider, György; Emődy, Levente

2017-06-01

Bactericidal effect against non-dividing bacteria is a very advantageous, but rare characteristic among antimicrobial agents, mostly possessed by those affecting the cell membrane. These kinds of agents can kill bacterial cells without lysis. We assessed these characteristics on primycin, a topical anti-staphylococcal agent highly effective against prevalent multiresistant strains, as it also acts on the cell membrane. In time-kill studies, primycin preserved its bactericidal activity against growth-arrested Staphylococcus aureus cultures. The bactericidal action was slower against growth-arrested cultures compared to the exponentially growing ones to different extents depending on the manner of arrest. The bactericidal effect was less influenced by stringent response and by protein synthesis inhibition, proving that it does not depend on metabolic activity. In contrast, uncoupling of the membrane potential predominantly slowed, and low temperature almost stopped killing of bacteria. In consideration of published data, these facts suggest that the antibacterial action of primycin involves disrupting of the membrane potential, and is predominantly influenced by the membrane fluidity. Optical density measurements and transmission electron microscopy verified that primycin kills bacterial cells without lysis. These results reveal favorable characteristics of primycin and point to, and broaden the knowledge on its membrane-targeted effect.

Effects of Vanadium-Containing Compounds on Membrane Lipids and on Microdomains Used in Receptor-Mediated Signaling

PubMed Central

Roess, Deborah A.; Smith, Steven M. L.; Winter, Peter; Zhou, Jun; Dou, Ping; Baruah, Bharat; Trujillo, Alejandro M.; Levinger, Nancy E.; Yang, Xioda; Barisas, B. George; Crans, Debbie C.

2011-01-01

There is increasing evidence for the involvement of plasma membrane microdomains in insulin receptor function. Moreover, disruption of these structures, which are typically enriched in sphingomyelin and cholesterol, results in insulin resistance. Treatment strategies for insulin resistance include the use of vanadium compounds which have been shown in animal models to enhance insulin responsiveness. One possible mechanism for insulin-enhancing effects might involve direct effects of vanadium compounds on membrane lipid organization. These changes in lipid organization promote the partitioning of insulin receptors and other receptors into membrane microdomains where receptors are optimally functional. To explore this possibility, we have used several strategies involving vanadium complexes such as [VO2dipic]− (pyridin-2,6-dicarboxylatodioxovanadium(V)), decavanadate (V10O286−, V10), BMOV (bis(maltolato)oxovanadium(IV)) and [VO(saltris)]2 (2-salicylideniminato-2-(hydroxymethyl)-1,3-dihydroxypropane-oxovanadium(V)). Our strategies include an evaluation of interactions between vanadium-containing compounds and model lipid systems, an evaluation of the effects of vanadium compounds on lipid fluidity in erythrocyte membranes, and studies of the effects of vanadium-containing compounds on signaling events initiated by receptors known to use membrane microdomains as signaling platforms. PMID:18729092

Theta frequency background tunes transmission but not summation of spiking responses.

PubMed

Parameshwaran, Dhanya; Bhalla, Upinder S

2013-01-01

Hippocampal neurons are known to fire as a function of frequency and phase of spontaneous network rhythms, associated with the animal's behaviour. This dependence is believed to give rise to precise rate and temporal codes. However, it is not well understood how these periodic membrane potential fluctuations affect the integration of synaptic inputs. Here we used sinusoidal current injection to the soma of CA1 pyramidal neurons in the rat brain slice to simulate background oscillations in the physiologically relevant theta and gamma frequency range. We used a detailed compartmental model to show that somatic current injection gave comparable results to more physiological synaptically driven theta rhythms incorporating excitatory input in the dendrites, and inhibitory input near the soma. We systematically varied the phase of synaptic inputs with respect to this background, and recorded changes in response and summation properties of CA1 neurons using whole-cell patch recordings. The response of the cell was dependent on both the phase of synaptic inputs and frequency of the background input. The probability of the cell spiking for a given synaptic input was up to 40% greater during the depolarized phases between 30-135 degrees of theta frequency current injection. Summation gain on the other hand, was not affected either by the background frequency or the phasic afferent inputs. This flat summation gain, coupled with the enhanced spiking probability during depolarized phases of the theta cycle, resulted in enhanced transmission of summed inputs during the same phase window of 30-135 degrees. Overall, our study suggests that although oscillations provide windows of opportunity to selectively boost transmission and EPSP size, summation of synaptic inputs remains unaffected during membrane oscillations.

G-protein-coupled estrogen receptor 1 is anatomically positioned to modulate synaptic plasticity in the mouse hippocampus.

PubMed

Waters, Elizabeth M; Thompson, Louisa I; Patel, Parth; Gonzales, Andreina D; Ye, Hector Zhiyu; Filardo, Edward J; Clegg, Deborah J; Gorecka, Jolanta; Akama, Keith T; McEwen, Bruce S; Milner, Teresa A

2015-02-11

Both estrous cycle and sex affect the numbers and types of neuronal and glial profiles containing the classical estrogen receptors α and β, and synaptic levels in the rodent dorsal hippocampus. Here, we examined whether the membrane estrogen receptor, G-protein-coupled estrogen receptor 1 (GPER1), is anatomically positioned in the dorsal hippocampus of mice to regulate synaptic plasticity. By light microscopy, GPER1-immunoreactivity (IR) was most noticeable in the pyramidal cell layer and interspersed interneurons, especially those in the hilus of the dentate gyrus. Diffuse GPER1-IR was found in all lamina but was most dense in stratum lucidum of CA3. Ultrastructural analysis revealed discrete extranuclear GPER1-IR affiliated with the plasma membrane and endoplasmic reticulum of neuronal perikarya and dendritic shafts, synaptic specializations in dendritic spines, and clusters of vesicles in axon terminals. Moreover, GPER1-IR was found in unmyelinated axons and glial profiles. Overall, the types and amounts of GPER1-labeled profiles were similar between males and females; however, in females elevated estrogen levels generally increased axonal labeling. Some estradiol-induced changes observed in previous studies were replicated by the GPER agonist G1: G1 increased PSD95-IR in strata oriens, lucidum, and radiatum of CA3 in ovariectomized mice 6 h after administration. In contrast, estradiol but not G1 increased Akt phosphorylation levels. Instead, GPER1 actions in the synapse may be due to interactions with synaptic scaffolding proteins, such as SAP97. These results suggest that although estrogen's actions via GPER1 may converge on the same synaptic elements, different pathways are used to achieve these actions. Copyright © 2015 the authors 0270-6474/15/352384-14$15.00/0.

Role of Water in Proton-Hydroxide Conductance Across Membranes

DTIC Science & Technology

1988-06-28

a~a,:v %~ ’ diffusion , rather than hopping along water wires, and there should be little or no deuterium effect. References Bangham , A.D...CONTRACT TITLE: Role of water in proton-hydroxide conductance across model and biological membranes. START DATE: October 1, 1987 RESEARCH OBJECTIVE: To...hypothesis of Bangham and Mason (1980) who suggested that general anesthetics might introduce defects into bilayers of synaptic vesicle membranes which lead

In vitro assay using engineered yeast vacuoles for neuronal SNARE-mediated membrane fusion

PubMed Central

Ko, Young-Joon; Lee, Miriam; Kang, KyeongJin; Song, Woo Keun; Jun, Youngsoo

2014-01-01

Intracellular membrane fusion requires not only SNARE proteins but also other regulatory proteins such as the Rab and Sec1/Munc18 (SM) family proteins. Although neuronal SNARE proteins alone can drive the fusion between synthetic liposomes, it remains unclear whether they are also sufficient to induce the fusion of biological membranes. Here, through the use of engineered yeast vacuoles bearing neuronal SNARE proteins, we show that neuronal SNAREs can induce membrane fusion between yeast vacuoles and that this fusion does not require the function of the Rab protein Ypt7p or the SM family protein Vps33p, both of which are essential for normal yeast vacuole fusion. Although excess vacuolar SNARE proteins were also shown to mediate Rab-bypass fusion, this fusion required homotypic fusion and vacuole protein sorting complex, which bears Vps33p and was accompanied by extensive membrane lysis. We also show that this neuronal SNARE-driven vacuole fusion can be stimulated by the neuronal SM protein Munc18 and blocked by botulinum neurotoxin serotype E, a well-known inhibitor of synaptic vesicle fusion. Taken together, our results suggest that neuronal SNARE proteins are sufficient to induce biological membrane fusion, and that this new assay can be used as a simple and complementary method for investigating synaptic vesicle fusion mechanisms. PMID:24821814

Membrane fluidization by alcohols inhibits DesK-DesR signalling in Bacillus subtilis.

PubMed

Vaňousová, Kateřina; Beranová, Jana; Fišer, Radovan; Jemioła-Rzemińska, Malgorzata; Matyska Lišková, Petra; Cybulski, Larisa; Strzałka, Kazimierz; Konopásek, Ivo

2018-03-01

After cold shock, the Bacillus subtilis desaturase Des introduces double bonds into the fatty acids of existing membrane phospholipids. The synthesis of Des is regulated exclusively by the two-component system DesK/DesR; DesK serves as a sensor of the state of the membrane and triggers Des synthesis after a decrease in membrane fluidity. The aim of our work is to investigate the biophysical changes in the membrane that are able to affect the DesK signalling state. Using linear alcohols (ethanol, propanol, butanol, hexanol, octanol) and benzyl alcohol, we were able to suppress Des synthesis after a temperature downshift. The changes in the biophysical properties of the membrane caused by alcohol addition were followed using membrane fluorescent probes and differential scanning calorimetry. We found that the membrane fluidization induced by alcohols was reflected in an increased hydration at the lipid-water interface. This is associated with a decrease in DesK activity. The addition of alcohol mimics a temperature increase, which can be measured isothermically by fluorescence anisotropy. The effect of alcohols on the membrane periphery is in line with the concept of the mechanism by which two hydrophilic motifs located at opposite ends of the transmembrane region of DesK, which work as a molecular caliper, sense temperature-dependent variations in membrane properties. Copyright © 2017 Elsevier B.V. All rights reserved.

Giving uridine and/or docosahexaenoic acid orally to rat dams during gestation and nursing increases synaptic elements in brains of weanling pups.

PubMed

Cansev, Mehmet; Marzloff, George; Sakamoto, Toshimasa; Ulus, Ismail H; Wurtman, Richard J

2009-01-01

Developing neurons synthesize substantial quantities of membrane phospholipids in producing new synapses. We investigated the effects of maternal uridine (as uridine-5'-monophosphate) and docosahexaenoic acid supplementation on pups' brain phospholipids, synaptic proteins and dendritic spine densities. Dams consumed neither, 1 or both compounds for 10 days before parturition and 20 days while nursing. By day 21, brains of weanlings receiving both exhibited significant increases in membrane phosphatides, various pre- and postsynaptic proteins (synapsin-1, mGluR1, PSD-95), and in hippocampal dendritic spine densities. Administering these phosphatide precursors to lactating mothers or infants could be useful for treating developmental disorders characterized by deficient synapses. 2009 S. Karger AG, Basel.

Affinity-purified tetanus neurotoxin interaction with synaptic membranes: properties of a protease-sensitive receptor component

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lazarovici, P.; Yavin, E.

1986-11-04

The pharmacokinetic interaction of an affinity-purified /sup 125/I-labeled tetanotoxin fraction with guinea pig brain synaptosomal preparations was investigated. Binding of tetanotoxin was time- and temperature-dependent, was proportional to protein concentration, and was saturable at about 8 x 10/sup -9/ M as estimated by a solid-surface binding assay. Binding was optimal at pH 6.5 under low ionic strength buffer and was almost entirely blocked by gangliosides or antitoxin. In analogy to intact nerve cells, binding of toxin to membranes resulted in a tight association operationally defined as sequestration. Binding and sequestration were abolished after membrane pretreatment with sialidase. The enzyme couldmore » not dissociate the membrane-bound toxin formed at 4 or 37/sup 0/C under low ionic strength conditions, which is in part compatible with internalization as defined in nerve cell cultures. In the latter system the toxin could be removed at 4/sup 0/C but not at 37/sup 0/C. Binding was significantly reduced upon pretreatment of guinea pig brain membranes by a variety of hydrolytic enzymes. It is proposed that, in addition to a ganglioside, interaction of tetanotoxin with synaptic membranes is facilitated by a protein and may also require an appropriate lipid environment. These latter membrane constituents may play a pivotal role in the sequestration of the toxin.« less

Tip-enhanced Raman spectroscopy of lipid bilayers in water with an alumina- and silver-coated tungsten tip.

PubMed

Nakata, Atsushi; Nomoto, Tomonori; Toyota, Taro; Fujinami, Masanori

2013-01-01

Tip-enhanced Raman spectroscopy (TERS) of supported phospholipid bilayers in an aqueous environment is discussed in this paper. Two bilayer membranes were examined: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). We fabricated alumina- and silver-coated tungsten tips that are very robust in water. There was a large time-dependence in the TERS spectra for the DOPC bilayers, whereas no such time-dependence was observed in the DPPC bilayer spectra under the probe tip. The spectral changes of DOPC bilayers are discussed in terms of the fluidity of the liquid crystalline phase. Time-resolved TERS thus has the potential to characterize inhomogeneity and diffusion in fluidic phospholipid bilayer membranes.

Toward a Neurocentric View of Learning.

PubMed

Titley, Heather K; Brunel, Nicolas; Hansel, Christian

2017-07-05

Synaptic plasticity (e.g., long-term potentiation [LTP]) is considered the cellular correlate of learning. Recent optogenetic studies on memory engram formation assign a critical role in learning to suprathreshold activation of neurons and their integration into active engrams ("engram cells"). Here we review evidence that ensemble integration may result from LTP but also from cell-autonomous changes in membrane excitability. We propose that synaptic plasticity determines synaptic connectivity maps, whereas intrinsic plasticity-possibly separated in time-amplifies neuronal responsiveness and acutely drives engram integration. Our proposal marks a move away from an exclusively synaptocentric toward a non-exclusive, neurocentric view of learning. Copyright © 2017 Elsevier Inc. All rights reserved.

The membrane effects, and sensitivity to strychnine, of neural inhibition of the Mauthner cell, and its inhibition by glycine and GABA

PubMed Central

Diamond, J.; Roper, S.; Yasargil, G. M.

1973-01-01

1. Anionic conductance changes in Mauthner neurones of goldfish were measured during synaptically evoked inhibition and inhibition caused by iontophoretic application of the putative inhibitory transmitters glycine and γ-aminobutyric acid (GABA). 2. The effects of either amino acid were indistinguishable from those of the neural inhibitory transmitter(s). The membrane permeability during the neural or drug response was increased to Br-, Cl-, I-, SCN-, NO3-, ClO3-, and formate (HCOO-), but not to HCO3-, BrO3-, IO3-, SO4-, HPO4-, H2PO4-, acetate and citrate. 3. Strychnine was injected intramuscularly, iontophoretically, or applied topically to the exposed brain in order to compare quantitatively its ability to prevent inhibition evoked by synaptic activation and by pharmacological means. Inhibitions were measured by the increase in membrane conductance. 4. Strychnine, at concentrations just adequate to block completely the late collateral inhibition (LCI) and crossed VIII nerve inhibition, had little effect on the pharmacological inhibition caused by glycine, and sometimes there was no detectable effect at all. In one experiment even a local iontophoretic application of strychnine in a sufficient dose to diffuse over the cell and block the LCI almost completely, merely halved the effect of a small dose of glycine applied to the same localized region of the membrane. 5. Higher concentrations of strychnine than those necessary to block synaptically evoked inhibition would reduce the effect of glycine but not that of GABA. The evidence indicated that any apparent effect of strychnine upon GABA could be explained by displacement of the GABA-containing iontophoretic pipette. 6. The glycine-blocking action of iontophoretic pulses of strychnine was of relatively very slow onset and long duration compared to the effects of pulses of glycine and GABA. 7. These findings can be interpreted as either (1) strychnine has a presynaptic action, preventing the release of inhibitory neurotransmitter, in addition to its less potent post-synaptic one in blocking pharmacological inhibition, or (2) strychnine acts entirely post-synaptically, but the physiological transmitter action differs from that of glycine and GABA in being considerably more sensitive to strychnine antagonism. In either case, the use of strychnine as evidence for the claim that glycine is an inhibitory neurotransmitter at the Mauthner cell is questionable. PMID:4354770

Possible evolutionary origins of human female sexual fluidity.

PubMed

Kanazawa, Satoshi

2017-08-01

I propose an evolutionary theory of human female sexual fluidity and argue that women may have been evolutionarily designed to be sexually fluid in order to allow them to have sex with their cowives in polygynous marriage and thus reduce conflict and tension inherent in such marriage. In addition to providing an extensive definition and operationalization of the concept of sexual fluidity and specifying its ultimate function for women, the proposed theory can potentially solve several theoretical and empirical puzzles in evolutionary psychology and sex research. Analyses of the National Longitudinal Study of Adolescent Health (Add Health) confirm the theory's predictions that: (i) women (but not men) who experience increased levels of sexual fluidity have a larger number of children (suggesting that female sexual fluidity, if heritable, may be evolutionarily selected); (ii) women (but not men) who experience marriage or parenthood early in adult life subsequently experience increased levels of sexual fluidity; and (iii) sexual fluidity is significantly positively correlated with known markers of unrestricted sexual orientation among women whereas it is significantly negatively correlated with such markers among men. © 2016 Cambridge Philosophical Society.

Uptake and localization mechanisms of fluorescent and colored lipid probes. Part 2. QSAR models that predict localization of fluorescent probes used to identify ("specifically stain") various biomembranes and membranous organelles.

PubMed

Horobin, R W; Stockert, J C; Rashid-Doubell, F

2015-05-01

We discuss a variety of biological targets including generic biomembranes and the membranes of the endoplasmic reticulum, endosomes/lysosomes, Golgi body, mitochondria (outer and inner membranes) and the plasma membrane of usual fluidity. For each target, we discuss the access of probes to the target membrane, probe uptake into the membrane and the mechanism of selectivity of the probe uptake. A statement of the QSAR decision rule that describes the required physicochemical features of probes that enable selective staining also is provided, followed by comments on exceptions and limits. Examples of probes typically used to demonstrate each target structure are noted and decision rule tabulations are provided for probes that localize in particular targets; these tabulations show distribution of probes in the conceptual space defined by the relevant structure parameters ("parameter space"). Some general implications and limitations of the QSAR models for probe targeting are discussed including the roles of certain cell and protocol factors that play significant roles in lipid staining. A case example illustrates the predictive ability of QSAR models. Key limiting values of the head group hydrophilicity parameter associated with membrane-probe interactions are discussed in an appendix.

Biguanides inhibit complex I, II and IV of rat liver mitochondria and modify their functional properties.

PubMed

Drahota, Z; Palenickova, E; Endlicher, R; Milerova, M; Brejchova, J; Vosahlikova, M; Svoboda, P; Kazdova, L; Kalous, M; Cervinkova, Z; Cahova, M

2014-01-01

In this study, we focused on an analysis of biguanides effects on mitochondrial enzyme activities, mitochondrial membrane potential and membrane permeability transition pore function. We used phenformin, which is more efficient than metformin, and evaluated its effect on rat liver mitochondria and isolated hepatocytes. In contrast to previously published data, we found that phenformin, after a 5 min pre-incubation, dose-dependently inhibits not only mitochondrial complex I but also complex II and IV activity in isolated mitochondria. The enzymes complexes inhibition is paralleled by the decreased respiratory control index and mitochondrial membrane potential. Direct measurements of mitochondrial swelling revealed that phenformin increases the resistance of the permeability transition pore to Ca(2+) ions. Our data might be in agreement with the hypothesis of Schäfer (1976) that binding of biguanides to membrane phospholipids alters membrane properties in a non-specific manner and, subsequently, different enzyme activities are modified via lipid phase. However, our measurements of anisotropy of fluorescence of hydrophobic membrane probe diphenylhexatriene have not shown a measurable effect of membrane fluidity with the 1 mM concentration of phenformin that strongly inhibited complex I activity. Our data therefore suggest that biguanides could be considered as agents with high efficacy but low specifity.

Lateral assembly of the immunoglobulin protein SynCAM 1 controls its adhesive function and instructs synapse formation.

PubMed

Fogel, Adam I; Stagi, Massimiliano; Perez de Arce, Karen; Biederer, Thomas

2011-09-16

Synapses are specialized adhesion sites between neurons that are connected by protein complexes spanning the synaptic cleft. These trans-synaptic interactions can organize synapse formation, but their macromolecular properties and effects on synaptic morphology remain incompletely understood. Here, we demonstrate that the synaptic cell adhesion molecule SynCAM 1 self-assembles laterally via its extracellular, membrane-proximal immunoglobulin (Ig) domains 2 and 3. This cis oligomerization generates SynCAM oligomers with increased adhesive capacity and instructs the interactions of this molecule across the nascent and mature synaptic cleft. In immature neurons, cis assembly promotes the adhesive clustering of SynCAM 1 at new axo-dendritic contacts. Interfering with the lateral self-assembly of SynCAM 1 in differentiating neurons strongly impairs its synaptogenic activity. At later stages, the lateral oligomerization of SynCAM 1 restricts synaptic size, indicating that this adhesion molecule contributes to the structural organization of synapses. These results support that lateral interactions assemble SynCAM complexes within the synaptic cleft to promote synapse induction and modulate their structure. These findings provide novel insights into synapse development and the adhesive mechanisms of Ig superfamily members.

Differential Roles of Postsynaptic Density-93 Isoforms in Regulating Synaptic Transmission

PubMed Central

Krüger, Juliane M.; Favaro, Plinio D.; Liu, Mingna; Kitlińska, Agata; Huang, Xiaojie; Raabe, Monika; Akad, Derya S.; Liu, Yanling; Urlaub, Henning; Dong, Yan; Xu, Weifeng

2013-01-01

In the postsynaptic density of glutamatergic synapses, the discs large (DLG)-membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins coordinates a multiplicity of signaling pathways to maintain and regulate synaptic transmission. Postsynaptic density-93 (PSD-93) is the most variable paralog in this family; it exists in six different N-terminal isoforms. Probably because of the structural and functional variability of these isoforms, the synaptic role of PSD-93 remains controversial. To accurately characterize the synaptic role of PSD-93, we quantified the expression of all six isoforms in the mouse hippocampus and examined them individually in hippocampal synapses. Using molecular manipulations, including overexpression, gene knockdown, PSD-93 knock-out mice combined with biochemical assays, and slice electrophysiology both in rat and mice, we demonstrate that PSD-93 is required at different developmental synaptic states to maintain the strength of excitatory synaptic transmission. This strength is differentially regulated by the six isoforms of PSD-93, including regulations of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-active and inactive synapses, and activity-dependent modulations. Collectively, these results demonstrate that alternative combinations of N-terminal PSD-93 isoforms and DLG-MAGUK paralogs can fine-tune signaling scaffolds to adjust synaptic needs to regulate synaptic transmission. PMID:24068818

Pannexin 1 regulates bidirectional hippocampal synaptic plasticity in adult mice.

PubMed

Ardiles, Alvaro O; Flores-Muñoz, Carolina; Toro-Ayala, Gabriela; Cárdenas, Ana M; Palacios, Adrian G; Muñoz, Pablo; Fuenzalida, Marco; Sáez, Juan C; Martínez, Agustín D

2014-01-01

The threshold for bidirectional modification of synaptic plasticity is known to be controlled by several factors, including the balance between protein phosphorylation and dephosphorylation, postsynaptic free Ca(2+) concentration and NMDA receptor (NMDAR) composition of GluN2 subunits. Pannexin 1 (Panx1), a member of the integral membrane protein family, has been shown to form non-selective channels and to regulate the induction of synaptic plasticity as well as hippocampal-dependent learning. Although Panx1 channels have been suggested to play a role in excitatory long-term potentiation (LTP), it remains unknown whether these channels also modulate long-term depression (LTD) or the balance between both types of synaptic plasticity. To study how Panx1 contributes to excitatory synaptic efficacy, we examined the age-dependent effects of eliminating or blocking Panx1 channels on excitatory synaptic plasticity within the CA1 region of the mouse hippocampus. By using different protocols to induce bidirectional synaptic plasticity, Panx1 channel blockade or lack of Panx1 were found to enhance LTP, whereas both conditions precluded the induction of LTD in adults, but not in young animals. These findings suggest that Panx1 channels restrain the sliding threshold for the induction of synaptic plasticity and underlying brain mechanisms of learning and memory.

Pannexin 1 regulates bidirectional hippocampal synaptic plasticity in adult mice

PubMed Central

Ardiles, Alvaro O.; Flores-Muñoz, Carolina; Toro-Ayala, Gabriela; Cárdenas, Ana M.; Palacios, Adrian G.; Muñoz, Pablo; Fuenzalida, Marco; Sáez, Juan C.; Martínez, Agustín D.

2014-01-01

The threshold for bidirectional modification of synaptic plasticity is known to be controlled by several factors, including the balance between protein phosphorylation and dephosphorylation, postsynaptic free Ca2+ concentration and NMDA receptor (NMDAR) composition of GluN2 subunits. Pannexin 1 (Panx1), a member of the integral membrane protein family, has been shown to form non-selective channels and to regulate the induction of synaptic plasticity as well as hippocampal-dependent learning. Although Panx1 channels have been suggested to play a role in excitatory long-term potentiation (LTP), it remains unknown whether these channels also modulate long-term depression (LTD) or the balance between both types of synaptic plasticity. To study how Panx1 contributes to excitatory synaptic efficacy, we examined the age-dependent effects of eliminating or blocking Panx1 channels on excitatory synaptic plasticity within the CA1 region of the mouse hippocampus. By using different protocols to induce bidirectional synaptic plasticity, Panx1 channel blockade or lack of Panx1 were found to enhance LTP, whereas both conditions precluded the induction of LTD in adults, but not in young animals. These findings suggest that Panx1 channels restrain the sliding threshold for the induction of synaptic plasticity and underlying brain mechanisms of learning and memory. PMID:25360084

Effect of phloretin on the permeability of thin lipid membranes

PubMed Central

1976-01-01

Phloretin dramatically increases cation conductances and decreases anion conductances of membranes treated with ion carriers (nonactin, valinomycin, carbonyl-cyanide-m-chlorophenylhydrazone [CCCP], and Hg(C6F5)2) or lipophilic ions (tetraphenylarsonium [tphAs+] and tetraphenylborate [TPhB-]). For example, on phosphatidylethanolamine membranes, 10(-4) M phloretin increases K+ -nonactin and TPhAs+ conductances and decreases CCCP- and TPhB- conductances 10(3)-fold; on lecithin: cholesterol membranes, it increases K+-nonactin conductance 10(5)-fold and decreases CCCP- conductance 10(3)-fold. Similar effects are obtained with p- and m-nitrophenol at 10(-2) M. These effects are produced by the un-ionized form of phloretin and the nitrophenols. We believe that phloretin, which possesses a large dipole moment, adsorbs and orients at the membrane surface to introduce a dipole potential of opposite polarity to the preexisting positive one, thus increasing the partition coefficient of cations into the membrane interior and decreasing the partition coefficient of anions. (Phloretin may also increase the fluidity of cholesterol-containing membranes; this is manifested by its two- to three-fold increase in nonelectrolyte permeability and its asymmetrical effect on cation and anion conductances in cholesterol-containing membranes.) It is possible that pholoretin's inhibition of chloride, urea, and glucose transport in biological membranes results from the effects of these intense intrafacial dipole fields on the translocator(s) of these molecules. PMID:946975

Detecting Subtle Plasma Membrane Perturbation in Living Cells Using Second Harmonic Generation Imaging

PubMed Central

Moen, Erick K.; Ibey, Bennett L.; Beier, Hope T.

2014-01-01

The requirement of center asymmetry for the creation of second harmonic generation (SHG) signals makes it an attractive technique for visualizing changes in interfacial layers such as the plasma membrane of biological cells. In this article, we explore the use of lipophilic SHG probes to detect minute perturbations in the plasma membrane. Three candidate probes, Di-4-ANEPPDHQ (Di-4), FM4-64, and all-trans-retinol, were evaluated for SHG effectiveness in Jurkat cells. Di-4 proved superior with both strong SHG signal and limited bleaching artifacts. To test whether rapid changes in membrane symmetry could be detected using SHG, we exposed cells to nanosecond-pulsed electric fields, which are believed to cause formation of nanopores in the plasma membrane. Upon nanosecond-pulsed electric fields exposure, we observed an instantaneous drop of ∼50% in SHG signal from the anodic pole of the cell. When compared to the simultaneously acquired fluorescence signals, it appears that the signal change was not due to the probe diffusing out of the membrane or changes in membrane potential or fluidity. We hypothesize that this loss in SHG signal is due to disruption in the interfacial nature of the membrane. The results show that SHG imaging has great potential as a tool for measuring rapid and subtle plasma membrane disturbance in living cells. PMID:24853757

Detecting subtle plasma membrane perturbation in living cells using second harmonic generation imaging.

PubMed

Moen, Erick K; Ibey, Bennett L; Beier, Hope T

2014-05-20

The requirement of center asymmetry for the creation of second harmonic generation (SHG) signals makes it an attractive technique for visualizing changes in interfacial layers such as the plasma membrane of biological cells. In this article, we explore the use of lipophilic SHG probes to detect minute perturbations in the plasma membrane. Three candidate probes, Di-4-ANEPPDHQ (Di-4), FM4-64, and all-trans-retinol, were evaluated for SHG effectiveness in Jurkat cells. Di-4 proved superior with both strong SHG signal and limited bleaching artifacts. To test whether rapid changes in membrane symmetry could be detected using SHG, we exposed cells to nanosecond-pulsed electric fields, which are believed to cause formation of nanopores in the plasma membrane. Upon nanosecond-pulsed electric fields exposure, we observed an instantaneous drop of ~50% in SHG signal from the anodic pole of the cell. When compared to the simultaneously acquired fluorescence signals, it appears that the signal change was not due to the probe diffusing out of the membrane or changes in membrane potential or fluidity. We hypothesize that this loss in SHG signal is due to disruption in the interfacial nature of the membrane. The results show that SHG imaging has great potential as a tool for measuring rapid and subtle plasma membrane disturbance in living cells. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Phospholipase A2 activity-dependent and -independent fusogenic activity of Naja nigricollis CMS-9 on zwitterionic and anionic phospholipid vesicles.

PubMed

Chiou, Yi-Ling; Chen, Ying-Jung; Lin, Shinne-Ren; Chang, Long-Sen

2011-11-01

CMS-9, a phospholipase A(2) (PLA(2)) from Naja nigricollis venom, induced the death of human breast cancer MCF-7 cells accompanied with the formation of cell clumps without clear boundaries between cells. Annexin V-FITC staining indicated that abundant phosphatidylserine appeared on the outer membrane of MCF-7 cell clumps, implying the possibility that CMS-9 may promote membrane fusion via anionic phospholipids. To validate this proposition, fusogenic activity of CMS-9 on vesicles composed of zwitterionic phospholipid alone or a combination of zwitterionic and anionic phospholipids was examined. Although CMS-9-induced fusion of zwitterionic phospholipid vesicles depended on PLA(2) activity, CMS-9-induced fusion of vesicles containing anionic phospholipids could occur without the involvement of PLA(2) activity. Membrane-damaging activity of CMS-9 was associated with its fusogenicity. Moreover, CMS-9 induced differently membrane leakage and membrane fusion of vesicles with different compositions. Membrane fluidity and binding capability with phospholipid vesicles were not related to the fusogenicity of CMS-9. However, membrane-bound conformation and mode of CMS-9 depended on phospholipid compositions. Collectively, our data suggest that PLA(2) activity-dependent and -independent fusogenicity of CMS-9 are closely related to its membrane-bound modes and targeted membrane compositions. Copyright © 2011 Elsevier Ltd. All rights reserved.

The actions of volatile anaesthetics on synaptic transmission in the dentate gyrus.

PubMed Central

Richards, C D; White, A E

1975-01-01

1. The action of four volatile anaesthetics on the evoked synaptic potentials of in vitro preparations of the hippocampus were examined. 2. All four anaesthetics (ether, halothane, methoxyflurane and trichloroethylene) depressed the synaptic transmission between the perforant path and the granule cells at concentrations lower than those required to maintain anaesthesia in intact animals. 3. The population excitatory post-synaptic potential (e.p.s.p.) and massed discharge of the cortical cells (population spike) were depressed at concentrations of the anaesthetics lower than those required to depress the compound action potential of the perforant path nerve fibres. None of the anaesthetics studied increased the threshold depolarization required for granule cell discharge. Furthermore, frequency potentiation of the evoked cortical e.p.s.p.s was not impaired by any of the anaesthetics studied. 4. It is concluded that all four anaesthetics depress synaptic transmission in the dentate gyrus either by reducing the amount of transmitter released from each nerve terminal in response to an afferent volley, or by decreasing the sensitivity of the post-synaptic membrane to released transmitted or by both effects together. PMID:1202196

Synaptic membrane rafts: traffic lights for local neurotrophin signaling?

PubMed

Zonta, Barbara; Minichiello, Liliana

2013-10-18

Lipid rafts, cholesterol and lipid rich microdomains, are believed to play important roles as platforms for the partitioning of transmembrane and synaptic proteins involved in synaptic signaling, plasticity, and maintenance. There is increasing evidence of a physical interaction between post-synaptic densities and post-synaptic lipid rafts. Localization of proteins within lipid rafts is highly regulated, and therefore lipid rafts may function as traffic lights modulating and fine-tuning neuronal signaling. The tyrosine kinase neurotrophin receptors (Trk) and the low-affinity p75 neurotrophin receptor (p75(NTR)) are enriched in neuronal lipid rafts together with the intermediates of downstream signaling pathways, suggesting a possible role of rafts in neurotrophin signaling. Moreover, neurotrophins and their receptors are involved in the regulation of cholesterol metabolism. Cholesterol is an important component of lipid rafts and its depletion leads to gradual loss of synapses, underscoring the importance of lipid rafts for proper neuronal function. Here, we review and discuss the idea that translocation of neurotrophin receptors in synaptic rafts may account for the selectivity of their transduced signals.

LTP-triggered cholesterol redistribution activates Cdc42 and drives AMPA receptor synaptic delivery

PubMed Central

Brachet, Anna; Norwood, Stephanie; Brouwers, Jos F.; Palomer, Ernest; Helms, J. Bernd

2015-01-01

Neurotransmitter receptor trafficking during synaptic plasticity requires the concerted action of multiple signaling pathways and the protein transport machinery. However, little is known about the contribution of lipid metabolism during these processes. In this paper, we addressed the question of the role of cholesterol in synaptic changes during long-term potentiation (LTP). We found that N-methyl-d-aspartate–type glutamate receptor (NMDAR) activation during LTP induction leads to a rapid and sustained loss or redistribution of intracellular cholesterol in the neuron. A reduction in cholesterol, in turn, leads to the activation of Cdc42 and the mobilization of GluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid–type glutamate receptors (AMPARs) from Rab11-recycling endosomes into the synaptic membrane, leading to synaptic potentiation. This process is accompanied by an increase of NMDAR function and an enhancement of LTP. These results imply that cholesterol acts as a sensor of NMDAR activation and as a trigger of downstream signaling to engage small GTPase (guanosine triphosphatase) activation and AMPAR synaptic delivery during LTP. PMID:25753037

Synaptic shot noise and conductance fluctuations affect the membrane voltage with equal significance.

PubMed

Richardson, Magnus J E; Gerstner, Wulfram

2005-04-01

The subthreshold membrane voltage of a neuron in active cortical tissue is a fluctuating quantity with a distribution that reflects the firing statistics of the presynaptic population. It was recently found that conductance-based synaptic drive can lead to distributions with a significant skew. Here it is demonstrated that the underlying shot noise caused by Poissonian spike arrival also skews the membrane distribution, but in the opposite sense. Using a perturbative method, we analyze the effects of shot noise on the distribution of synaptic conductances and calculate the consequent voltage distribution. To first order in the perturbation theory, the voltage distribution is a gaussian modulated by a prefactor that captures the skew. The gaussian component is identical to distributions derived using current-based models with an effective membrane time constant. The well-known effective-time-constant approximation can therefore be identified as the leading-order solution to the full conductance-based model. The higher-order modulatory prefactor containing the skew comprises terms due to both shot noise and conductance fluctuations. The diffusion approximation misses these shot-noise effects implying that analytical approaches such as the Fokker-Planck equation or simulation with filtered white noise cannot be used to improve on the gaussian approximation. It is further demonstrated that quantities used for fitting theory to experiment, such as the voltage mean and variance, are robust against these non-Gaussian effects. The effective-time-constant approximation is therefore relevant to experiment and provides a simple analytic base on which other pertinent biological details may be added.

MPP1 directly interacts with flotillins in erythrocyte membrane - Possible mechanism of raft domain formation.

PubMed

Biernatowska, Agnieszka; Augoff, Katarzyna; Podkalicka, Joanna; Tabaczar, Sabina; Gajdzik-Nowak, Weronika; Czogalla, Aleksander; Sikorski, Aleksander F

2017-11-01

Flotillins are prominent, oligomeric protein components of erythrocyte (RBC) membrane raft domains and are considered to play an important structural role in lateral organization of the plasma membrane. In our previous work on erythroid membranes and giant plasma membrane vesicles (GPMVs) derived from them we have shown that formation of functional domains (resting state rafts) depends on the presence of membrane palmitoylated protein 1 (MPP1/p55), pointing to its new physiological role. Exploration of the molecular mechanism of MPP1 function in organizing membrane domains described here, through searching for its molecular partners in RBC membrane by using different methods, led to the identification of the raft-marker proteins, flotillin 1 and flotillin 2, as hitherto unreported direct MPP1 binding-partners in the RBC membrane. These proteins are found in high molecular-weight complexes in native RBC membrane and, significantly, their presence was shown to be separate from the well-known protein 4.1-dependent interactions of MPP1 with membrane proteins. Furthermore, FLIM analysis revealed that loss of the endogenous MPP1-flotillins interactions resulted in significant changes in RBC membrane-fluidity, emphasizing the physiological importance of such interactions in vivo. Therefore, our data establish a new perspective on the role of MPP1 in erythroid cells and suggests that direct MPP1-flotillins interactions could be the major driving-force behind the formation of raft domains in RBC. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

Diet and cognition: interplay between cell metabolism and neuronal plasticity.

PubMed

Gomez-Pinilla, Fernando; Tyagi, Ethika

2013-11-01

To discuss studies in humans and animals revealing the ability of foods to benefit the brain: new information with regards to mechanisms of action and the treatment of neurological and psychiatric disorders. Dietary factors exert their effects on the brain by affecting molecular events related to the management of energy metabolism and synaptic plasticity. Energy metabolism influences neuronal function, neuronal signaling, and synaptic plasticity, ultimately affecting mental health. Epigenetic regulation of neuronal plasticity appears as an important mechanism by which foods can prolong their effects on long-term neuronal plasticity. The prime focus of the discussion is to emphasize the role of cell metabolism as a mediator for the action of foods on the brain. Oxidative stress promotes damage to phospholipids present in the plasma membrane such as the omega-3 fatty acid docosahexenoic acid, disrupting neuronal signaling. Thus, dietary docosahexenoic acid seems crucial for supporting plasma membrane function, interneuronal signaling, and cognition. The dual action of brain-derived neurotrophic factor in neuronal metabolism and synaptic plasticity is crucial for activating signaling cascades under the action of diet and other environmental factors, using mechanisms of epigenetic regulation.

Lengthening of the Stargazin Cytoplasmic Tail Increases Synaptic Transmission by Promoting Interaction to Deeper Domains of PSD-95.

PubMed

Hafner, Anne-Sophie; Penn, Andrew C; Grillo-Bosch, Dolors; Retailleau, Natacha; Poujol, Christel; Philippat, Amandine; Coussen, Françoise; Sainlos, Matthieu; Opazo, Patricio; Choquet, Daniel

2015-04-22

PSD-95 is a prominent organizer of the postsynaptic density (PSD) that can present a filamentous orientation perpendicular to the plasma membrane. Interactions between PSD-95 and transmembrane proteins might be particularly sensitive to this orientation, as "long" cytoplasmic tails might be required to reach deeper PSD-95 domains. Extension/retraction of transmembrane protein C-tails offer a new way of regulating binding to PSD-95. Using stargazin as a model, we found that enhancing the apparent length of stargazin C-tail through phosphorylation or by an artificial linker was sufficient to potentiate binding to PSD-95, AMPAR anchoring, and synaptic transmission. A linear extension of stargazin C-tail facilitates binding to PSD-95 by preferentially engaging interaction with the farthest located PDZ domains regarding to the plasma membrane, which present a greater affinity for the stargazin PDZ-domain-binding motif. Our study reveals that the concerted orientation of the stargazin C-tail and PSD-95 is a major determinant of synaptic strength. Copyright © 2015 Elsevier Inc. All rights reserved.

Partially overlapping distribution of epsin1 and HIP1 at the synapse: analysis by immunoelectron microscopy.

PubMed

Yao, Pamela J; Bushlin, Ittai; Petralia, Ronald S

2006-01-10

Synapses of neurons use clathrin-mediated endocytic pathways for recycling of synaptic vesicles and trafficking of neurotransmitter receptors. Epsin 1 and huntingtin-interacting protein 1 (HIP1) are endocytic accessory proteins. Both proteins interact with clathrin and the AP2 adaptor complex and also bind to the phosphoinositide-containing plasma membrane via an epsin/AP180 N-terminal homology (ENTH/ANTH) domain. Epsin1 and HIP1 are found in neurons; however, their precise roles in synapses remain largely unknown. Using immunogold electron microscopy, we examine and compare the synaptic distribution of epsin1 and HIP1 in rat CA1 hippocampal synapse. We find that epsin1 is located across both sides of the synapse, whereas HIP1 displays a preference for the postsynaptic compartment. Within the synaptic compartments, espin1 is distributed similarly throughout, whereas postsynaptic HIP1 is concentrated near the plasma membrane. Our results suggest a dual role for epsin1 and HIP1 in the synapse: as broadly required factors for promoting clathrin assembly and as adaptors for specific endocytic pathways.

Generation of field potentials and modulation of their dynamics through volume integration of cortical activity.

PubMed

Kajikawa, Yoshinao; Schroeder, Charles E

2015-01-01

Field potentials (FPs) recorded within the brain, often called "local field potentials" (LFPs), are useful measures of net synaptic activity in a neuronal ensemble. However, due to volume conduction, FPs spread beyond regions of underlying synaptic activity, and thus an "LFP" signal may not accurately reflect the temporal patterns of synaptic activity in the immediately surrounding neuron population. To better understand the physiological processes reflected in FPs, we explored the relationship between the FP and its membrane current generators using current source density (CSD) analysis in conjunction with a volume conductor model. The model provides a quantitative description of the spatiotemporal summation of immediate local and more distant membrane currents to produce the FP. By applying the model to FPs in the macaque auditory cortex, we have investigated a critical issue that has broad implications for FP research. We have shown that FP responses in particular cortical layers are differentially susceptible to activity in other layers. Activity in the supragranular layers has the strongest contribution to FPs in other cortical layers, and infragranular FPs are most susceptible to contributions from other layers. To define the physiological processes generating FPs recorded in loci of relatively weak synaptic activity, strong effects produced by synaptic events in the vicinity have to be taken into account. While outlining limitations and caveats inherent to FP measurements, our results also suggest specific peak and frequency band components of FPs can be related to activity in specific cortical layers. These results may help improving the interpretability of FPs. Copyright © 2015 the American Physiological Society.

Generation of field potentials and modulation of their dynamics through volume integration of cortical activity

PubMed Central

Schroeder, Charles E.

2014-01-01

Field potentials (FPs) recorded within the brain, often called “local field potentials” (LFPs), are useful measures of net synaptic activity in a neuronal ensemble. However, due to volume conduction, FPs spread beyond regions of underlying synaptic activity, and thus an “LFP” signal may not accurately reflect the temporal patterns of synaptic activity in the immediately surrounding neuron population. To better understand the physiological processes reflected in FPs, we explored the relationship between the FP and its membrane current generators using current source density (CSD) analysis in conjunction with a volume conductor model. The model provides a quantitative description of the spatiotemporal summation of immediate local and more distant membrane currents to produce the FP. By applying the model to FPs in the macaque auditory cortex, we have investigated a critical issue that has broad implications for FP research. We have shown that FP responses in particular cortical layers are differentially susceptible to activity in other layers. Activity in the supragranular layers has the strongest contribution to FPs in other cortical layers, and infragranular FPs are most susceptible to contributions from other layers. To define the physiological processes generating FPs recorded in loci of relatively weak synaptic activity, strong effects produced by synaptic events in the vicinity have to be taken into account. While outlining limitations and caveats inherent to FP measurements, our results also suggest specific peak and frequency band components of FPs can be related to activity in specific cortical layers. These results may help improving the interpretability of FPs. PMID:25274348

IFITM3 requires an amphipathic helix for antiviral activity.

PubMed

Chesarino, Nicholas M; Compton, Alex A; McMichael, Temet M; Kenney, Adam D; Zhang, Lizhi; Soewarna, Victoria; Davis, Matthew; Schwartz, Olivier; Yount, Jacob S

2017-10-01

Interferon-induced transmembrane protein 3 (IFITM3) is a cellular factor that blocks virus fusion with cell membranes. IFITM3 has been suggested to alter membrane curvature and fluidity, though its exact mechanism of action is unclear. Using a bioinformatic approach, we predict IFITM3 secondary structures and identify a highly conserved, short amphipathic helix within a hydrophobic region of IFITM3 previously thought to be a transmembrane domain. Consistent with the known ability of amphipathic helices to alter membrane properties, we show that this helix and its amphipathicity are required for the IFITM3-dependent inhibition of influenza virus, Zika virus, vesicular stomatitis virus, Ebola virus, and human immunodeficiency virus infections. The homologous amphipathic helix within IFITM1 is also required for the inhibition of infection, indicating that IFITM proteins possess a conserved mechanism of antiviral action. We further demonstrate that the amphipathic helix of IFITM3 is required to block influenza virus hemagglutinin-mediated membrane fusion. Overall, our results provide evidence that IFITM proteins utilize an amphipathic helix for inhibiting virus fusion. © 2017 The Authors.

Flavonoids in Microheterogeneous Media, Relationship between Their Relative Location and Their Reactivity towards Singlet Oxygen

PubMed Central

Günther, Germán; Berríos, Eduardo; Pizarro, Nancy; Valdés, Karina; Montero, Guillermo; Arriagada, Francisco; Morales, Javier

2015-01-01

In this work, the relationship between the molecular structure of three flavonoids (kaempferol, quercetin and morin), their relative location in microheterogeneous media (liposomes and erythrocyte membranes) and their reactivity against singlet oxygen was studied. The changes observed in membrane fluidity induced by the presence of these flavonoids and the influence of their lipophilicity/hydrophilicity on the antioxidant activity in lipid membranes were evaluated by means of fluorescent probes such as Laurdan and diphenylhexatriene (DPH). The small differences observed for the value of generalized polarization of Laurdan (GP) curves in function of the concentration of flavonoids, indicate that these three compounds promote similar alterations in liposomes and erythrocyte membranes. In addition, these compounds do not produce changes in fluorescence anisotropy of DPH, discarding their location in deeper regions of the lipid bilayer. The determined chemical reactivity sequence is similar in all the studied media (kaempferol < quercetin < morin). Morin is approximately 10 times more reactive than quercetin and 20 to 30 times greater than kaempferol, depending on the medium. PMID:26098745

Kinematics of red cell aspiration by fluorescence-imaged microdeformation.

PubMed

Discher, D E; Mohandas, N

1996-10-01

Maps of fluorescing red cell membrane components on a pipette-aspirated projection are quantitated in an effort to elucidate and unify the heterogeneous kinematics of deformation. Transient gradients of diffusing fluorescent lipid first demonstrate the fluidity of an otherwise uniform-density bilayer and corroborate a "universal" calibration scale for relative surface density. A steep but smooth and stable gradient in the densities of the skeleton components spectrin, actin, and protein 4.1 is used to estimate large elastic strains along the aspirated skeleton. The deformation fields are argued to be an unhindered response to loading in the surface normal direction. Density maps intermediate to those of the compressible skeleton and fluid bilayer are exhibited by particular transmembrane proteins (e.g., Band 3) and yield estimates for the skeleton-connected fractions. Such connected proteins appear to occupy a significant proportion of the undeformed membrane surface and can lead to steric exclusion of unconnected integral membrane proteins from regions of network condensation. Consistent with membrane repatterning kinematics in reversible deformation, final vesiculation of the projection tip produces a cell fragment concentrated in freely diffusing proteins but depleted of skeleton.

Interaction of Soybean 7S Globulin Peptide with Cell Membrane Model via Isothermal Titration Calorimetry, Quartz Crystal Microbalance with Dissipation, and Langmuir Monolayer Study.

PubMed

Zou, Yuan; Pan, Runting; Ruan, Qijun; Wan, Zhili; Guo, Jian; Yang, Xiaoquan

2018-05-16

To understand the underlying molecular mechanism of the cholesterol-lowering effect of soybean 7S globulins, the interactions of their pepsin-released peptides (7S-peptides) with cell membrane models consisting of dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), and cholesterol (CHOL) were systematically studied. The results showed that 7S-peptides were bound to DPPC/DOPC/CHOL liposomes mainly through van der Waals forces and hydrogen bonds, and the presence of higher CHOL concentrations enhanced the binding affinity (e.g., DPPC/DOPC/CHOL = 1:1:0, binding ratio = 0.114; DPPC/DOPC/CHOL = 1:1:1, binding ratio = 2.02). Compression isotherms indicated that the incorporation of 7S-peptides increased the DPPC/DOPC/CHOL monolayer fluidity and the lipid raft size. The presence of CHOL accelerated the 7S-peptide accumulation on lipid rafts, which could serve as platforms for peptides to develop into β-sheet rich structures. These results allow us to hypothesize that 7S-peptides may indirectly influence membrane protein functions via altering the membrane organization in the enterocytes.

Synaptic dynamics and neuronal network connectivity are reflected in the distribution of times in Up states.

PubMed

Dao Duc, Khanh; Parutto, Pierre; Chen, Xiaowei; Epsztein, Jérôme; Konnerth, Arthur; Holcman, David

2015-01-01

The dynamics of neuronal networks connected by synaptic dynamics can sustain long periods of depolarization that can last for hundreds of milliseconds such as Up states recorded during sleep or anesthesia. Yet the underlying mechanism driving these periods remain unclear. We show here within a mean-field model that the residence time of the neuronal membrane potential in cortical Up states does not follow a Poissonian law, but presents several peaks. Furthermore, the present modeling approach allows extracting some information about the neuronal network connectivity from the time distribution histogram. Based on a synaptic-depression model, we find that these peaks, that can be observed in histograms of patch-clamp recordings are not artifacts of electrophysiological measurements, but rather are an inherent property of the network dynamics. Analysis of the equations reveals a stable focus located close to the unstable limit cycle, delimiting a region that defines the Up state. The model further shows that the peaks observed in the Up state time distribution are due to winding around the focus before escaping from the basin of attraction. Finally, we use in vivo recordings of intracellular membrane potential and we recover from the peak distribution, some information about the network connectivity. We conclude that it is possible to recover the network connectivity from the distribution of times that the neuronal membrane voltage spends in Up states.

Spatially Distributed Dendritic Resonance Selectively Filters Synaptic Input

PubMed Central

Segev, Idan; Shamma, Shihab

2014-01-01

An important task performed by a neuron is the selection of relevant inputs from among thousands of synapses impinging on the dendritic tree. Synaptic plasticity enables this by strenghtening a subset of synapses that are, presumably, functionally relevant to the neuron. A different selection mechanism exploits the resonance of the dendritic membranes to preferentially filter synaptic inputs based on their temporal rates. A widely held view is that a neuron has one resonant frequency and thus can pass through one rate. Here we demonstrate through mathematical analyses and numerical simulations that dendritic resonance is inevitably a spatially distributed property; and therefore the resonance frequency varies along the dendrites, and thus endows neurons with a powerful spatiotemporal selection mechanism that is sensitive both to the dendritic location and the temporal structure of the incoming synaptic inputs. PMID:25144440

Synaptic dynamics contribute to long-term single neuron response fluctuations.

PubMed

Reinartz, Sebastian; Biro, Istvan; Gal, Asaf; Giugliano, Michele; Marom, Shimon

2014-01-01

Firing rate variability at the single neuron level is characterized by long-memory processes and complex statistics over a wide range of time scales (from milliseconds up to several hours). Here, we focus on the contribution of non-stationary efficacy of the ensemble of synapses-activated in response to a given stimulus-on single neuron response variability. We present and validate a method tailored for controlled and specific long-term activation of a single cortical neuron in vitro via synaptic or antidromic stimulation, enabling a clear separation between two determinants of neuronal response variability: membrane excitability dynamics vs. synaptic dynamics. Applying this method we show that, within the range of physiological activation frequencies, the synaptic ensemble of a given neuron is a key contributor to the neuronal response variability, long-memory processes and complex statistics observed over extended time scales. Synaptic transmission dynamics impact on response variability in stimulation rates that are substantially lower compared to stimulation rates that drive excitability resources to fluctuate. Implications to network embedded neurons are discussed.

Clarinet (CLA-1), a novel active zone protein required for synaptic vesicle clustering and release

PubMed Central

Nelson, Jessica; Richmond, Janet E; Colón-Ramos, Daniel A; Shen, Kang

2017-01-01

Active zone proteins cluster synaptic vesicles at presynaptic terminals and coordinate their release. In forward genetic screens, we isolated a novel Caenorhabditis elegans active zone gene, clarinet (cla-1). cla-1 mutants exhibit defects in synaptic vesicle clustering, active zone structure and synapse number. As a result, they have reduced spontaneous vesicle release and increased synaptic depression. cla-1 mutants show defects in vesicle distribution near the presynaptic dense projection, with fewer undocked vesicles contacting the dense projection and more docked vesicles at the plasma membrane. cla-1 encodes three isoforms containing common C-terminal PDZ and C2 domains with homology to vertebrate active zone proteins Piccolo and RIM. The C-termini of all isoforms localize to the active zone. Specific loss of the ~9000 amino acid long isoform results in vesicle clustering defects and increased synaptic depression. Our data indicate that specific isoforms of clarinet serve distinct functions, regulating synapse development, vesicle clustering and release. PMID:29160205

Surface diffusion of astrocytic glutamate transporters shapes synaptic transmission.

PubMed

Murphy-Royal, Ciaran; Dupuis, Julien P; Varela, Juan A; Panatier, Aude; Pinson, Benoît; Baufreton, Jérôme; Groc, Laurent; Oliet, Stéphane H R

2015-02-01

Control of the glutamate time course in the synapse is crucial for excitatory transmission. This process is mainly ensured by astrocytic transporters, high expression of which is essential to compensate for their slow transport cycle. Although molecular mechanisms regulating transporter intracellular trafficking have been identified, the relationship between surface transporter dynamics and synaptic function remains unexplored. We found that GLT-1 transporters were highly mobile on rat astrocytes. Surface diffusion of GLT-1 was sensitive to neuronal and glial activities and was strongly reduced in the vicinity of glutamatergic synapses, favoring transporter retention. Notably, glutamate uncaging at synaptic sites increased GLT-1 diffusion, displacing transporters away from this compartment. Functionally, impairing GLT-1 membrane diffusion through cross-linking in vitro and in vivo slowed the kinetics of excitatory postsynaptic currents, indicative of a prolonged time course of synaptic glutamate. These data provide, to the best of our knowledge, the first evidence for a physiological role of GLT-1 surface diffusion in shaping synaptic transmission.

Additive effects on the energy barrier for synaptic vesicle fusion cause supralinear effects on the vesicle fusion rate.

PubMed

Schotten, Sebastiaan; Meijer, Marieke; Walter, Alexander Matthias; Huson, Vincent; Mamer, Lauren; Kalogreades, Lawrence; ter Veer, Mirelle; Ruiter, Marvin; Brose, Nils; Rosenmund, Christian; Sørensen, Jakob Balslev; Verhage, Matthijs; Cornelisse, Lennart Niels

2015-04-14

The energy required to fuse synaptic vesicles with the plasma membrane ('activation energy') is considered a major determinant in synaptic efficacy. From reaction rate theory, we predict that a class of modulations exists, which utilize linear modulation of the energy barrier for fusion to achieve supralinear effects on the fusion rate. To test this prediction experimentally, we developed a method to assess the number of releasable vesicles, rate constants for vesicle priming, unpriming, and fusion, and the activation energy for fusion by fitting a vesicle state model to synaptic responses induced by hypertonic solutions. We show that complexinI/II deficiency or phorbol ester stimulation indeed affects responses to hypertonic solution in a supralinear manner. An additive vs multiplicative relationship between activation energy and fusion rate provides a novel explanation for previously observed non-linear effects of genetic/pharmacological perturbations on synaptic transmission and a novel interpretation of the cooperative nature of Ca(2+)-dependent release.

Extracellular chelation of zinc does not affect hippocampal excitability and seizure-induced cell death in rats

PubMed Central

Lavoie, Nathalie; Peralta, Modesto R; Chiasson, Marilou; Lafortune, Kathleen; Pellegrini, Luca; Seress, László; Tóth, Katalin

2007-01-01

In the nervous system, zinc can influence synaptic responses and at extreme concentrations contributes to epileptic and ischaemic neuronal injury. Zinc can originate from synaptic vesicles, the extracellular space and from intracellular stores. In this study, we aimed to determine which of these zinc pools is responsible for the increased hippocampal excitability observed in zinc-depleted animals or following zinc chelation. Also, we investigated the source of intracellularly accumulating zinc in vulnerable neurons. Our data show that membrane-permeable and membrane-impermeable zinc chelators had little or no effect on seizure activity in the CA3 region. Furthermore, extracellular zinc chelation could not prevent the accumulation of lethal concentrations of zinc in dying neurons following epileptic seizures. At the electron microscopic level, zinc staining significantly increased at the presynaptic membrane of mossy fibre terminals in kainic acid-treated animals. These data indicate that intracellular but not extracellular zinc chelators could influence neuronal excitability and seizure-induced zinc accumulation observed in the cytosol of vulnerable neurons. PMID:17095563

[Changes of the neuronal membrane excitability as cellular mechanisms of learning and memory].

PubMed

Gaĭnutdinov, Kh L; Andrianov, V V; Gaĭnutdinova, T Kh

2011-01-01

In the presented review given literature and results of own studies of dynamics of electrical characteristics of neurons, which change are included in processes both an elaboration of learning, and retention of the long-term memory. Literary datas and our results allow to conclusion, that long-term retention of behavioural reactions during learning is accompanied not only by changing efficiency of synaptic transmission, as well as increasing of excitability of command neurons of the defensive reflex. This means, that in the process of learning are involved long-term changes of the characteristics a membrane of certain elements of neuronal network, dependent from the metabolism of the cells. see text). Thou phenomena possible mark as cellular (electrophysiological) correlates of long-term plastic modifications of the behaviour. The analyses of having results demonstrates an important role of membrane characteristics of neurons (their excitability) and parameters an synaptic transmission not only in initial stage of learning, as well as in long-term modifications of the behaviour (long-term memory).

Synaptic Vesicle Endocytosis Occurs on Multiple Timescales and Is Mediated by Formin-Dependent Actin Assembly.

PubMed

Soykan, Tolga; Kaempf, Natalie; Sakaba, Takeshi; Vollweiter, Dennis; Goerdeler, Felix; Puchkov, Dmytro; Kononenko, Natalia L; Haucke, Volker

2017-02-22

Neurotransmission is based on the exocytic fusion of synaptic vesicles (SVs) followed by endocytic membrane retrieval and the reformation of SVs. Recent data suggest that at physiological temperature SVs are internalized via clathrin-independent ultrafast endocytosis (UFE) within hundreds of milliseconds, while other studies have postulated a key role for clathrin-mediated endocytosis (CME) of SV proteins on a timescale of seconds to tens of seconds. Here we demonstrate using cultured hippocampal neurons as a model that at physiological temperature SV endocytosis occurs on several timescales from less than a second to several seconds, yet, is largely independent of clathrin. Clathrin-independent endocytosis (CIE) of SV membranes is mediated by actin-nucleating formins such as mDia1, which are required for the formation of presynaptic endosome-like vacuoles from which SVs reform. Our results resolve previous discrepancies in the field and suggest that SV membranes are predominantly retrieved via CIE mediated by formin-dependent actin assembly. Copyright © 2017 Elsevier Inc. All rights reserved.

Single-molecule studies of the neuronal SNARE fusion machinery.

PubMed

Brunger, Axel T; Weninger, Keith; Bowen, Mark; Chu, Steven

2009-01-01

SNAREs are essential components of the machinery for Ca(2+)-triggered fusion of synaptic vesicles with the plasma membrane, resulting in neurotransmitter release into the synaptic cleft. Although much is known about their biophysical and structural properties and their interactions with accessory proteins such as the Ca(2+) sensor synaptotagmin, their precise role in membrane fusion remains an enigma. Ensemble studies of liposomes with reconstituted SNAREs have demonstrated that SNAREs and accessory proteins can trigger lipid mixing/fusion, but the inability to study individual fusion events has precluded molecular insights into the fusion process. Thus, this field is ripe for studies with single-molecule methodology. In this review, we discuss applications of single-molecule approaches to observe reconstituted SNAREs, their complexes, associated proteins, and their effect on biological membranes. Some of the findings are provocative, such as the possibility of parallel and antiparallel SNARE complexes or of vesicle docking with only syntaxin and synaptobrevin, but have been confirmed by other experiments.

HAMLET interacts with lipid membranes and perturbs their structure and integrity.

PubMed

Mossberg, Ann-Kristin; Puchades, Maja; Halskau, Øyvind; Baumann, Anne; Lanekoff, Ingela; Chao, Yinxia; Martinez, Aurora; Svanborg, Catharina; Karlsson, Roger

2010-02-23

Cell membrane interactions rely on lipid bilayer constituents and molecules inserted within the membrane, including specific receptors. HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded alpha-lactalbumin (HLA) and oleic acid that is internalized by tumor cells, suggesting that interactions with the phospholipid bilayer and/or specific receptors may be essential for the tumoricidal effect. This study examined whether HAMLET interacts with artificial membranes and alters membrane structure. We show by surface plasmon resonance that HAMLET binds with high affinity to surface adherent, unilamellar vesicles of lipids with varying acyl chain composition and net charge. Fluorescence imaging revealed that HAMLET accumulates in membranes of vesicles and perturbs their structure, resulting in increased membrane fluidity. Furthermore, HAMLET disrupted membrane integrity at neutral pH and physiological conditions, as shown by fluorophore leakage experiments. These effects did not occur with either native HLA or a constitutively unfolded Cys-Ala HLA mutant (rHLA(all-Ala)). HAMLET also bound to plasma membrane vesicles formed from intact tumor cells, with accumulation in certain membrane areas, but the complex was not internalized by these vesicles or by the synthetic membrane vesicles. The results illustrate the difference in membrane affinity between the fatty acid bound and fatty acid free forms of partially unfolded HLA and suggest that HAMLET engages membranes by a mechanism requiring both the protein and the fatty acid. Furthermore, HAMLET binding alters the morphology of the membrane and compromises its integrity, suggesting that membrane perturbation could be an initial step in inducing cell death.

Sterol Regulation of Voltage-Gated K+ Channels.

PubMed

Balajthy, Andras; Hajdu, Peter; Panyi, Gyorgy; Varga, Zoltan

2017-01-01

Cholesterol is an essential lipid building block of the cellular plasma membrane. In addition to its structural role, it regulates the fluidity and raft structure of the membrane and influences the course of numerous membrane-linked signaling pathways and the function of transmembrane proteins, including ion channels. This is supported by a vast body of scientific data, which demonstrates the modulation of ion channels with a great variety of ion selectivity, gating, and tissue distribution by changes in membrane cholesterol. Here, we review what is currently known about the modulation of voltage-gated K + (Kv) channels by changes in membrane cholesterol content, considering raft association of the channels, the roles of cholesterol recognition sites, and those of adaptor proteins in cholesterol-Kv channel interactions. We specifically focus on Kv1.3, the dominant K + channel of human T cells. Effects of cholesterol depletion and enrichment and 7-dehydrocholesterol enrichment on Kv1.3 gating are discussed in the context of the immunological synapse and the comparison of the in vitro effects of sterol modifications on Kv1.3 function with ex vivo effects on cells from hypercholesterolemic and Smith-Lemli-Opitz patients. © 2017 Elsevier Inc. All rights reserved.

Bioeffects of low-energy continuous ultrasound on isolated sarcoma 180 cells.

PubMed

Wang, Xiaobing; Liu, Quanhong; Wang, Zhezhi; Wang, Pan; Hao, Qiao; Li, Chendi

2009-01-01

The aim of this study was to investigate the mechanism underlying bioeffects of low-intensity continuous ultrasound on isolated sarcoma 180 (S180) cells and cellular responses to these effects. After sonication, several structural and functional parameters were examined to elucidate ultrasound-induced cell damage. Instant disruption of the cell membrane might be caused by acoustic cavitation, producing mechanical and chemical effects that acted simultaneously on S180 cells; this could be reflected by immediate (morphological) changes such as membrane permeability, membrane fluidity, lipid peroxidation and the generation of hydroxyl radicals in culture medium. Our results of the delayed effects also indicated S180 cells were sensitive to ultrasound-induced apoptosis, and the rate of apoptosis rose gradually with a prolonged incubation time. The presence of apoptotic cells was identified by a distinct morphological form characterized by membrane blebbing, cell shrinkage, chromatin condensation and DNA fragmentation. Moreover, delayed cytotoxicity was accompanied by an increase in intracellular reactive oxygen species (ROS) and a decrease in the mitochondrial membrane potential, and the two events presented obviously a negative correlation. ROS secondarily generated from damaged mitochondria may play a role in the induction of apoptosis. Copyright 2009 S. Karger AG, Basel.

Fullerene inhibits benzo(a)pyrene Efflux from Cyprinus carpio hepatocytes by affecting cell membrane fluidity and P-glycoprotein expression.

PubMed

Chen, Qiqing; Hu, Xialin; Wang, Rui; Yuan, Jin; Yin, Daqiang

2016-05-01

P-Glycoprotein (P-gp) can protect cells by pumping out toxic compounds, and has been found widely expressed in fish tissues. Here, we illustrate the P-gp efflux ability for benzo(a)pyrene (BaP) in the hepatocytes of common carp (Cyprinus carpio) after exposing to fullerene aqueous suspension (nC60). The results revealed that nC60 increased the membrane fluidity by decreasing the ratio of saturated to unsaturated fatty acids, and increased the cholesterol contents. These findings, combined with 10-38% and 70-75% down-regulation of P-gp mRNA and protein respectively, suggested that nC60 caused inhibition on P-gp efflux transport system. Therefore, we further investigated the cellular efflux ability for BaP. Results showed unequivocally that nC60 is a potent P-gp inhibitor. The retaining BaP amounts after efflux were elevated by 1.7-2.8 fold during the 10 day exposure. Meanwhile, 5mg/L humic acid (one of the important fractions of natural organic matter, which is ubiquitous in aquatic environment) alleviated the nC60 damage to hepatocytes in terms of oxidative damage, cholesterol increment, and P-gp content reduction; and finally attenuated the suppressed P-gp efflux ability. Collectively, this study provides the first evidence of nC60 toxicity to P-gp functionality in fish and illustrates the possible mechanism of the suppressed P-gp efflux ability for BaP. Copyright © 2016 Elsevier B.V. All rights reserved.

ELKS active zone proteins as multitasking scaffolds for secretion

PubMed Central

Held, Richard G.

2018-01-01

Synaptic vesicle exocytosis relies on the tethering of release ready vesicles close to voltage-gated Ca2+ channels and specific lipids at the future site of fusion. This enables rapid and efficient neurotransmitter secretion during presynaptic depolarization by an action potential. Extensive research has revealed that this tethering is mediated by an active zone, a protein dense structure that is attached to the presynaptic plasma membrane and opposed to postsynaptic receptors. Although roles of individual active zone proteins in exocytosis are in part understood, the molecular mechanisms that hold the protein scaffold at the active zone together and link it to the presynaptic plasma membrane have remained unknown. This is largely due to redundancy within and across scaffolding protein families at the active zone. Recent studies, however, have uncovered that ELKS proteins, also called ERC, Rab6IP2 or CAST, act as active zone scaffolds redundant with RIMs. This redundancy has led to diverse synaptic phenotypes in studies of ELKS knockout mice, perhaps because different synapses rely to a variable extent on scaffolding redundancy. In this review, we first evaluate the need for presynaptic scaffolding, and we then discuss how the diverse synaptic and non-synaptic functional roles of ELKS support the hypothesis that ELKS provides molecular scaffolding for organizing vesicle traffic at the presynaptic active zone and in other cellular compartments. PMID:29491150

Clathrin and synaptic vesicle endocytosis: studies at the squid giant synapse

PubMed Central

Augustine, G.J.; Morgan, J.R.; Villalba-Galea, C.A.; Jin, S.; Prasad, K.; Lafer, E.M.

2015-01-01

The role of clathrin-mediated endocytosis in SV (synaptic vesicle) recycling has been studied by combining molecular biology, physiology and electron microscopy at the squid giant synapse. Procedures that prevent clathrin from assembling into membrane coats, such as impairment of binding of the AP180 and AP-2 adaptor proteins, completely prevent membrane budding during endocytosis. These procedures also reduce exocytosis, presumably an indirect effect of a reduction in the number of SVs following block of endocytosis. Disrupting the binding of auxilin to Hsc70 (heat-shock cognate 70) prevents clathrin-coated vesicles from uncoating and also disrupts SV recycling. Taken together, these results indicate that a clathrin-dependent pathway is the primary means of SV recycling at this synapse under physiological conditions. PMID:16417485

Glutamatergic postsynaptic block by Pamphobeteus spider venoms in crayfish.

PubMed

Araque, A; Ferreira, W; Lucas, S; Buño, W

1992-01-31

The effects of toxins from venom glands of two south american spiders (Pamphobeteus platyomma and P. soracabae) on glutamatergic excitatory synaptic transmission were studied in the neuromuscular junction of the opener muscle of crayfish. The toxins selectively and reversibly blocked both excitatory postsynaptic currents and potentials in a dose-dependent manner. They also reversibly abolished glutamate-induced postsynaptic membrane depolarization. They had no effect on resting postsynaptic membrane conductance nor on postsynaptic voltage-gated currents. The synaptic facilitation and the frequency of miniature postsynaptic potentials were unaffected by the toxins, indicating that presynaptic events were not modified. Picrotoxin, a selective antagonist of the gamma-aminobutyric acid (GABA)A receptor, did not modify toxin effects. We conclude that both toxins specifically block the postsynaptic glutamate receptor-channel complex.

Mutation K42E in dehydrodolichol diphosphate synthase (DHDDS) causes recessive retinitis pigmentosa.

PubMed

Lam, Byron L; Züchner, Stephan L; Dallman, Julia; Wen, Rong; Alfonso, Eduardo C; Vance, Jeffery M; Peričak-Vance, Margaret A

2014-01-01

A single-nucleotide mutation in the gene that encodes DHDDS has been identified by whole exome sequencing as the cause of the non-syndromic recessive retinitis pigmentosa (RP) in a family of Ashkenazi Jewish origin in which three of the four siblings have early onset retinal degeneration. The peripheral retinal degeneration in the affected siblings was evident in the initial examination in 1992 and only one had detectable electroretinogram (ERG) that suggested cone-rod dysfunction. The pigmentary retinal degeneration subsequently progressed rapidly. The identified mutation changes the highly conserved residue Lys42 to Glu, resulting in lower catalytic efficiency. Patterns of plasma transferrin isoelectric focusing gel were normal in all family members, indicating no significant abnormality in protein glycosylation. Dolichols have been shown to influence the fluidity and of the membrane and promote vesicle fusion. Considering that photoreceptor outer segments contain stacks of membrane discs, we believe that the mutation may lead to low dolichol levels in photoreceptor outer segments, resulting in unstable membrane structure that leads to photoreceptor degeneration.

Activation of TRPV2 and BKCa channels by the LL-37 enantiomers stimulates calcium entry and migration of cancer cells.

PubMed

Gambade, Audrey; Zreika, Sami; Guéguinou, Maxime; Chourpa, Igor; Fromont, Gaëlle; Bouchet, Ana Maria; Burlaud-Gaillard, Julien; Potier-Cartereau, Marie; Roger, Sébastien; Aucagne, Vincent; Chevalier, Stéphan; Vandier, Christophe; Goupille, Caroline; Weber, Günther

2016-04-26

Expression of the antimicrobial peptide hCAP18/LL-37 is associated to malignancy in various cancer forms, stimulating cell migration and metastasis. We report that LL-37 induces migration of three cancer cell lines by activating the TRPV2 calcium-permeable channel and recruiting it to pseudopodia through activation of the PI3K/AKT pathway. Ca2+ entry through TRPV2 cooperated with a K+ efflux through the BKCa channel. In a panel of human breast tumors, the expression of TRPV2 and LL-37 was found to be positively correlated. The D-enantiomer of LL-37 showed identical effects as the L-peptide, suggesting that no binding to a specific receptor was involved. LL-37 attached to caveolae and pseudopodia membranes and decreased membrane fluidity, suggesting that a modification of the physical properties of the lipid membrane bilayer was the underlying mechanism of its effects.

Activation of TRPV2 and BKCa channels by the LL-37 enantiomers stimulates calcium entry and migration of cancer cells

PubMed Central

Guéguinou, Maxime; Chourpa, Igor; Fromont, Gaëlle; Bouchet, Ana Maria; Burlaud-Gaillard, Julien; Potier-Cartereau, Marie; Roger, Sébastien; Aucagne, Vincent; Chevalier, Stéphan; Vandier, Christophe

2016-01-01

Expression of the antimicrobial peptide hCAP18/LL-37 is associated to malignancy in various cancer forms, stimulating cell migration and metastasis. We report that LL-37 induces migration of three cancer cell lines by activating the TRPV2 calcium-permeable channel and recruiting it to pseudopodia through activation of the PI3K/AKT pathway. Ca2+ entry through TRPV2 cooperated with a K+ efflux through the BKCa channel. In a panel of human breast tumors, the expression of TRPV2 and LL-37 was found to be positively correlated. The D-enantiomer of LL-37 showed identical effects as the L-peptide, suggesting that no binding to a specific receptor was involved. LL-37 attached to caveolae and pseudopodia membranes and decreased membrane fluidity, suggesting that a modification of the physical properties of the lipid membrane bilayer was the underlying mechanism of its effects. PMID:26993604

Modulating membrane fluidity corrects Batten disease phenotypes in vitro and in vivo.

PubMed

Schultz, Mark L; Tecedor, Luis; Lysenko, Elena; Ramachandran, Shyam; Stein, Colleen S; Davidson, Beverly L

2018-07-01

The neuronal ceroid lipofuscinoses are a class of inherited neurodegenerative diseases characterized by the accumulation of autofluorescent storage material. The most common neuronal ceroid lipofuscinosis has juvenile onset with rapid onset blindness and progressive degeneration of cognitive processes. The juvenile form is caused by mutations in the CLN3 gene, which encodes the protein CLN3. While mouse models of Cln3 deficiency show mild disease phenotypes, it is apparent from patient tissue- and cell-based studies that its loss impacts many cellular processes. Using Cln3 deficient mice, we previously described defects in mouse brain endothelial cells and blood-brain barrier (BBB) permeability. Here we expand on this to other components of the BBB and show that Cln3 deficient mice have increased astrocyte endfeet area. Interestingly, this phenotype is corrected by treatment with a commonly used GAP junction inhibitor, carbenoxolone (CBX). In addition to its action on GAP junctions, CBX has also been proposed to alter lipid microdomains. In this work, we show that CBX modifies lipid microdomains and corrects membrane fluidity alterations in Cln3 deficient endothelial cells, which in turn improves defects in endocytosis, caveolin-1 distribution at the plasma membrane, and Cdc42 activity. In further work using the NIH Library of Integrated Network-based Cellular Signatures (LINCS), we discovered other small molecules whose impact was similar to CBX in that they improved Cln3-deficient cell phenotypes. Moreover, Cln3 deficient mice treated orally with CBX exhibited recovery of impaired BBB responses and reduced autofluorescence. CBX and the compounds identified by LINCS, many of which have been used in humans or approved for other indications, may find therapeutic benefit in children suffering from CLN3 deficiency through mechanisms independent of their original intended use. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Receptor-mediated membrane adhesion of lipid-polymer hybrid (LPH) nanoparticles studied by dissipative particle dynamics simulations

NASA Astrophysics Data System (ADS)

Li, Zhenlong; Gorfe, Alemayehu A.

2014-12-01

Lipid-polymer hybrid (LPH) nanoparticles represent a novel class of targeted drug delivery platforms that combine the advantages of liposomes and biodegradable polymeric nanoparticles. However, the molecular details of the interaction between LPHs and their target cell membranes remain poorly understood. We have investigated the receptor-mediated membrane adhesion process of a ligand-tethered LPH nanoparticle using extensive dissipative particle dynamics (DPD) simulations. We found that the spontaneous adhesion process follows a first-order kinetics characterized by two distinct stages: a rapid nanoparticle-membrane engagement, followed by a slow growth in the number of ligand-receptor pairs coupled with structural re-organization of both the nanoparticle and the membrane. The number of ligand-receptor pairs increases with the dynamic segregation of ligands and receptors toward the adhesion zone causing an out-of-plane deformation of the membrane. Moreover, the fluidity of the lipid shell allows for strong nanoparticle-membrane interactions to occur even when the ligand density is low. The LPH-membrane avidity is enhanced by the increased stability of each receptor-ligand pair due to the geometric confinement and the cooperative effect arising from multiple binding events. Thus, our results reveal the unique advantages of LPH nanoparticles as active cell-targeting nanocarriers and provide some general principles governing nanoparticle-cell interactions that may aid future design of LPHs with improved affinity and specificity for a given target of interest.

Flying-fox (Pteropus spp.) sperm membrane fatty acid composition, its relationship to cold shock injury and implications for cryopreservation success.

PubMed

Melville, D F; Johnston, S D; Miller, R R

2012-12-01

The very large acrosome of Pteropus species spermatozoa is prone to damage during cooling procedures. Cryogenic succuss has been linked to membrane composition, therefore the lipid composition of five Pteropus species sperm acrosomal and plasma membranes were investigated to provide insight into reasons for cold shock susceptibility. Rapid chilling and re-warming of spermatozoa from three Pteropus species resulted in a decrease (P<0.05) in acrosomal integrity. Biochemical analysis of lipids revealed that stearic acid (18:0) was the predominant saturated fatty acid and oleic acid (18:1, n-9) the predominant unsaturated fatty acid in both acrosomal and plasma membranes. Linolenic acid (18:3, n-3) was only detected in plasma membranes of Pteropus hypomelanus and was detected in acrosomal membranes of all Pteropus spp. studied (except Pteropus giganteus). Although detected in both plasma and acrosomal membranes of Pteropus vampyrus, docosahexaenoic acid (22:6) was not detected at all in Pteropus poliocephalus, only in trace levels in the acrosomal and plasma membranes of P. giganteus and P. hypomelanus and not in acrosomal membranes of Pteropus rodricensis. No difference was seen in the levels of polyunsaturated fatty acids (PUFAs) within plasma membranes, however PUFAs were lower (P<0.05) in acrosomal membranes of P. giganteus compared with P. vampyrus. Pteropus spp. spermatozoa have a very low ratio of unsaturated/saturated membrane fatty acids (<0.5). Membranes containing more PUFAs are more fluid, so the use of cryogenic media which improves membrane fluidity should improve Pteropus spp. spermatozoal viability post-thaw. Copyright © 2012 Elsevier Inc. All rights reserved.

Diet fat alters synaptosomal phosphatidylethanolaminemethyl-transferase activity and phosphatidylcholine synthesis in brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hargreaves, K.M.; Clandinin, M.T.

1986-03-05

Phosphatidylcholine (PC) can be synthesized via three routes, each having potentially different metabolic fates. One route for PC synthesis is methylation of phosphatidylethanolamine (PE). To examine if dietary fat affects membrane PE composition and phosphatidylethanolaminemethyltransferase (PEMT) activity, male weanling rats were fed semi-purified diets containing 20% (w/w) fat of differing fatty acid composition for 24 days. Microsomal and synaptic plasma membranes were isolated and phospholipid composition analyzed. PEMT activity was measured by incorporation of the methyl group from /sup 3/H-S-adenosylmethionine into PE. Polyunsaturated diets high in omega 6 fatty acids produce a high ratio of omega 6/omega 3 fatty acidsmore » in synaptic plasma membranes. Dietary omega 3 and omega 6 fatty acid levels are reflected in membrane phospholipid content of 22:6(3), 20:4(6), 22:4(6) and 22:5(6). Diet-induced increase in these longer chain homologues of omega 6 and omega 3 fatty acids and a high ratio of omega 6/omega 3 fatty acids in PE are both associated with increased PEMT activity. These results suggest that diet-fat induced change in fatty acid composition of membrane PE results in transition in PEMT activity and synthesis of PC in brain, by providing preferred species of PE for methylation.« less

Spine Calcium Transients Induced by Synaptically-Evoked Action Potentials Can Predict Synapse Location and Establish Synaptic Democracy

PubMed Central

Meredith, Rhiannon M.; van Ooyen, Arjen

2012-01-01

CA1 pyramidal neurons receive hundreds of synaptic inputs at different distances from the soma. Distance-dependent synaptic scaling enables distal and proximal synapses to influence the somatic membrane equally, a phenomenon called “synaptic democracy”. How this is established is unclear. The backpropagating action potential (BAP) is hypothesised to provide distance-dependent information to synapses, allowing synaptic strengths to scale accordingly. Experimental measurements show that a BAP evoked by current injection at the soma causes calcium currents in the apical shaft whose amplitudes decay with distance from the soma. However, in vivo action potentials are not induced by somatic current injection but by synaptic inputs along the dendrites, which creates a different excitable state of the dendrites. Due to technical limitations, it is not possible to study experimentally whether distance information can also be provided by synaptically-evoked BAPs. Therefore we adapted a realistic morphological and electrophysiological model to measure BAP-induced voltage and calcium signals in spines after Schaffer collateral synapse stimulation. We show that peak calcium concentration is highly correlated with soma-synapse distance under a number of physiologically-realistic suprathreshold stimulation regimes and for a range of dendritic morphologies. Peak calcium levels also predicted the attenuation of the EPSP across the dendritic tree. Furthermore, we show that peak calcium can be used to set up a synaptic democracy in a homeostatic manner, whereby synapses regulate their synaptic strength on the basis of the difference between peak calcium and a uniform target value. We conclude that information derived from synaptically-generated BAPs can indicate synapse location and can subsequently be utilised to implement a synaptic democracy. PMID:22719238

Identification of PSD-95 Depalmitoylating Enzymes.

PubMed

Yokoi, Norihiko; Fukata, Yuko; Sekiya, Atsushi; Murakami, Tatsuro; Kobayashi, Kenta; Fukata, Masaki

2016-06-15

Postsynaptic density (PSD)-95, the most abundant postsynaptic scaffolding protein, plays a pivotal role in synapse development and function. Continuous palmitoylation cycles on PSD-95 are essential for its synaptic clustering and regulation of AMPA receptor function. However, molecular mechanisms for palmitate cycling on PSD-95 remain incompletely understood, as PSD-95 depalmitoylating enzymes remain unknown. Here, we isolated 38 mouse or rat serine hydrolases and found that a subset specifically depalmitoylated PSD-95 in heterologous cells. These enzymes showed distinct substrate specificity. α/β-Hydrolase domain-containing protein 17 members (ABHD17A, 17B, and 17C), showing the strongest depalmitoylating activity to PSD-95, showed different localization from other candidates in rat hippocampal neurons, and were distributed to recycling endosomes, the dendritic plasma membrane, and the synaptic fraction. Expression of ABHD17 in neurons selectively reduced PSD-95 palmitoylation and synaptic clustering of PSD-95 and AMPA receptors. Furthermore, taking advantage of the acyl-PEGyl exchange gel shift (APEGS) method, we quantitatively monitored the palmitoylation stoichiometry and the depalmitoylation kinetics of representative synaptic proteins, PSD-95, GluA1, GluN2A, mGluR5, Gαq, and HRas. Unexpectedly, palmitate on all of them did not turn over in neurons. Uniquely, most of the PSD-95 population underwent rapid palmitoylation cycles, and palmitate cycling on PSD-95 decelerated accompanied by its increased stoichiometry as synapses developed, probably contributing to postsynaptic receptor consolidation. Finally, inhibition of ABHD17 expression dramatically delayed the kinetics of PSD-95 depalmitoylation. This study suggests that local palmitoylation machinery composed of synaptic DHHC palmitoylating enzymes and ABHD17 finely controls the amount of synaptic PSD-95 and synaptic function. Protein palmitoylation, the most common lipid modification, dynamically regulates neuronal protein localization and function. Its unique reversibility is conferred by DHHC-type palmitoyl acyl transferases (palmitoylating enzymes) and still controversial palmitoyl-protein thioesterases (depalmitoylating enzymes). Here, we identified the membrane-anchored serine hydrolases, ABHD17A, 17B, and 17C, as the physiological PSD-95 depalmitoylating enzymes that regulate PSD-95 palmitoylation cycles in neurons. This study describes the first direct evidence for the neuronal depalmitoylating enzyme and provides a new aspect of the dynamic regulatory mechanisms of synaptic development and synaptic plasticity. In addition, our established APEGS assay, which provides unbiased and quantitative information about the palmitoylation state and dynamics, revealed the distinct regulatory mechanisms for synaptic palmitoylation. Copyright © 2016 Yokoi, Fukata et al.

Identification of PSD-95 Depalmitoylating Enzymes

PubMed Central

Yokoi, Norihiko; Sekiya, Atsushi; Murakami, Tatsuro; Kobayashi, Kenta

2016-01-01

Postsynaptic density (PSD)-95, the most abundant postsynaptic scaffolding protein, plays a pivotal role in synapse development and function. Continuous palmitoylation cycles on PSD-95 are essential for its synaptic clustering and regulation of AMPA receptor function. However, molecular mechanisms for palmitate cycling on PSD-95 remain incompletely understood, as PSD-95 depalmitoylating enzymes remain unknown. Here, we isolated 38 mouse or rat serine hydrolases and found that a subset specifically depalmitoylated PSD-95 in heterologous cells. These enzymes showed distinct substrate specificity. α/β-Hydrolase domain-containing protein 17 members (ABHD17A, 17B, and 17C), showing the strongest depalmitoylating activity to PSD-95, showed different localization from other candidates in rat hippocampal neurons, and were distributed to recycling endosomes, the dendritic plasma membrane, and the synaptic fraction. Expression of ABHD17 in neurons selectively reduced PSD-95 palmitoylation and synaptic clustering of PSD-95 and AMPA receptors. Furthermore, taking advantage of the acyl-PEGyl exchange gel shift (APEGS) method, we quantitatively monitored the palmitoylation stoichiometry and the depalmitoylation kinetics of representative synaptic proteins, PSD-95, GluA1, GluN2A, mGluR5, Gαq, and HRas. Unexpectedly, palmitate on all of them did not turn over in neurons. Uniquely, most of the PSD-95 population underwent rapid palmitoylation cycles, and palmitate cycling on PSD-95 decelerated accompanied by its increased stoichiometry as synapses developed, probably contributing to postsynaptic receptor consolidation. Finally, inhibition of ABHD17 expression dramatically delayed the kinetics of PSD-95 depalmitoylation. This study suggests that local palmitoylation machinery composed of synaptic DHHC palmitoylating enzymes and ABHD17 finely controls the amount of synaptic PSD-95 and synaptic function. SIGNIFICANCE STATEMENT Protein palmitoylation, the most common lipid modification, dynamically regulates neuronal protein localization and function. Its unique reversibility is conferred by DHHC-type palmitoyl acyl transferases (palmitoylating enzymes) and still controversial palmitoyl-protein thioesterases (depalmitoylating enzymes). Here, we identified the membrane-anchored serine hydrolases, ABHD17A, 17B, and 17C, as the physiological PSD-95 depalmitoylating enzymes that regulate PSD-95 palmitoylation cycles in neurons. This study describes the first direct evidence for the neuronal depalmitoylating enzyme and provides a new aspect of the dynamic regulatory mechanisms of synaptic development and synaptic plasticity. In addition, our established APEGS assay, which provides unbiased and quantitative information about the palmitoylation state and dynamics, revealed the distinct regulatory mechanisms for synaptic palmitoylation. PMID:27307232

Antioxidant and membrane effects of procyanidin dimers and trimers isolated from peanut and cocoa.

PubMed

Verstraeten, Sandra V; Hammerstone, John F; Keen, Carl L; Fraga, César G; Oteiza, Patricia I

2005-06-15

The antioxidant and membrane effects of dimer (Dim) and trimer (Trim) procyanidins isolated from cocoa (Theobroma cacao) (B- and C-bonded) and peanut (Arachis hypogea L.) skin (A-bonded) were evaluated in phosphatidyl choline liposomes. When liposomes were oxidized with a steady source of oxidants, the above dimers and trimers inhibited to a similar extent lipid oxidation in a concentration (0.33-5 microM)-dependent manner. With respect to membrane effects, Dim A1, Dim B, Trim A, and Trim C increased (Dim A1 = Dim B and Trim A = Trim C), while Dim A2 decreased, membrane surface potential. All of the procyanidins tested decreased membrane fluidity as determined by fluorescent probes at the water-lipid interface, an effect that extended into the hydrophobic region of the bilayer. Both dimers and trimers protected the lipid bilayer from disruption by Triton X-100. The magnitude of the protection was Dim A1 > Dim A2 > Dim B and Trim C > Trim A. Thus, dimers and trimers can interact with membrane phospholipids, presumably with their polar headgroup. As a consequence of this interaction, they can provide protection against the attack of oxidants and other molecules that challenge the integrity of the bilayer.

Interaction of Viscotoxins A3 and B with Membrane Model Systems: Implications to Their Mechanism of Action

PubMed Central

Giudici, Marcela; Pascual, Roberto; de la Canal, Laura; Pfüller, Karola; Pfüller, Uwe; Villalaín, José

2003-01-01

Viscotoxins are small proteins that are thought to interact with biomembranes, displaying different toxic activities against a varied number of cell types, being viscotoxin A3 (VtA3) the most cytotoxic whereas viscotoxin B (VtB) is the less potent. By using infrared and fluorescence spectroscopies, we have studied the interaction of VtA3 and VtB, both wild and reduced ones, with model membranes containing negatively charged phospholipids. Both VtA3 and VtB present a high conformational stability, and a similar conformation both in solution and when bound to membranes. In solution, the infrared spectra of the reduced proteins show an increase in bandwidth compared to the nonreduced ones indicating a greater flexibility. VtA3 and VtB bind with high affinity to membranes containing negatively charged phospholipids and are motional restricted, their binding being dependent on phospholipid composition. Whereas nonreduced proteins maintain their structure when bound to membranes, reduced ones aggregate. Furthermore, leakage experiments show that wild proteins were capable of disrupting membranes whereas reduced proteins were not. The effect of VtA3 and VtB on membranes having different phospholipid composition is diverse, affecting the cooperativity and fluidity of the membranes. Viscotoxins interact with membranes in a complex way, most likely organizing themselves at the surface inducing the appearance of defects that lead to the destabilization and disruption of the membrane bilayer. PMID:12885644

Emergent spatial synaptic structure from diffusive plasticity.

PubMed

Sweeney, Yann; Clopath, Claudia

2017-04-01

Some neurotransmitters can diffuse freely across cell membranes, influencing neighbouring neurons regardless of their synaptic coupling. This provides a means of neural communication, alternative to synaptic transmission, which can influence the way in which neural networks process information. Here, we ask whether diffusive neurotransmission can also influence the structure of synaptic connectivity in a network undergoing plasticity. We propose a form of Hebbian synaptic plasticity which is mediated by a diffusive neurotransmitter. Whenever a synapse is modified at an individual neuron through our proposed mechanism, similar but smaller modifications occur in synapses connecting to neighbouring neurons. The effects of this diffusive plasticity are explored in networks of rate-based neurons. This leads to the emergence of spatial structure in the synaptic connectivity of the network. We show that this spatial structure can coexist with other forms of structure in the synaptic connectivity, such as with groups of strongly interconnected neurons that form in response to correlated external drive. Finally, we explore diffusive plasticity in a simple feedforward network model of receptive field development. We show that, as widely observed across sensory cortex, the preferred stimulus identity of neurons in our network become spatially correlated due to diffusion. Our proposed mechanism of diffusive plasticity provides an efficient mechanism for generating these spatial correlations in stimulus preference which can flexibly interact with other forms of synaptic organisation. © 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Cholecystokinin octapeptide analogues stable to brain proteolysis.

PubMed

Knight, M; Barone, P; Tamminga, C A; Steardo, L; Chase, T N

1985-01-01

Based on recent findings identifying the initial degradative cleavage of CCK-8 at the Met3-Gly4 bond by a metalloendopeptidase, two analogues of CCK-8 with D-Ala and D-Trp substitutions at the Gly4 position were synthesized as stable analogues. Their stability to proteolysis by brain membranes and their binding potency at central CCK receptors were quantified. Both peptides are stable to degradation by peptidases in cortical synaptic membrane preparations. The analogues are nearly equipotent to CCK-8 in their affinities for inhibition of 125I-CCK-33 binding to guinea pig cortical membranes. L-Ala and L-Trp substituted peptides were synthesized for comparison. Both these peptides are degraded by synaptic membranes and the L-Trp substituted peptide possesses a greatly reduced affinity for central CCK receptors. Therefore, the structure of CCK due to the D conformation of Gly is more capable of interacting with brain CCK receptors. Further conformational analysis will establish whether the stabilized structure is a beta-bend or a beta-turn. Since these peptides are highly potent and stable to brain proteolysis they may be useful as stable CCK analogues for in vivo application.

Cytochrome b5 reductase is the component from neuronal synaptic plasma membrane vesicles that generates superoxide anion upon stimulation by cytochrome c.

PubMed

Samhan-Arias, Alejandro K; Fortalezas, Sofia; Cordas, Cristina M; Moura, Isabel; Moura, José J G; Gutierrez-Merino, Carlos

2018-05-01

In this work, we measured the effect of cytochrome c on the NADH-dependent superoxide anion production by synaptic plasma membrane vesicles from rat brain. In these membranes, the cytochrome c stimulated NADH-dependent superoxide anion production was inhibited by antibodies against cytochrome b 5 reductase linking the production to this enzyme. Measurement of the superoxide anion radical generated by purified recombinant soluble and membrane cytochrome b 5 reductase corroborates the production of the radical by different enzyme isoforms. In the presence of cytochrome c, a burst of superoxide anion as well as the reduction of cytochrome c by cytochrome b 5 reductase was measured. Complex formation between both proteins suggests that cytochrome b 5 reductase is one of the major partners of cytochrome c upon its release from mitochondria to the cytosol during apoptosis. Superoxide anion production and cytochrome c reduction are the consequences of the stimulated NADH consumption by cytochrome b 5 reductase upon complex formation with cytochrome c and suggest a major role of this enzyme as an anti-apoptotic protein during cell death. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Dynamic Control of Synaptic Adhesion and Organizing Molecules in Synaptic Plasticity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rudenko, Gabby

Synapses play a critical role in establishing and maintaining neural circuits, permitting targeted information transfer throughout the brain. A large portfolio of synaptic adhesion/organizing molecules (SAMs) exists in the mammalian brain involved in synapse development and maintenance. SAMs bind protein partners, formingtrans-complexes spanning the synaptic cleft orcis-complexes attached to the same synaptic membrane. SAMs play key roles in cell adhesion and in organizing protein interaction networks; they can also provide mechanisms of recognition, generate scaffolds onto which partners can dock, and likely take part in signaling processes as well. SAMs are regulated through a portfolio of different mechanisms that affectmore » their protein levels, precise localization, stability, and the availability of their partners at synapses. Interaction of SAMs with their partners can further be strengthened or weakened through alternative splicing, competing protein partners, ectodomain shedding, or astrocytically secreted factors. Given that numerous SAMs appear altered by synaptic activity, in vivo, these molecules may be used to dynamically scale up or scale down synaptic communication. Many SAMs, including neurexins, neuroligins, cadherins, and contactins, are now implicated in neuropsychiatric and neurodevelopmental diseases, such as autism spectrum disorder, schizophrenia, and bipolar disorder and studying their molecular mechanisms holds promise for developing novel therapeutics.« less

Dynamic Control of Synaptic Adhesion and Organizing Molecules in Synaptic Plasticity

PubMed Central

2017-01-01

Synapses play a critical role in establishing and maintaining neural circuits, permitting targeted information transfer throughout the brain. A large portfolio of synaptic adhesion/organizing molecules (SAMs) exists in the mammalian brain involved in synapse development and maintenance. SAMs bind protein partners, forming trans-complexes spanning the synaptic cleft or cis-complexes attached to the same synaptic membrane. SAMs play key roles in cell adhesion and in organizing protein interaction networks; they can also provide mechanisms of recognition, generate scaffolds onto which partners can dock, and likely take part in signaling processes as well. SAMs are regulated through a portfolio of different mechanisms that affect their protein levels, precise localization, stability, and the availability of their partners at synapses. Interaction of SAMs with their partners can further be strengthened or weakened through alternative splicing, competing protein partners, ectodomain shedding, or astrocytically secreted factors. Given that numerous SAMs appear altered by synaptic activity, in vivo, these molecules may be used to dynamically scale up or scale down synaptic communication. Many SAMs, including neurexins, neuroligins, cadherins, and contactins, are now implicated in neuropsychiatric and neurodevelopmental diseases, such as autism spectrum disorder, schizophrenia, and bipolar disorder and studying their molecular mechanisms holds promise for developing novel therapeutics. PMID:28255461

Learning and memory: Steroids and epigenetics.

PubMed

Colciago, Alessandra; Casati, Lavinia; Negri-Cesi, Paola; Celotti, Fabio

2015-06-01

Memory formation and utilization is a complex process involving several brain structures in conjunction as the hippocampus, the amygdala and the adjacent cortical areas, usually defined as medial temporal lobe structures (MTL). The memory processes depend on the formation and modulation of synaptic connectivity affecting synaptic strength, synaptic plasticity and synaptic consolidation. The basic neurocognitive mechanisms of learning and memory are shortly recalled in the initial section of this paper. The effect of sex hormones (estrogens, androgens and progesterone) and of adrenocortical steroids on several aspects of memory processes are then analyzed on the basis of animal and human studies. A specific attention has been devoted to the different types of steroid receptors (membrane or nuclear) involved and on local metabolic transformations when required. The review is concluded by a short excursus on the steroid activated epigenetic mechanisms involved in memory formation. Copyright © 2015 Elsevier Ltd. All rights reserved.

PSD-95 family MAGUKs are essential for anchoring AMPA and NMDA receptor complexes at the postsynaptic density

PubMed Central

Chen, Xiaobing; Levy, Jonathan M.; Hou, Austin; Winters, Christine; Azzam, Rita; Sousa, Alioscka A.; Leapman, Richard D.; Nicoll, Roger A.; Reese, Thomas S.

2015-01-01

The postsynaptic density (PSD)-95 family of membrane-associated guanylate kinases (MAGUKs) are major scaffolding proteins at the PSD in glutamatergic excitatory synapses, where they maintain and modulate synaptic strength. How MAGUKs underlie synaptic strength at the molecular level is still not well understood. Here, we explore the structural and functional roles of MAGUKs at hippocampal excitatory synapses by simultaneous knocking down PSD-95, PSD-93, and synapse-associated protein (SAP)102 and combining electrophysiology and transmission electron microscopic (TEM) tomography imaging to analyze the resulting changes. Acute MAGUK knockdown greatly reduces synaptic transmission mediated by α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) and N-methyl-d-aspartate receptors (NMDARs). This knockdown leads to a significant rise in the number of silent synapses, diminishes the size of PSDs without changes in pre- or postsynaptic membrane, and depletes the number of membrane-associated PSD-95–like vertical filaments and transmembrane structures, identified as AMPARs and NMDARs by EM tomography. The differential distribution of these receptor-like structures and dependence of their abundance on PSD size matches that of AMPARs and NMDARs in the hippocampal synapses. The loss of these structures following MAGUK knockdown tracks the reduction in postsynaptic AMPAR and NMDAR transmission, confirming the structural identities of these two types of receptors. These results demonstrate that MAGUKs are required for anchoring both types of glutamate receptors at the PSD and are consistent with a structural model where MAGUKs, corresponding to membrane-associated vertical filaments, are the essential structural proteins that anchor and organize both types of glutamate receptors and govern the overall molecular organization of the PSD. PMID:26604311

PSD-95 family MAGUKs are essential for anchoring AMPA and NMDA receptor complexes at the postsynaptic density.

PubMed

Chen, Xiaobing; Levy, Jonathan M; Hou, Austin; Winters, Christine; Azzam, Rita; Sousa, Alioscka A; Leapman, Richard D; Nicoll, Roger A; Reese, Thomas S

2015-12-15

The postsynaptic density (PSD)-95 family of membrane-associated guanylate kinases (MAGUKs) are major scaffolding proteins at the PSD in glutamatergic excitatory synapses, where they maintain and modulate synaptic strength. How MAGUKs underlie synaptic strength at the molecular level is still not well understood. Here, we explore the structural and functional roles of MAGUKs at hippocampal excitatory synapses by simultaneous knocking down PSD-95, PSD-93, and synapse-associated protein (SAP)102 and combining electrophysiology and transmission electron microscopic (TEM) tomography imaging to analyze the resulting changes. Acute MAGUK knockdown greatly reduces synaptic transmission mediated by α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) and N-methyl-d-aspartate receptors (NMDARs). This knockdown leads to a significant rise in the number of silent synapses, diminishes the size of PSDs without changes in pre- or postsynaptic membrane, and depletes the number of membrane-associated PSD-95-like vertical filaments and transmembrane structures, identified as AMPARs and NMDARs by EM tomography. The differential distribution of these receptor-like structures and dependence of their abundance on PSD size matches that of AMPARs and NMDARs in the hippocampal synapses. The loss of these structures following MAGUK knockdown tracks the reduction in postsynaptic AMPAR and NMDAR transmission, confirming the structural identities of these two types of receptors. These results demonstrate that MAGUKs are required for anchoring both types of glutamate receptors at the PSD and are consistent with a structural model where MAGUKs, corresponding to membrane-associated vertical filaments, are the essential structural proteins that anchor and organize both types of glutamate receptors and govern the overall molecular organization of the PSD.

Biochemistry of snake venom neurotoxins and their application to the study of synapse. [Neurotoxins isolated from venom of the Formosan banded krait

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hanley, M.R.

1978-11-01

The crude venom of the Formosan banded krait, Bungarus multicinctus, was separated into eleven lethal protein fractions. Nine fractions were purified to final homogeneous toxins, designated ..cap alpha..-bungarotoxin, ..beta..-bungarotoxin, and toxins 7, 8, 9A, 11, 12, 13, and 14. Three of the toxins, ..cap alpha..-bungarotoxin, 7, and 8, were identified as post-synaptic curarimimetic neurotoxins. The remaining toxins were identified as pre-synaptic neurotoxins. ..cap alpha..-Bungarotoxin, toxin 7, and toxin 8 are all highly stable basic polypeptides of approx. 8000 daltons molecular weight. The pre-synaptic toxins fell into two structural groups: toxin 9A and 14 which were single basic chains of approx.more » 14,000 daltons, and ..beta..-bungarotoxin, and toxins 11 thru 13 which were composed of two chains of approx. 8000 and approx. 13,000 daltons covalently linked by disulfides. All the pre-synaptic neurotoxins were shown to have intrinsic calcium-dependent phospholipase A activities. Under certain conditions, intact synaptic membranes were hydrolyzed more rapidly than protein-free extracted synaptic-lipid liposomes which, in turn, were hydrolyzed more rapidly than any other tested liposomes. It was speculated that cell-surface arrays of phosphatidyl serine/glycolipids created high affinity target sites for ..beta..-bungarotoxin. Single-chain toxins were found to be qualitatively different from the two-chain toxins in their ability to block the functioning of acetylcholine receptors, and were quantitatively different in their enzymatic and membrane disruptive activities. ..beta..-Bungarotoxin was shown to be an extremely potent neuronal lesioning agent. There was no apparent selectivity for cholinergic over non-cholinergic neurons, nor for nerve terminals over cell bodies. It was suggested that ..beta..-bungarotoxin can be considered a useful new histological tool, which may exhibit some regional selectivity.« less

Determinants of HDL Cholesterol Efflux Capacity after Virgin Olive Oil Ingestion: Interrelationships with Fluidity of HDL Monolayer.

PubMed

Fernández-Castillejo, Sara; Rubió, Laura; Hernáez, Álvaro; Catalán, Úrsula; Pedret, Anna; Valls, Rosa-M; Mosele, Juana I; Covas, Maria-Isabel; Remaley, Alan T; Castañer, Olga; Motilva, Maria-José; Solá, Rosa

2017-12-01

Cholesterol efflux capacity of HDL (CEC) is inversely associated with cardiovascular risk. HDL composition, fluidity, oxidation, and size are related with CEC. We aimed to assess which HDL parameters were CEC determinants after virgin olive oil (VOO) ingestion. Post-hoc analyses from the VOHF study, a crossover intervention with three types of VOO. We assessed the relationship of 3-week changes in HDL-related variables after intervention periods with independence of the type of VOO. After univariate analyses, mixed linear models were fitted with variables related with CEC and fluidity. Fluidity and Apolipoprotein (Apo)A-I content in HDL was directly associated, and HDL oxidative status inversely, with CEC. A reduction in free cholesterol, an increase in triglycerides in HDL, and a decrease in small HDL particle number or an increase in HDL mean size, were associated to HDL fluidity. HDL fluidity, ApoA-I concentration, and oxidative status are major determinants for CEC after VOO. The impact on CEC of changes in free cholesterol and triglycerides in HDL, and those of small HDL or HDL mean size, could be mechanistically linked through HDL fluidity. Our work points out novel therapeutic targets to improve HDL functionality in humans through nutritional or pharmacological interventions. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Prolonged Preservation of Human Platelets for Combat Casualty Care.

DTIC Science & Technology

1994-04-15

membrane fluidity. 5) Flurbiprofen : Flurbiprofen is an inhibitor of the cyclooxygenase enzyme and thus blocks this arm of the arachidonic acid cascade...were previously demonstrated individually to effectively block agonist stimulated activation. These agents are quinacrine, flurbiprofen and...1 0 02 1 Quinacrine 0 0 25 13 0 67 28 5nM 0 25 38 0 67 56 50nM 0 25 37 0 44 61 .5M 0 6 25 0 38 28 Flurbiprofen 0 0 6 0 0 63 13 10nM 0 6 0 0 56 19 .lM

Spike threshold dynamics in spinal motoneurons during scratching and swimming.

PubMed

Grigonis, Ramunas; Alaburda, Aidas

2017-09-01

Action potential threshold can vary depending on firing history and synaptic inputs. We used an ex vivo carapace-spinal cord preparation from adult turtles to study spike threshold dynamics in motoneurons during two distinct types of functional motor behaviour - fictive scratching and fictive swimming. The threshold potential depolarizes by about 10 mV within each burst of spikes generated during scratch and swim network activity and recovers between bursts to a slightly depolarized level. Slow synaptic integration resulting in a wave of membrane potential depolarization is the factor influencing the threshold potential within firing bursts during motor behaviours. Depolarization of the threshold potential decreases the excitability of motoneurons and may provide a mechanism for stabilization of the response of a motoneuron to intense synaptic inputs to maintain the motor commands within an optimal range for muscle activation. During functional spinal neural network activity motoneurons receive intense synaptic input, and this could modulate the threshold for action potential generation, providing the ability to dynamically adjust the excitability and recruitment order for functional needs. In the present study we investigated the dynamics of action potential threshold during motor network activity. Intracellular recordings from spinal motoneurons in an ex vivo carapace-spinal cord preparation from adult turtles were performed during two distinct types of motor behaviour - fictive scratching and fictive swimming. We found that the threshold of the first spike in episodes of scratching and swimming was the lowest. The threshold potential depolarizes by about 10 mV within each burst of spikes generated during scratch and swim network activity and recovers between bursts to a slightly depolarized level. Depolarization of the threshold potential results in decreased excitability of motoneurons. Synaptic inputs do not modulate the threshold of the first action potential during episodes of scratching or of swimming. There is no correlation between changes in spike threshold and interspike intervals within bursts. Slow synaptic integration that results in a wave of membrane potential depolarization rather than fast synaptic events preceding each spike is the factor influencing the threshold potential within firing bursts during motor behaviours. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

Mouse VAP33 is associated with the endoplasmic reticulum and microtubules

PubMed Central

Skehel, P. A.; Fabian-Fine, R.; Kandel, E. R.

2000-01-01

VAMP/synaptobrevin is a synaptic vesicle protein that is essential for neurotransmitter release. Intracellular injection of antisera against the Aplysia californica VAMP/synaptobrevin-binding protein ApVAP33 inhibited evoked excitatory postsynaptic potentials (EPSPs) in cultured cells, suggesting that this association may regulate the function of VAMP/synaptobrevin. We have identified and characterized a mouse homologue of ApVAP33, mVAP33. The overall domain structure of the proteins is conserved, and they have similar biochemical properties. mVAP33 mRNA is detectable in all mouse tissues examined, in contrast to the more restricted expression seen in A. californica. We analyzed the cellular distribution of mVAP33 protein in brain slices and cultured cortical cells by light and electron microscopy. Although present at higher levels in neurons, immunoreactivity was detected throughout both neurons and glia in a reticular pattern similar to that of endoplasmic reticulum-resident proteins. mVAP33 does not colocalize with VAMP/synaptobrevin at synaptic structures, but expression overlaps with lower levels of VAMP/synaptobrevin in the soma. Ultrastructural analysis revealed mVAP33 associated with microtubules and intracellular vesicles of heterogeneous size. In primary neuronal cultures, large aggregates of mVAP33 are also detected in short filamentous structures, which are occasionally associated with intracellular membranes. There is no evidence for accumulation of mVAP33 on synaptic vesicles or at the plasma membrane. These data suggest that mVAP33 is an endoplasmic-reticulum–resident protein that associates with components of the cytoskeleton. Any functional interaction between mVAP33 and VAMP/synaptobrevin, therefore, most likely involves the delivery of components to synaptic terminals rather than a direct participation in synaptic vesicle exocytosis. PMID:10655491

Nuclear and membrane estrogen receptor antagonists induce similar mTORC2 activation-reversible changes in synaptic protein expression and actin polymerization in the mouse hippocampus.

PubMed

Xing, Fang-Zhou; Zhao, Yan-Gang; Zhang, Yuan-Yuan; He, Li; Zhao, Ji-Kai; Liu, Meng-Ying; Liu, Yan; Zhang, Ji-Qiang

2018-06-01

Estrogens play pivotal roles in hippocampal synaptic plasticity through nuclear receptors (nERs; including ERα and ERβ) and the membrane receptor (mER; also called GPR30), but the underlying mechanism and the contributions of nERs and mER remain unclear. Mammalian target of rapamycin complex 2 (mTORC2) is involved in actin cytoskeleton polymerization and long-term memory, but whether mTORC2 is involved in the regulation of hippocampal synaptic plasticity by ERs is unclear. We treated animals with nER antagonists (MPP/PHTPP) or the mER antagonist (G15) alone or in combination with A-443654, an activator of mTORC2. Then, we examined the changes in hippocampal SRC-1 expression, mTORC2 signaling (rictor and phospho-AKTSer473), actin polymerization (phospho-cofilin and profilin-1), synaptic protein expression (GluR1, PSD95, spinophilin, and synaptophysin), CA1 spine density, and synapse density. All of the examined parameters except synaptophysin expression were significantly decreased by MPP/PHTPP and G15 treatment. MPP/PHTPP and G15 induced a similar decrease in most parameters except p-cofilin, GluR1, and spinophilin expression. The ER antagonist-induced decreases in these parameters were significantly reversed by mTORC2 activation, except for the change in SRC-1, rictor, and synaptophysin expression. nERs and mER contribute similarly to the changes in proteins and structures associated with synaptic plasticity, and mTORC2 may be a novel target of hippocampal-dependent dementia such as Alzheimer's disease as proposed by previous studies. © 2018 John Wiley & Sons Ltd.

Ankyrins: Roles in synaptic biology and pathology.

PubMed

Smith, Katharine R; Penzes, Peter

2018-05-03

Ankyrins are broadly expressed adaptors that organize diverse membrane proteins into specialized domains and link them to the sub-membranous cytoskeleton. In neurons, ankyrins are known to have essential roles in organizing the axon initial segment and nodes of Ranvier. However, recent studies have revealed novel functions for ankyrins at synapses, where they organize and stabilize neurotransmitter receptors, modulate dendritic spine morphology and control adhesion to the presynaptic site. Ankyrin genes have also been highly associated with a range of neurodevelopmental and psychiatric diseases, including bipolar disorder, schizophrenia and autism, which all demonstrate overlap in their genetics, mechanisms and phenotypes. This review discusses the novel synaptic functions of ankyrin proteins in neurons, and places these exciting findings in the context of ANK genes as key neuropsychiatric disorder risk-factors. Copyright © 2018 Elsevier Inc. All rights reserved.

Membrane Properties and the Balance between Excitation and Inhibition Control Gamma-Frequency Oscillations Arising from Feedback Inhibition

PubMed Central

Economo, Michael N.; White, John A.

2012-01-01

Computational studies as well as in vivo and in vitro results have shown that many cortical neurons fire in a highly irregular manner and at low average firing rates. These patterns seem to persist even when highly rhythmic signals are recorded by local field potential electrodes or other methods that quantify the summed behavior of a local population. Models of the 30–80 Hz gamma rhythm in which network oscillations arise through ‘stochastic synchrony’ capture the variability observed in the spike output of single cells while preserving network-level organization. We extend upon these results by constructing model networks constrained by experimental measurements and using them to probe the effect of biophysical parameters on network-level activity. We find in simulations that gamma-frequency oscillations are enabled by a high level of incoherent synaptic conductance input, similar to the barrage of noisy synaptic input that cortical neurons have been shown to receive in vivo. This incoherent synaptic input increases the emergent network frequency by shortening the time scale of the membrane in excitatory neurons and by reducing the temporal separation between excitation and inhibition due to decreased spike latency in inhibitory neurons. These mechanisms are demonstrated in simulations and in vitro current-clamp and dynamic-clamp experiments. Simulation results further indicate that the membrane potential noise amplitude has a large impact on network frequency and that the balance between excitatory and inhibitory currents controls network stability and sensitivity to external inputs. PMID:22275859

Chemical synapses without synaptic vesicles: Purinergic neurotransmission through a CALHM1 channel-mitochondrial signaling complex.

PubMed

Romanov, Roman A; Lasher, Robert S; High, Brigit; Savidge, Logan E; Lawson, Adam; Rogachevskaja, Olga A; Zhao, Haitian; Rogachevsky, Vadim V; Bystrova, Marina F; Churbanov, Gleb D; Adameyko, Igor; Harkany, Tibor; Yang, Ruibiao; Kidd, Grahame J; Marambaud, Philippe; Kinnamon, John C; Kolesnikov, Stanislav S; Finger, Thomas E

2018-05-08

Conventional chemical synapses in the nervous system involve a presynaptic accumulation of neurotransmitter-containing vesicles, which fuse with the plasma membrane to release neurotransmitters that activate postsynaptic receptors. In taste buds, type II receptor cells do not have conventional synaptic features but nonetheless show regulated release of their afferent neurotransmitter, ATP, through a large-pore, voltage-gated channel, CALHM1. Immunohistochemistry revealed that CALHM1 was localized to points of contact between the receptor cells and sensory nerve fibers. Ultrastructural and super-resolution light microscopy showed that the CALHM1 channels were consistently associated with distinctive, large (1- to 2-μm) mitochondria spaced 20 to 40 nm from the presynaptic membrane. Pharmacological disruption of the mitochondrial respiratory chain limited the ability of taste cells to release ATP, suggesting that the immediate source of released ATP was the mitochondrion rather than a cytoplasmic pool of ATP. These large mitochondria may serve as both a reservoir of releasable ATP and the site of synthesis. The juxtaposition of the large mitochondria to areas of membrane displaying CALHM1 also defines a restricted compartment that limits the influx of Ca 2+ upon opening of the nonselective CALHM1 channels. These findings reveal a distinctive organelle signature and functional organization for regulated, focal release of purinergic signals in the absence of synaptic vesicles. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Electrophysiology of neurones of the inferior mesenteric ganglion of the cat.

PubMed Central

Julé, Y; Szurszewski, J H

1983-01-01

Intracellular recordings were obtained from cells in vitro in the inferior mesenteric ganglia of the cat. Neurones could be classified into three types: non-spontaneous, irregular discharging and regular discharging neurones. Non-spontaneous neurones had a stable resting membrane potential and responded with action potentials to indirect preganglionic nerve stimulation and to intracellular injection of depolarizing current. Irregular discharging neurones were characterized by a discharge of excitatory post-synaptic potentials (e.p.s.p.s.) which sometimes gave rise to action potentials. This activity was abolished by hexamethonium bromide, chlorisondamine and d-tubocurarine chloride. Tetrodotoxin and a low Ca2+ -high Mg2+ solution also blocked on-going activity in irregular discharging neurones. Regular discharging neurones were characterized by a rhythmic discharge of action potentials. Each action potential was preceded by a gradual depolarization of the intracellularly recorded membrane potential. Intracellular injection of hyperpolarizing current abolished the regular discharge of action potential. No synaptic potentials were observed during hyperpolarization of the membrane potential. Nicotinic, muscarinic and adrenergic receptor blocking drugs did not modify the discharge of action potentials in regular discharging neurones. A low Ca2+ -high Mg2+ solution also had no effect on the regular discharge of action potentials. Interpolation of an action potential between spontaneous action potentials in regular discharging neurones reset the rhythm of discharge. It is suggested that regular discharging neurones were endogenously active and that these neurones provided synaptic input to irregular discharging neurones. PMID:6140310

Electrophysiology of neurones of the inferior mesenteric ganglion of the cat.

PubMed

Julé, Y; Szurszewski, J H

1983-11-01

Intracellular recordings were obtained from cells in vitro in the inferior mesenteric ganglia of the cat. Neurones could be classified into three types: non-spontaneous, irregular discharging and regular discharging neurones. Non-spontaneous neurones had a stable resting membrane potential and responded with action potentials to indirect preganglionic nerve stimulation and to intracellular injection of depolarizing current. Irregular discharging neurones were characterized by a discharge of excitatory post-synaptic potentials (e.p.s.p.s.) which sometimes gave rise to action potentials. This activity was abolished by hexamethonium bromide, chlorisondamine and d-tubocurarine chloride. Tetrodotoxin and a low Ca2+ -high Mg2+ solution also blocked on-going activity in irregular discharging neurones. Regular discharging neurones were characterized by a rhythmic discharge of action potentials. Each action potential was preceded by a gradual depolarization of the intracellularly recorded membrane potential. Intracellular injection of hyperpolarizing current abolished the regular discharge of action potential. No synaptic potentials were observed during hyperpolarization of the membrane potential. Nicotinic, muscarinic and adrenergic receptor blocking drugs did not modify the discharge of action potentials in regular discharging neurones. A low Ca2+ -high Mg2+ solution also had no effect on the regular discharge of action potentials. Interpolation of an action potential between spontaneous action potentials in regular discharging neurones reset the rhythm of discharge. It is suggested that regular discharging neurones were endogenously active and that these neurones provided synaptic input to irregular discharging neurones.

Properties of Fiber Cell Plasma Membranes Isolated from the Cortex and Nucleus of the Porcine Eye Lens

PubMed Central

Mainali, Laxman; Raguz, Marija; O’Brien, William J.; Subczynski, Witold K.

2012-01-01

The organization and physical properties of the lipid bilayer portion of intact cortical and nuclear fiber cell plasma membranes isolated from the eyes lenses of two-year-old pigs were studied using electron paramagnetic resonance (EPR) spin-labeling. Membrane fluidity, hydrophobicity, and the oxygen transport parameter (OTP) were assessed from the EPR spectra of precisely positioned spin labels. Intact cortical and nuclear membranes, which include membrane proteins, were found to contain three distinct lipid environments. These lipid environments were termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain (lipids in protein aggregates). The amount of boundary and trapped lipids was greater in intact nuclear membranes than in cortical membranes. The properties of intact membranes were compared with the organization and properties of lens lipid membranes made of the total lipid extracts from the lens cortex or nucleus. In cortical lens lipid membranes, only one homogenous environment was detected, which was designated as a bulk lipid domain (phospholipid bilayer saturated with cholesterol). Lens lipid membranes prepared from the lens nucleus possessed two domains, assigned as a bulk lipid domain and a cholesterol bilayer domain (CBD). In intact nuclear membranes, it was difficult to discriminate the CBD, which was clearly detected in nuclear lens lipid membranes because the OTP measured in the CBD is the same as in the domain formed by trapped lipids. The two domains unique to intact membranes—namely, the domain formed by boundary lipids and the domain formed by trapped lipids—were most likely formed due to the presence of membrane proteins. It is concluded that formation of rigid and practically impermeable domains is enhanced in the lens nucleus, indicating changes in membrane composition that may help to maintain low oxygen concentration in this lens region. PMID:22326289

Exocytosis and Endocytosis: Modes, Functions, and Coupling Mechanisms*

PubMed Central

Wu, Ling-Gang; Hamid, Edaeni; Shin, Wonchul; Chiang, Hsueh-Cheng

2016-01-01

Vesicle exocytosis releases content to mediate many biological events, including synaptic transmission essential for brain functions. Following exocytosis, endocytosis is initiated to retrieve exocytosed vesicles within seconds to minutes. Decades of studies in secretory cells reveal three exocytosis modes coupled to three endocytosis modes: (a) full-collapse fusion, in which vesicles collapse into the plasma membrane, followed by classical endocytosis involving membrane invagination and vesicle reformation; (b) kiss-and-run, in which the fusion pore opens and closes; and (c) compound exocytosis, which involves exocytosis of giant vesicles formed via vesicle-vesicle fusion, followed by bulk endocytosis that retrieves giant vesicles. Here we review these exo- and endocytosis modes and their roles in regulating quantal size and synaptic strength, generating synaptic plasticity, maintaining exocytosis, and clearing release sites for vesicle replenishment. Furthermore, we highlight recent progress in understanding how vesicle endocytosis is initiated and is thus coupled to exocytosis. The emerging model is that calcium influx via voltage-dependent calcium channels at the calcium microdomain triggers endocytosis and controls endocytosis rate; calmodulin and synaptotagmin are the calcium sensors; and the exocytosis machinery, including SNARE proteins (synaptobrevin, SNAP25, and syntaxin), is needed to coinitiate endocytosis, likely to control the amount of endocytosis. PMID:24274740

Diet and cognition: interplay between cell metabolism and neuronal plasticity

PubMed Central

Gomez-Pinilla, Fernando; Tyagi, Ethika

2014-01-01

Purpose of Study To discuss studies in humans and animals revealing the ability of foods to benefit the brain: new information with regards to mechanisms of action and the treatment of neurological and psychiatric disorders. Recent Findings Dietary factors exert their effects on the brain by affecting molecular events related to the management of energy metabolism and synaptic plasticity. Energy metabolism influences neuronal function, neuronal signaling, and synaptic plasticity, ultimately affecting mental health. Epigenetic regulation of neuronal plasticity appears as an important mechanism by which foods can prolong their effects on long term neuronal plasticity. Summary The prime focus of the discussion is to emphasize the role of cell metabolism as a mediator for the action of foods on the brain. Oxidative stress promotes damage to phospholipids present in the plasma membrane such as the omega-3 fatty acid DHA, disrupting neuronal signaling. Thus, dietary DHA seems crucial for supporting plasma membrane function, interneuronal signaling, and cognition. The dual action of brain-derived neurotrophic factor (BDNF) in neuronal metabolism and synaptic plasticity is crucial for activating signaling cascades under the action of diet and other environmental factors, using mechanisms of epigenetic regulation. PMID:24071781

More than synaptic plasticity: Role of nonsynaptic plasticity in learning and memory

PubMed Central

Mozzachiodi, Riccardo; Byrne, John H.

2009-01-01

Decades of research on the cellular mechanisms of memory have led to the widely-held view that memories are stored as modifications of synaptic strength. These changes involve presynaptic processes, such as direct modulation of the release machinery, or postsynaptic processes, such as modulation of receptor properties. Parallel studies have revealed that memories may also be stored by nonsynaptic processes, such as modulation of voltage-dependent membrane conductances, which are expressed as changes in neuronal excitability. Although in some cases nonsynaptic changes may function as part of the engram itself, they may also serve as mechanisms through which a neural circuit is set to a permissive state to facilitate synaptic modifications that are necessary for memory storage. PMID:19889466

Electron paramagnetic resonance study of lipid and protein membrane components of erythrocytes oxidized with hydrogen peroxide

PubMed Central

Mendanha, S.A.; Anjos, J.L.V.; Silva, A.H.M.; Alonso, A.

2012-01-01

Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to monitor membrane dynamic changes in erythrocytes subjected to oxidative stress with hydrogen peroxide (H2O2). The lipid spin label, 5-doxyl stearic acid, responded to dramatic reductions in membrane fluidity, which was correlated with increases in the protein content of the membrane. Membrane rigidity, associated with the binding of hemoglobin (Hb) to the erythrocyte membrane, was also indicated by a spin-labeled maleimide, 5-MSL, covalently bound to the sulfhydryl groups of membrane proteins. At 2% hematocrit, these alterations in membrane occurred at very low concentrations of H2O2 (50 µM) after only 5 min of incubation at 37°C in azide phosphate buffer, pH 7.4. Lipid peroxidation, suggested by oxidative hemolysis and malondialdehyde formation, started at 300 µM H2O2 (for incubation of 3 h), which is a concentration about six times higher than those detected with the probes. Ascorbic acid and α-tocopherol protected the membrane against lipoperoxidation, but did not prevent the binding of proteins to the erythrocyte membrane. Moreover, the antioxidant (+)-catechin, which also failed to prevent the cross-linking of cytoskeletal proteins with Hb, was very effective in protecting erythrocyte ghosts from lipid peroxidation induced by the Fenton reaction. This study also showed that EPR spectroscopy can be useful to assess the molecular dynamics of red blood cell membranes in both the lipid and protein domains and examine oxidation processes in a system that is so vulnerable to oxidation. PMID:22473321

Additive effects on the energy barrier for synaptic vesicle fusion cause supralinear effects on the vesicle fusion rate

PubMed Central

Schotten, Sebastiaan; Meijer, Marieke; Walter, Alexander Matthias; Huson, Vincent; Mamer, Lauren; Kalogreades, Lawrence; ter Veer, Mirelle; Ruiter, Marvin; Brose, Nils; Rosenmund, Christian

2015-01-01

The energy required to fuse synaptic vesicles with the plasma membrane (‘activation energy’) is considered a major determinant in synaptic efficacy. From reaction rate theory, we predict that a class of modulations exists, which utilize linear modulation of the energy barrier for fusion to achieve supralinear effects on the fusion rate. To test this prediction experimentally, we developed a method to assess the number of releasable vesicles, rate constants for vesicle priming, unpriming, and fusion, and the activation energy for fusion by fitting a vesicle state model to synaptic responses induced by hypertonic solutions. We show that complexinI/II deficiency or phorbol ester stimulation indeed affects responses to hypertonic solution in a supralinear manner. An additive vs multiplicative relationship between activation energy and fusion rate provides a novel explanation for previously observed non-linear effects of genetic/pharmacological perturbations on synaptic transmission and a novel interpretation of the cooperative nature of Ca2+-dependent release. DOI: http://dx.doi.org/10.7554/eLife.05531.001 PMID:25871846

Sensing Exocytosis and Triggering Endocytosis at Synapses: Synaptic Vesicle Exocytosis–Endocytosis Coupling

PubMed Central

Lou, Xuelin

2018-01-01

The intact synaptic structure is critical for information processing in neural circuits. During synaptic transmission, rapid vesicle exocytosis increases the size of never terminals and endocytosis counteracts the increase. Accumulating evidence suggests that SV exocytosis and endocytosis are tightly connected in time and space during SV recycling, and this process is essential for synaptic function and structural stability. Research in the past has illustrated the molecular details of synaptic vesicle (SV) exocytosis and endocytosis; however, the mechanisms that timely connect these two fundamental events are poorly understood at central synapses. Here we discuss recent progress in SV recycling and summarize several emerging mechanisms by which synapses can “sense” the occurrence of exocytosis and timely initiate compensatory endocytosis. They include Ca2+ sensing, SV proteins sensing, and local membrane stress sensing. In addition, the spatial organization of endocytic zones adjacent to active zones provides a structural basis for efficient coupling between SV exocytosis and endocytosis. Through linking different endocytosis pathways with SV fusion, these mechanisms ensure necessary plasticity and robustness of nerve terminals to meet diverse physiological needs. PMID:29593500

Drive the Car(go)s-New Modalities to Control Cargo Trafficking in Live Cells.

PubMed

Mondal, Payel; Khamo, John S; Krishnamurthy, Vishnu V; Cai, Qi; Zhang, Kai

2017-01-01

Synaptic transmission is a fundamental molecular process underlying learning and memory. Successful synaptic transmission involves coupled interaction between electrical signals (action potentials) and chemical signals (neurotransmitters). Defective synaptic transmission has been reported in a variety of neurological disorders such as Autism and Alzheimer's disease. A large variety of macromolecules and organelles are enriched near functional synapses. Although a portion of macromolecules can be produced locally at the synapse, a large number of synaptic components especially the membrane-bound receptors and peptide neurotransmitters require active transport machinery to reach their sites of action. This spatial relocation is mediated by energy-consuming, motor protein-driven cargo trafficking. Properly regulated cargo trafficking is of fundamental importance to neuronal functions, including synaptic transmission. In this review, we discuss the molecular machinery of cargo trafficking with emphasis on new experimental strategies that enable direct modulation of cargo trafficking in live cells. These strategies promise to provide insights into a quantitative understanding of cargo trafficking, which could lead to new intervention strategies for the treatment of neurological diseases.

Equilibrium potential for the postsynaptic response in the squid giant synapse.

PubMed

Llinás, R; Joyner, R W; Nicholson, C

1974-11-01

The reversal potential for the EPSP in the squid giant synapse has been studied by means of an intracellular, double oil gap technique. This method allows the electrical isolation of a portion of the axon from the rest of the fiber and generates a quasi-isopotential segment. In order to make the input resistance of this nerve segment as constant as possible, the electroresponsive properties of the nerve membrane were blocked by intracellular injection of tetraethylammonium (TEA) and local extracellular application of tetrodotoxin (TTX). Thus, EPSP's could be evoked in the isolated segment with a minimal amount of electroresponsive properties. The reversal potential for the EPSP (EEPSP) was measured by recording the synaptic potential or the synaptic current during voltage clamping. The results indicate that EEPSP may vary from +15 to +25 mV, which is more positive than would be expected for a 1:1 conductance change for Na(+) and K(+) (approximately -15 mV) and too negative for a pure Na(+) conductance ((+)40 mV). This latter value (E(Na)) was directly determined in the voltage clamp experiments. The results suggest that the synaptic potential is probably produced by a permeability change to Na(+) to K(+) in a 4:1 ratio. No change in time-course was observed in the synaptic current at clamp levels of -100 and +90 mV. The implications of a variable ratio for Na(+)-K(+) permeability in subsynaptic-postsynaptic membranes are discussed.

Propagating waves can explain irregular neural dynamics.

PubMed

Keane, Adam; Gong, Pulin

2015-01-28

Cortical neurons in vivo fire quite irregularly. Previous studies about the origin of such irregular neural dynamics have given rise to two major models: a balanced excitation and inhibition model, and a model of highly synchronized synaptic inputs. To elucidate the network mechanisms underlying synchronized synaptic inputs and account for irregular neural dynamics, we investigate a spatially extended, conductance-based spiking neural network model. We show that propagating wave patterns with complex dynamics emerge from the network model. These waves sweep past neurons, to which they provide highly synchronized synaptic inputs. On the other hand, these patterns only emerge from the network with balanced excitation and inhibition; our model therefore reconciles the two major models of irregular neural dynamics. We further demonstrate that the collective dynamics of propagating wave patterns provides a mechanistic explanation for a range of irregular neural dynamics, including the variability of spike timing, slow firing rate fluctuations, and correlated membrane potential fluctuations. In addition, in our model, the distributions of synaptic conductance and membrane potential are non-Gaussian, consistent with recent experimental data obtained using whole-cell recordings. Our work therefore relates the propagating waves that have been widely observed in the brain to irregular neural dynamics. These results demonstrate that neural firing activity, although appearing highly disordered at the single-neuron level, can form dynamical coherent structures, such as propagating waves at the population level. Copyright © 2015 the authors 0270-6474/15/351591-15$15.00/0.

Membrane Diffusion Occurs by Continuous-Time Random Walk Sustained by Vesicular Trafficking.

PubMed

Goiko, Maria; de Bruyn, John R; Heit, Bryan

2018-06-19

Diffusion in cellular membranes is regulated by processes that occur over a range of spatial and temporal scales. These processes include membrane fluidity, interprotein and interlipid interactions, interactions with membrane microdomains, interactions with the underlying cytoskeleton, and cellular processes that result in net membrane movement. The complex, non-Brownian diffusion that results from these processes has been difficult to characterize, and moreover, the impact of factors such as membrane recycling on membrane diffusion remains largely unexplored. We have used a careful statistical analysis of single-particle tracking data of the single-pass plasma membrane protein CD93 to show that the diffusion of this protein is well described by a continuous-time random walk in parallel with an aging process mediated by membrane corrals. The overall result is an evolution in the diffusion of CD93: proteins initially diffuse freely on the cell surface but over time become increasingly trapped within diffusion-limiting membrane corrals. Stable populations of freely diffusing and corralled CD93 are maintained by an endocytic/exocytic process in which corralled CD93 is selectively endocytosed, whereas freely diffusing CD93 is replenished by exocytosis of newly synthesized and recycled CD93. This trafficking not only maintained CD93 diffusivity but also maintained the heterogeneous distribution of CD93 in the plasma membrane. These results provide insight into the nature of the biological and biophysical processes that can lead to significantly non-Brownian diffusion of membrane proteins and demonstrate that ongoing membrane recycling is critical to maintaining steady-state diffusion and distribution of proteins in the plasma membrane. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Safeguards of Neurotransmission: Endocytic Adaptors as Regulators of Synaptic Vesicle Composition and Function

PubMed Central

Kaempf, Natalie; Maritzen, Tanja

2017-01-01

Communication between neurons relies on neurotransmitters which are released from synaptic vesicles (SVs) upon Ca2+ stimuli. To efficiently load neurotransmitters, sense the rise in intracellular Ca2+ and fuse with the presynaptic membrane, SVs need to be equipped with a stringently controlled set of transmembrane proteins. In fact, changes in SV protein composition quickly compromise neurotransmission and most prominently give rise to epileptic seizures. During exocytosis SVs fully collapse into the presynaptic membrane and consequently have to be replenished to sustain neurotransmission. Therefore, surface-stranded SV proteins have to be efficiently retrieved post-fusion to be used for the generation of a new set of fully functional SVs, a process in which dedicated endocytic sorting adaptors play a crucial role. The question of how the precise reformation of SVs is achieved is intimately linked to how SV membranes are retrieved. For a long time both processes were believed to be two sides of the same coin since Clathrin-mediated endocytosis (CME), the proposed predominant SV recycling mode, will jointly retrieve SV membranes and proteins. However, with the recent proposal of Clathrin-independent SV recycling pathways SV membrane retrieval and SV reformation turn into separable events. This review highlights the progress made in unraveling the molecular mechanisms mediating the high-fidelity retrieval of SV proteins and discusses how the gathered knowledge about SV protein recycling fits in with the new notions of SV membrane endocytosis. PMID:29085282

Alzheimer's Therapeutics Targeting Amyloid Beta 1–42 Oligomers II: Sigma-2/PGRMC1 Receptors Mediate Abeta 42 Oligomer Binding and Synaptotoxicity

PubMed Central

Izzo, Nicholas J.; Xu, Jinbin; Zeng, Chenbo; Kirk, Molly J.; Mozzoni, Kelsie; Silky, Colleen; Rehak, Courtney; Yurko, Raymond; Look, Gary; Rishton, Gilbert; Safferstein, Hank; Cruchaga, Carlos; Goate, Alison; Cahill, Michael A.; Arancio, Ottavio; Mach, Robert H.; Craven, Rolf; Head, Elizabeth; LeVine, Harry; Spires-Jones, Tara L.; Catalano, Susan M.

2014-01-01

Amyloid beta (Abeta) 1–42 oligomers accumulate in brains of patients with Mild Cognitive Impairment (MCI) and disrupt synaptic plasticity processes that underlie memory formation. Synaptic binding of Abeta oligomers to several putative receptor proteins is reported to inhibit long-term potentiation, affect membrane trafficking and induce reversible spine loss in neurons, leading to impaired cognitive performance and ultimately to anterograde amnesia in the early stages of Alzheimer's disease (AD). We have identified a receptor not previously associated with AD that mediates the binding of Abeta oligomers to neurons, and describe novel therapeutic antagonists of this receptor capable of blocking Abeta toxic effects on synapses in vitro and cognitive deficits in vivo. Knockdown of sigma-2/PGRMC1 (progesterone receptor membrane component 1) protein expression in vitro using siRNA results in a highly correlated reduction in binding of exogenous Abeta oligomers to neurons of more than 90%. Expression of sigma-2/PGRMC1 is upregulated in vitro by treatment with Abeta oligomers, and is dysregulated in Alzheimer's disease patients' brain compared to age-matched, normal individuals. Specific, high affinity small molecule receptor antagonists and antibodies raised against specific regions on this receptor can displace synthetic Abeta oligomer binding to synaptic puncta in vitro and displace endogenous human AD patient oligomers from brain tissue sections in a dose-dependent manner. These receptor antagonists prevent and reverse the effects of Abeta oligomers on membrane trafficking and synapse loss in vitro and cognitive deficits in AD mouse models. These findings suggest sigma-2/PGRMC1 receptors mediate saturable oligomer binding to synaptic puncta on neurons and that brain penetrant, small molecules can displace endogenous and synthetic oligomers and improve cognitive deficits in AD models. We propose that sigma-2/PGRMC1 is a key mediator of the pathological effects of Abeta oligomers in AD and is a tractable target for small molecule disease-modifying therapeutics. PMID:25390692

[The synaptic proteins of the temporal associative area in the neocortex (field Ep) of cats with normal and decreased cognitive capacities].

PubMed

Zakharova (Orlova), E I; Mukhin, E I

1994-01-01

Fractions of light and heavy synaptosomes were prepared from associative temporal area of cat brain, which were previously tested behaviorally for ability to solve the generalization, gnostic and abstraction tasks. The synaptic membrane subfractions and synaptoplasma fractions were isolated and the content of the total protein and of the demasked protein sulfhydryl groups (SH groups) was investigated spectrophotometrically. The maximal content of the demasked SH groups was revealed in the upper subfractions (mainly the membranes of cholinergic synapses) and minimal content--in the lower subfractions (mainly noncholinergic synapses). Significantly smaller total protein content in the upper and middle subfractions of light synaptosomes was found, and more demasked SH groups in the membrane-bound proteins of the upper and middle subfractions of light and heavy synaptosomes was found in the cortex area of the "clever" then "silly" cats. Suggestion concerning characteristic for brains of "clever" cats relatively low total quantity of synapses in the area Ep of the cortex and significantly higher proportion of cholinergic ones is discussed.

Caffeine Controls Glutamatergic Synaptic Transmission and Pyramidal Neuron Excitability in Human Neocortex

PubMed Central

Kerkhofs, Amber; Xavier, Ana C.; da Silva, Beatriz S.; Canas, Paula M.; Idema, Sander; Baayen, Johannes C.; Ferreira, Samira G.; Cunha, Rodrigo A.; Mansvelder, Huibert D.

2018-01-01

Caffeine is the most widely used psychoactive drug, bolstering attention and normalizing mood and cognition, all functions involving cerebral cortical circuits. Whereas studies in rodents showed that caffeine acts through the antagonism of inhibitory A1 adenosine receptors (A1R), neither the role of A1R nor the impact of caffeine on human cortical neurons is known. We here provide the first characterization of the impact of realistic concentrations of caffeine experienced by moderate coffee drinkers (50 μM) on excitability of pyramidal neurons and excitatory synaptic transmission in the human temporal cortex. Moderate concentrations of caffeine disinhibited several of the inhibitory A1R-mediated effects of adenosine, similar to previous observations in the rodent brain. Thus, caffeine restored the adenosine-induced decrease of both intrinsic membrane excitability and excitatory synaptic transmission in the human pyramidal neurons through antagonism of post-synaptic A1R. Indeed, the A1R-mediated effects of endogenous adenosine were more efficient to inhibit synaptic transmission than neuronal excitability. This was associated with a distinct affinity of caffeine for synaptic versus extra-synaptic human cortical A1R, probably resulting from a different molecular organization of A1R in human cortical synapses. These findings constitute the first neurophysiological description of the impact of caffeine on pyramidal neuron excitability and excitatory synaptic transmission in the human temporal cortex, providing adequate ground for the effects of caffeine on cognition in humans. PMID:29354052

Improvement of Transmembrane Transport Mechanism Study of Imperatorin on P-Glycoprotein-Mediated Drug Transport.

PubMed

Liao, Zheng-Gen; Tang, Tao; Guan, Xue-Jing; Dong, Wei; Zhang, Jing; Zhao, Guo-Wei; Yang, Ming; Liang, Xin-Li

2016-11-24

P-glycoprotein (P-gp) affects the transport of many drugs; including puerarin and vincristine. Our previous study demonstrated that imperatorin increased the intestinal absorption of puerarin and vincristine by inhibiting P-gp-mediated drug efflux. However; the underlying mechanism was not known. The present study investigated the mechanism by which imperatorin promotes P-gp-mediated drug transport. We used molecular docking to predict the binding force between imperatorin and P-gp and the effect of imperatorin on P-gp activity. P-gp efflux activity and P-gp ATPase activity were measured using a rhodamine 123 (Rh-123) accumulation assay and a Pgp-Glo™ assay; respectively. The fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to assess cellular membrane fluidity in MDCK-MDR1 cells. Western blotting was used to analyze the effect of imperatorin on P-gp expression; and P-gp mRNA levels were assessed by qRT-PCR. Molecular docking results demonstrated that the binding force between imperatorin and P-gp was much weaker than the force between P-gp and verapamil (a P-gp substrate). Imperatorin activated P-gp ATPase activity; which had a role in the inhibition of P-gp activity. Imperatorin promoted Rh-123 accumulation in MDCK-MDR1 cells and decreased cellular membrane fluidity. Western blotting demonstrated that imperatorin inhibited P-gp expression; and qRT-PCR revealed that imperatorin down-regulated P-gp (MDR1) gene expression. Imperatorin decreased P-gp-mediated drug efflux by inhibiting P-gp activity and the expression of P-gp mRNA and protein. Our results suggest that imperatorin could down-regulate P-gp expression to overcome multidrug resistance in tumors.

Effects of in vitro supplementation with Syzygium cumini (L.) on platelets from subjects affected by diabetes mellitus.

PubMed

Raffaelli, Francesca; Borroni, Francesca; Alidori, Alessandro; Tirabassi, Giacomo; Faloia, Emanuela; Rabini, Rosa Anna; Giulietti, Alessia; Mazzanti, Laura; Nanetti, Laura; Vignini, Arianna

2015-01-01

The aim of this study was to assess the in vitro effects of Syzygium cumini (L.) (Sc) incubation on platelets from patients with diabetes, in order to test its efficacy as a potential adjuvant therapy. This study was performed on 77 patients with diabetes [29 in good (DMgc) and 48 in poor glycemic control (DMpc)] and 85 controls. In patients, platelets were analyzed at recruitment and after in vitro Sc incubation (final concentration of 200 µg/ml for 3 hours at 37 °C), whereas in controls only basal evaluation was performed. Lipoperoxide and nitric oxide (NO) levels, superoxide dismutase (SOD) and Na(+)/K(+) ATPase activities, total antioxidant capacity (TAC), and membrane fluidity tested by anisotropy of fluorescent probes 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 1-6-phenyl-1,3,5-hexatriene (DPH) were determined. Collagen-induced platelet aggregation was also evaluated. In vitro Sc activity counteracts oxidative damage, by improving platelet function through augmented membrane fluidity and Na(+)/K(+) ATPase activity; it also enhances antioxidant system functionality by increasing NO levels, SOD activity, and TAC and by decreasing lipoperoxide levels both in whole samples and in DMgc and DMpc. In addition, a slight tendency towards collagen-induced platelet aggregation decrease after Sc was observed. However, all these parameters, even after improvement, did not reach the levels of control subjects. Our results suggest that Sc may have a preventive and protective effect in oxidative damage progression associated with diabetes mellitus and its complications. If our data will be confirmed, Sc supplementation might become a further tool in the management of this disease, especially in view of its easy availability, safety, low cost, and absence of side effects.

A comparison study between different molecular weight polysaccharides derived from Lentinus edodes and their antioxidant activities in vivo.

PubMed

You, Ruxu; Wang, Kaiping; Liu, Jinyu; Liu, Maochang; Luo, Li; Zhang, Yu

2011-12-01

Polysaccharide purified Lentinus edodes (Berk.) Sing (Tricholomataceae) has been reported to attenuate oxidative stress in vitro. This study investigated whether polysaccharides from L. edodes with different molecular weight have protective effects against oxidative stress induced by D-galactose (D-gal) in vivo, and determined the specific relationship between molecular weight and antioxidant activity. In the present study, we successfully obtained three purified polysaccharides, coded as LT1, LT2, and LT3, and their molecular weights were 25.5, 306.2, and 605.4 kDa, respectively. The D-gal-treated mice received three polysaccharides once daily for 60 days. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), the content of malondialdehyde (MDA), and erythrocyte membrane fluidity were measured to evaluate the changes of the antioxidant ability. It was demonstrated that the administration of LT1, LT2, and LT3 could improve the antioxidant status to different levels. Furthermore, LT2 exhibited the highest antioxidant ability among these samples in vivo. Indeed, LT2 significantly decreased the content of MDA in liver (15.91 ± 0.31 versus 23.79 ± 1.18 nmol/mg protein for the model group, p < 0.05), enhanced the fluidity of erythrocyte membrane (2.458 ± 0.023 versus 2.167 ± 0.024 for the model group, p < 0.05), and increased the activities of SOD (147.19 ± 4.90 versus 82.26 ± 5.55 units/mg protein for the model group, p < 0.05) and GSH-Px (310.91 ± 6.24 versus 243.64 ± 6.77 units/mg protein for the model group, p < 0.05) in liver. The LT2 had a potential to be used as a novel natural antioxidant.