Migraine and Its Association with Hyperactivity of Cell Membranes in the Course of Latent Magnesium Deficiency-Preliminary Study of the Importance of the Latent Tetany Presence in the Migraine Pathogenesis.

So far, there is no consistent and convincing theory explaining the pathogenesis of migraines. Vascular disorders, the effect of oxidative stress on neurons, and the contribution of magnesium-calcium deficiencies in triggering cortical depression and abnormal glutaminergic neurotransmission are taken into account. However, there are no reliable publications confirming the role of dietary deficits of magnesium and latent tetany as factors triggering migraine attacks. The aim of the study was to evaluate the influence of latent magnesium deficiency assessed with the electrophysiological tetany test on the course of migraine. The study included: a group of 35 patients (29 women and six men; in mean age 41 years) with migraine and a control group of 24 (17 women and seven men; in mean age 39 years) healthy volunteers. Migraine diagnosis was based on the International Headache Society criteria, 3rd edition. All patients and controls after full general and neurological examination were subjected to a standard electrophysiological ischemic tetany test. Moreover, the level of magnesium in blood serum was tested and was in the normal range in all patients. Then, the incidence of a positive tetany EMG test results in the migraine group and the results in the subgroups with and without aura were compared to the results in the control group. Moreover, the relationship between clinical markers of spasmophilia and the results of the tetany test was investigated in the migraine group. As well as the relationship between migraine frequency and tetany test results. There was no statistically significant difference in the occurrence of the electrophysiological exponent of spasmophilia between the migraine and control group. Neither correlation between the occurrence of clinical symptoms nor the frequency of migraine attacks and the results of the tetany test was stated (p > 0.05). However, there was an apparent statistical difference between the subgroup of migraine patients with aura in relation to the control group (p < 0.05). The result raises hope to find a trigger for migraine attacks of this clinical form, the more that this factor may turn out to be easy to supplement with dietary supplementation.

The protein kinase Ire1 impacts pathogenicity of Candida albicans by regulating homeostatic adaptation to endoplasmic reticulum stress.

The unfolded protein response (UPR), crucial for the maintenance of endoplasmic reticulum (ER) homeostasis, is tied to the regulation of multiple cellular processes in pathogenic fungi. Here, we show that Candida albicans relies on an ER-resident protein, inositol-requiring enzyme 1 (Ire1) for sensing ER stress and activating the UPR. Compromised Ire1 function impacts cellular processes that are dependent on functional secretory homeostasis, as inferred from transcriptional profiling. Concordantly, an Ire1-mutant strain exhibits pleiotropic roles in ER stress response, antifungal tolerance, cell wall regulation and virulence-related traits. Hac1 is the downstream target of C. albicans Ire1 as it initiates the unconventional splicing of the 19 bp intron from HAC1 mRNA during tunicamycin-induced ER stress. Ire1 also activates the UPR in response to perturbations in cell wall integrity and cell membrane homeostasis in a manner that does not necessitate the splicing of HAC1 mRNA. Furthermore, the Ire1-mutant strain is severely defective in hyphal morphogenesis and biofilm formation as well as in establishing a successful infection in vivo. Together, these findings demonstrate that C. albicans Ire1 functions to regulate traits that are essential for virulence and suggest its importance in responding to multiple stresses, thus integrating various stress signals to maintain ER homeostasis.

Evaluation of the Effectiveness of Crotoxin as an Antiseptic against Candida spp. Biofilms.

The growing number of oral infections caused by the Candida species are becoming harder to treat as the commonly used antibiotics become less effective. This drawback has led to the search for alternative strategies of treatment, which include the use of antifungal molecules derived from natural products. Herein, crotoxin (CTX), the main toxin of Crotalus durissus terrificus venom, was challenged against Candida tropicalis (CBS94) and Candida dubliniensis (CBS7987) strains by in vitro antimicrobial susceptibility tests. Minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), and inhibition of biofilm formation were evaluated after CTX treatment. In addition, CTX-induced cytotoxicity in HaCaT cells was assessed by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) colorimetric assay. Native CTX showed a higher antimicrobial activity (MIC = 47 µg/mL) when compared to CTX-containing mouthwash (MIC = 750 µg/mL) and nystatin (MIC = 375 µg/mL). Candida spp biofilm formation was more sensitive to both CTX and CTX-containing mouthwash (IC100 = 12 µg/mL) when compared to nystatin (IC100 > 47 µg/mL). Moreover, significant membrane permeabilization at concentrations of 1.5 and 47 µg/mL was observed. Native CTX was less cytotoxic to HaCaT cells than CTX-containing mouthwash or nystatin between 24 and 48 h. These preliminary findings highlight the potential use of CTX in the treatment of oral candidiasis caused by resistant strains.

Induction of Daptomycin Tolerance in Enterococcus faecalis by Fatty Acid Combinations.

Enterococcus faecalis is a Gram-positive bacterium that normally exists as an intestinal commensal in humans but is also a leading cause of nosocomial infections. Previous work noted that growth supplementation with serum induced tolerance to membrane-damaging agents, including the antibiotic daptomycin. Specific fatty acids found within serum could independently provide tolerance to daptomycin (protective fatty acids), yet some fatty acids found in serum did not and had negative effects on enterococcal physiology (nonprotective fatty acids). Here, we measured a wide array of physiological responses after supplementation with combinations of protective and nonprotective fatty acids to better understand how serum induces daptomycin tolerance. When cells were supplemented with either nonprotective fatty acid, palmitic acid, or stearic acid, there were marked defects in growth and morphology, but these defects were rescued upon supplementation with either protective fatty acid, oleic acid, or linoleic acid. Membrane fluidity decreased with growth in either palmitic or stearic acid alone but returned to basal levels when a protective fatty acid was supplied. Daptomycin tolerance could be induced if a protective fatty acid was provided with a nonprotective fatty acid, and some specific combinations protected as well as serum supplementation. While cell envelope charge has been associated with tolerance to daptomycin in other Gram-positive bacteria, we concluded that it does not correlate with the fatty acid-induced protection we observed. Based on these observations, we conclude that daptomycin tolerance by serum is driven by specific, protective fatty acids found within the fluid.IMPORTANCE With an increasing prevalence of antibiotic resistance in the clinic, we strive to understand more about microbial defensive mechanisms. A nongenetic tolerance to the antibiotic daptomycin was discovered in Enterococcus faecalis that results in the increased survival of bacterial populations after treatment with the drug. This tolerance mechanism likely synergizes with antibiotic resistance in the clinic. Given that this tolerance phenotype is induced by incorporation of fatty acids present in the host, it can be assumed that infections by this organism require a higher dose of antibiotic for successful eradication. The mixture of fatty acids in human fluids is quite diverse, with little understanding between the interplay of fatty acid combinations and the tolerance phenotype we observe. It is crucial to understand the effects of fatty acid combinations on E. faecalis physiology if we are to suppress the tolerance physiology in the clinic.

Profiling of myristoylation in Toxoplasma gondii reveals an N-myristoylated protein important for host cell penetration.

N-myristoylation is a ubiquitous class of protein lipidation across eukaryotes and N-myristoyl transferase (NMT) has been proposed as an attractive drug target in several pathogens. Myristoylation often primes for subsequent palmitoylation and stable membrane attachment, however, growing evidence suggests additional regulatory roles for myristoylation on proteins. Here we describe the myristoylated proteome of Toxoplasma gondii using chemoproteomic methods and show that a small-molecule NMT inhibitor developed against related Plasmodium spp. is also functional in Toxoplasma. We identify myristoylation on a transmembrane protein, the microneme protein 7 (MIC7), which enters the secretory pathway in an unconventional fashion with the myristoylated N-terminus facing the lumen of the micronemes. MIC7 and its myristoylation play a crucial role in the initial steps of invasion, likely during the interaction with and penetration of the host cell. Myristoylation of secreted eukaryotic proteins represents a substantial expansion of the functional repertoire of this co-translational modification.

A microscopic parasite known as Toxoplasma gondii infects around 30% of the human population. Most infections remain asymptomatic, but in people with a compromised immune system, developing fetuses and people infected with particular virulent strains of the parasite, infection can be fatal. T. gondii is closely related to other parasites that also infect humans, including the one that causes malaria. These parasites have complex lifecycles that involve successive rounds of invading the cells of their hosts, growing and then exiting these cells. Signaling proteins found at specific locations within parasite cells regulate the ability of the parasites to interact with and invade host cells. Sometimes these signaling proteins are attached to membranes using lipid anchors, for example through a molecule called myristic acid. An enzyme called NMT can attach myristic acid to one end of its target proteins. The myristic acid tag can influence the ability of target proteins to bind to other proteins, or to membranes. Previous studies have found that drugs that inhibit the NMT enzyme prevent the malaria parasite from successfully invading and growing inside host cells. The NMT enzyme from T. gondii is very similar to that of the malaria parasite. Broncel et al. have shown that the drug developed against P. falciparum also inhibits the ability of T. gondii to grow. These findings suggest that drugs against the NMT enzyme may be useful to treat diseases caused by T. gondii and other closely-related parasites. Broncel et al. also identified 65 proteins in T. gondii that contain a myristic acid tag using an approach called proteomics. One of the unexpected 'myristoylated' proteins identified in the experiments is known as MIC7. This protein was found to be transported onto the surface of T. gondii parasites and is required in its myristoylated form for the parasite to successfully invade host cells. This was surprising as myristoylated proteins are generally thought to not enter the pathway that brings proteins to the outside of cell. These findings suggest that myristic acid on proteins that are secreted can facilitate interactions between cells, maybe by inserting the myristic acid into the cell membrane.

Variations in the content of tonoplast lipids under abiotic stress.

MAIN CONCLUSION: The vacuolar membrane is an essential component in protecting the plant cell from stress factors. Different variations in the tonoplast lipid content, which depend on the type of stress, have been reviewed. The lipid content of vacuolar membranes of beet roots (Beta vulgaris L.) under hypoosmotic, hyperosmotic and oxidative types of stress has been studied. These types of stress induce variations in the content of almost all the classes of studied lipids (phospholipids, glycoglycerolipids, sterols and fatty acids). The variations, which are characteristic of a single stress, include the variations (i) in the content of individual glycoglycerolipids and in their total content, (ii) in the total content of sterols, and (iii) in the ratio of content of phosphatidylcholine/phosphatidylethanolamine in the scope of tonoplast phospholipids. Variations observed under all of the types of stress under scrutiny include (i) variations in the content of fatty acids of tonoplast lipids, (ii) some decrease in the content of phosphatidic acid and phosphatidylethanolamine, and (iii) variations in the content of individual sterols. Stigmasterol, campesterol, as well as the stigmasterol/sitosterol ratio increased in varying degrees under all of the types of stress. The most substantial variations have been observed in the content of sterols under abiotic stress. This is probably due to role of sterols in regulation of such membrane characteristics as permeability and microviscosity. In our opinion, sterols may represent one of the main components of tonoplast adaptive mechanisms.

Intracellular Recording of Cardiomyocyte Action Potentials with Nanopatterned Volcano-Shaped Microelectrode Arrays.

Micronanotechnology-based multielectrode arrays have led to remarkable progress in the field of transmembrane voltage recording of excitable cells. However, providing long-term optoporation- or electroporation-free intracellular access remains a considerable challenge. In this study, a novel type of nanopatterned volcano-shaped microelectrode (nanovolcano) is described that spontaneously fuses with the cell membrane and permits stable intracellular access. The complex nanostructure was manufactured following a simple and scalable fabrication process based on ion beam etching redeposition. The resulting ring-shaped structure provided passive intracellular access to neonatal rat cardiomyocytes. Intracellular action potentials were successfully recorded in vitro from different devices, and continuous recording for more than 1 h was achieved. By reporting transmembrane action potentials at potentially high spatial resolution without the need to apply physical triggers, the nanovolcanoes show distinct advantages over multielectrode arrays for the assessment of electrophysiological characteristics of cardiomyocyte networks at the transmembrane voltage level over time.

Glucose stimulates somatostatin secretion in pancreatic δ-cells by cAMP-dependent intracellular Ca2+ release.

Somatostatin secretion from pancreatic islet δ-cells is stimulated by elevated glucose levels, but the underlying mechanisms have only partially been elucidated. Here we show that glucose-induced somatostatin secretion (GISS) involves both membrane potential-dependent and -independent pathways. Although glucose-induced electrical activity triggers somatostatin release, the sugar also stimulates GISS via a cAMP-dependent stimulation of CICR and exocytosis of somatostatin. The latter effect is more quantitatively important and in mouse islets depolarized by 70 mM extracellular K+ , increasing glucose from 1 mM to 20 mM produced an ∼3.5-fold stimulation of somatostatin secretion, an effect that was mimicked by the application of the adenylyl cyclase activator forskolin. Inhibiting cAMP-dependent pathways with PKI or ESI-05, which inhibit PKA and exchange protein directly activated by cAMP 2 (Epac2), respectively, reduced glucose/forskolin-induced somatostatin secretion. Ryanodine produced a similar effect that was not additive to that of the PKA or Epac2 inhibitors. Intracellular application of cAMP produced a concentration-dependent stimulation of somatostatin exocytosis and elevation of cytoplasmic Ca2+ ([Ca2+]i). Both effects were inhibited by ESI-05 and thapsigargin (an inhibitor of SERCA). By contrast, inhibition of PKA suppressed δ-cell exocytosis without affecting [Ca2+]i Simultaneous recordings of electrical activity and [Ca2+]i in δ-cells expressing the genetically encoded Ca2+ indicator GCaMP3 revealed that the majority of glucose-induced [Ca2+]i spikes did not correlate with δ-cell electrical activity but instead reflected Ca2+ release from the ER. These spontaneous [Ca2+]i spikes are resistant to PKI but sensitive to ESI-05 or thapsigargin. We propose that cAMP links an increase in plasma glucose to stimulation of somatostatin secretion by promoting CICR, thus evoking exocytosis of somatostatin-containing secretory vesicles in the δ-cell.

A 15-minute session of direct current stimulation does not produce lasting changes in axonal excitability.

OBJECTIVE: To assess the applicability and lasting effects on axonal membrane excitability of transcutaneous peripheral nerve direct current stimulation (pDCS). METHODS: We included 15 healthy subjects. pDCS was performed with the active electrode placed over the left forearm and the reference electrode on the back of the hand. We used 5×5cm rubber electrodes and the current applied was 2.5mA during 15minutes. Three pDCS sessions were performed on the same day with a 20-minute interval between them: first a sham stimulation, followed by cathodal and anodal stimulations in random order. Motor nerve excitability measurements were performed immediately after each pDCS session using the TRONDNF nerve excitability protocol of the QTRAC program. RESULTS: The protocol was completed and well tolerated in all subjects. There were no consistent significant differences in excitability measurements between the three sessions. CONCLUSIONS: No consistent long-lasting effects were noted on peripheral nerve excitability beyond the period of application of pDCS. We showed that a 15-minute session of DCS is not able to produce lasting changes in axonal excitability, supporting the hypothesis that the functional and clinical impact of DCS protocols applied to the central nervous system is related to long-term synaptic changes rather sustained local changes in axonal rest membrane potential.

Synergistic Antimicrobial Activity by Light or Thermal Treatment and Lauric Arginate: Membrane Damage and Oxidative Stress.

The need for more effective antimicrobials is critical for the food industry to improve food safety and reduce spoilage of minimally processed foods. The present study was initiated to develop an efficient and novel antimicrobial approach which combines physical treatments (UV-A or mild heat) and generally recognized as safe lauroyl arginate ethyl (LAE) to inactivate surrogate strains, including Escherichia coli and Listeria innocua Synergistic inactivation of bacteria resulted in an ∼6-log reduction of target bacteria, while individual treatments resulted in <1.5-log inactivation under the same set of conditions. In addition, the synergistic mechanism between LAE and UV-A/mild heat was evaluated by supplementing with a variety of antioxidants for suppressing oxidative stress and measurement of cell membrane damage by nucleic acid release. These results demonstrate that the synergistic antimicrobial activity of LAE and mild physical stresses was suppressed by supplementation with antioxidants. The research also compared LAE with another membrane-targeting lipopeptide antimicrobial agent, polymyxin B, to understand the uniqueness of LAE-induced synergy. Briefly, differences in modes of action between LAE and polymyxin B were characterized by comparing the MIC, damage to liposomes, and oxidative stress generation. These differences in the mode of action between LAE and polymyxin B suggested that both compounds target cell membrane but significantly differ in mechanisms, including membrane disruption and oxidative stress generation. Overall, this study illustrates synergistic antimicrobial activity of LAE with light or mild heat and indicates a novel oxidative stress pathway that enhances the activity of LAE beyond membrane damage.IMPORTANCE This study highlights an effective antimicrobial processing approach using a novel combination of lauroyl arginate ethyl (LAE) and two different physical treatments, light (UV-A) and mild heat. Both combinations demonstrated synergistic inactivation against a model Gram-negative bacterium or a Gram-positive bacterium or both by a >5-log reduction. Further mechanistic study revealed that oxidative stress is responsible for synergistic inactivation between LAE and UV-A, while both membrane damage and oxidative stress are responsible for the synergistic combination between LAE and mild heat. The mode of action of LAE was further compared to that of polymyxin B and analyzed using artificial membrane model systems and the addition of antioxidants. The proposed combination of LAE and common physical treatments may improve food preservation, food safety, and current sanitation processes for the food industry and the inactivation of pathogenic strains in biomedical environments.

Does fennel extract ameliorate oxidative stress frozen-thawed ram sperm?

The aim of this study was to evaluate the quality of ram semen after cryopreservation with different levels of fennel (Foeniculum vulgare) extract (0 (F0), 5 (F5), 10 (F10) and 15 (F15) mg/L) and sperm concentrations (200 (C200) and 400 (C400) × 106 sperm/mL) in a soy lecithin (SL)-based extender. Twenty ejaculates were collected from four ghezel rams and diluted with eight sperm concentrations/fennel combinations: F0C200, F5C200, F10C200, F15C200, F0C400, F5C400, F10C400 and F15C400. Sperm motility, abnormality, plasma membrane, viability, mitochondrial activity, lipid peroxidation (LPO), mitochondrial activity and apoptotic changes were evaluated after freeze-thawing process. It was observed that F10C400 significantly improved total and progressive motility, VSL, membrane integrity of post-thawed ram sperm. MDA level was lower in F5C200 and F10C400 compared to other treatments. The higher percentage of live sperm and the lower percentage of apoptotic sperm were obtained in F10C200 compared to F0C200, F5C200 F15C400, F0C400, F5C400 and F15C400. Extender F10C200 resulted in the highest mitochondria activity compared to the rest of the extenders except F10C400. We conclude that a combination of 10 mg/mL fennel (Foeniculum vulgare) extract and sperm concentration of 200 × 106 sperm/mL can improve the ram semen quality cryopreserved in a soybean lecithin based extender.

A preliminary study on the use of jenny colostrum to improve quality in extenders for freezing donkey semen.

Sperm quality in donkeys (Equus asinus) after freezing thawing is still considered lower than that from other animals, including horses. The aim of this study was to test a new freezing extender supplemented with jenny colostrum on donkey sperm. After thawing, we evaluated sperm motility by means of computer-assisted analysis, viability by SYBR-14 and propidium iodide (PI), membrane functional integrity by HOS-test and acrosome integrity by isothiocyanate conjugated with peanut agglutinin (FITC-PNA) and PI. Ejaculates were collected from five fertile Donkeys. Sperm samples were pooled, diluted and cryopreserved into three experimental extender groups: BotuCrio®, lactose-extender supplemented with egg yolk (20%) and lactose-extender supplemented with jenny colostrum (20%). The results demonstrated that lactose-jenny colostrum samples displayed significantly higher values in almost all parameters evaluated (p < 0.05) compared with the other two extenders after thawing (BotuCrio® and lactose-egg yolk based extender, respectively) -Total Motility, Viability, HOS test, VCL, VSL and VAP. Acrosome status, LIN, STR and WOB despite showing lower values, none of them were statistically significant (p > 0.05). In conclusion, the extender containing jenny colostrum can be successfully used for donkey semen crypreservation and could effectively improve donkey sperm qualities after freezing -thawing.

Exogenous fatty acids alter phospholipid composition, membrane permeability, capacity for biofilm formation, and antimicrobial peptide susceptibility in Klebsiella pneumoniae.

Klebsiella pneumoniae represents a major threat to human health due to a combination of its nosocomial emergence and a propensity for acquiring antibiotic resistance. Dissemination of the bacteria from its native intestinal location creates severe, complicated infections that are particularly problematic in healthcare settings. Thus, there is an urgency for identifying novel treatment regimens as the incidence of highly antibiotic-resistant bacteria rises. Recent findings have highlighted the ability of some Gram-negative bacteria to utilize exogenous fatty acids in ways that modify membrane phospholipids and influence virulence phenotypes, such as biofilm formation and antibiotic resistance. This study explores the ability of K. pneumoniae to assimilate and respond to exogenous fatty acids. The combination of thin-layer chromatography liquid chromatography-mass spectrometry confirmed adoption of numerous exogenous polyunsaturated fatty acids (PUFAs) into the phospholipid species of K. pneumoniae. Membrane permeability was variably affected as determined by two dye uptake assays. Furthermore, the availability of many PUFAs lowered the MICs to the antimicrobial peptides polymyxin B and colistin. Biofilm formation was significantly affected depending upon the supplemented fatty acid.

Multimicrobial Kombucha Culture Tolerates Mars-Like Conditions Simulated on Low-Earth Orbit.

A kombucha multimicrobial culture (KMC) was exposed to simulated Mars-like conditions in low-Earth orbit (LEO). The study was part of the Biology and Mars Experiment (BIOMEX), which was accommodated in the European Space Agency's EXPOSE-R2 facility, outside the International Space Station. The aim of the study was to investigate the capability of a KMC microecosystem to survive simulated Mars-like conditions in LEO. During the 18-month exposure period, desiccated KMC samples, represented by living cellulose-based films, were subjected to simulated anoxic Mars-like conditions and ultraviolet (UV) radiation, as prevalent at the surface of present-day Mars. Postexposure analysis demonstrated that growth of both the bacterial and yeast members of the KMC community was observed after 60 days of incubation; whereas growth was detected after 2 days in the initial KMC. The KMC that was exposed to extraterrestrial UV radiation showed degradation of DNA, alteration in the composition and structure of the cellular membranes, and an inhibition of cellulose synthesis. In the "space dark control" (exposed to LEO conditions without the UV radiation), the diversity of the microorganisms that survived in the biofilm was reduced compared with the ground-based controls. This was accompanied by structural dissimilarities in the extracellular membrane vesicles. After a series of subculturing, the revived communities restored partially their structure and associated activities.

The antimicrobial activities and action-mechanism of tea tree oil against food-borne bacteria in fresh cucumber juice.

BACKGROUND: Foodborne diseases caused by foodborne pathogens have increasingly become a worldwide public health concern. Due to potential harmful effects of synthetic chemicals, there is a pressure for adoption of natural alternatives to obtain microbial safety of food. Tea tree oil (TTO) exhibited a wide range of pharmacological actions attribute to the broad spectrum activities. However, to the best of our knowledge, no systematic research on the mode of antibacterial actions of TTO against Listeria monocytogenes (L. monocytogenes) and Escherichia coli (E. coli) in vitro models have been conducted so far. RESULTS: The present investigation reported on the antimicrobial activities of TTO and examined its possible antimicrobial mode of action against L. monocytogenes and E. coli. Results showed that the susceptibility of L. monocytogenes were excellent with the lower minimal inhibitory concentration (MIC) values and larger inhibition zones. TTO changed the integrity of the membrane, as evidenced by the release of 260 nm absorbing intracellular materials and the alteration of membrane potential. The results of flow cytometry showed that TTO caused bacterial membrane permeabilization in a dose-dependent manner. The remarkable cellular morphological changes in bacteria caused by TTO were observed using the scanning electron microscope, indicating cell damage. In addition, antimicrobial preserving properties of TTO were evaluated by time-kill assay after its incorporation in cucumber juice, the results showed TTO successfully inhibited L. monocytogenes and E. coli development, at room temperature and in refrigerator (25 °C and 4 °C) respectively, demonstrating it had good preservative activities in food system. CONCLUSIONS: These findings suggested that TTO exhibited good antimicrobial effect against food-borne pathogens and could be potentially used in food industries as a food preservative.

Preparation of asymmetric phospholipid vesicles for use as cell membrane models.

Freely suspended liposomes are widely used as model membranes for studying lipid-lipid and protein-lipid interactions. Liposomes prepared by conventional methods have chemically identical bilayer leaflets. By contrast, living cells actively maintain different lipid compositions in the two leaflets of the plasma membrane, resulting in asymmetric membrane properties that are critical for normal cell function. Here, we present a protocol for the preparation of unilamellar asymmetric phospholipid vesicles that better mimic biological membranes. Asymmetry is generated by methyl-ß-cyclodextrin-catalyzed exchange of the outer leaflet lipids between vesicle pools of differing lipid composition. Lipid destined for the outer leaflet of the asymmetric vesicles is provided by heavy-donor multilamellar vesicles containing a dense sucrose core. Donor lipid is exchanged into extruded unilamellar acceptor vesicles that lack the sucrose core, facilitating the post-exchange separation of the donor and acceptor pools by centrifugation because of differences in vesicle size and density. We present two complementary assays allowing quantification of each leaflet's lipid composition: the overall lipid composition is determined by gas chromatography-mass spectrometry, whereas the lipid distribution between the two leaflets is determined by NMR, using the lanthanide shift reagent Pr3+. The preparation protocol and the chromatographic assay can be applied to any type of phospholipid bilayer, whereas the NMR assay is specific to lipids with choline-containing headgroups, such as phosphatidylcholine and sphingomyelin. In ~12 h, the protocol can produce a large yield of asymmetric vesicles (up to 20 mg) suitable for a wide range of biophysical studies.

Pouteria ramiflora (Mart.) Radlk. extract: Flavonoids quantification and chemopreventive effect on HepG2 cells.

Pouteria ramiflora (Mart.) Radlk., popularly known as curriola, is commonly used in Brazil as medicinal plant to treat worm infections, dysentery, pain, inflammation, hyperlipidemia, and obesity. At present the safety of this extract when used therapeutically in human remains to be determined. Thus, the aim of this study was to examine cytotoxicity, antiproliferative, and antimutagenic actions of this extract. The hydroalcoholic extract from P. ramiflora leaves consisted of flavonoids identified and quantified as myricetin-3-O-ß-D-galactopyranoside (13.55 mg/g) and myricetin-3-O-α-L-rhamnopyranoside (9.61 mg/g). The extract exhibited cytotoxicity at concentrations higher than 1.5 µg/ml in human hepatocarcinoma (HepG2)and 2.5 µg/ml in non-tumoral primary gastric (GAS) cells using the MTT assay, and at concentrations higher than 3 µg/ml in HepG2 and 3.5 µg/ml in GAS cells by the neutral red assay. The extract did not show antiproliferative effect as evidenced by the nuclear division index (NDI). However, in the presence of benzo[a]pyrene (BaP) (positive control), an enhanced cytostatic effect in the NDI and flow cytometry was noted. It is of interest that when the extract was co-incubated with BaP a significant decrease in DNA damage was observed indicating an antimutagenic action. This protective effect might be attributed to myricetin and gallic acid found in P. ramiflora extract. The low cytotoxicity action and protective effect observed in the present study encourage further studies regarding other biological effects of P. ramiflora, as well as its potential use as a chemopreventive agent.

ns Pulsed Electric Field-Induced Action Potentials in the Circuital Model of an Axon.

Pulsed electric fields with duration in the sub- and ns time scale (nsPEFs) increase the permeability of cell membranes, enabling the transport of normally impermeant molecules into or out of the cell (electroporation). Such effect is associated to intracellular alterations and indicates nsPEFs as a new stimulus to modulate cell functions. In particular, studies dealing with the application of nsPEFs to excitable cells suggest their use for the stimulation/inhibition of cell excitation. In this paper, the circuital model per surface unit of the plasma membrane of an axon was developed to implement the Hodgkin and Huxley equations, describing the action potential activation process. For the first time, a power electronics circuital simulator was adopted. The model was first validated with conventional microsecond stimuli, and then it was employed to identify the conditions for cell excitation by nsPEFs. The results demonstrated the possibility of electrostimulation by nsPEFs at depolarization levels far below those required for inducing electroporation, and with ionic current dynamics similar to that induced by conventional stimuli, confirming recent experimental findings. Moreover, by using a power electronics tool, easier integration of the cell modeling with the design and optimization of pulse generation systems can be gained.

New saliva secretion model based on the expression of Na+-K+ pump and K+ channels in the apical membrane of parotid acinar cells.

The plasma membrane of parotid acinar cells is functionally divided into apical and basolateral regions. According to the current model, fluid secretion is driven by transepithelial ion gradient, which facilitates water movement by osmosis into the acinar lumen from the interstitium. The osmotic gradient is created by the apical Cl- efflux and the subsequent paracellular Na+ transport. In this model, the Na+-K+ pump is located exclusively in the basolateral membrane and has essential role in salivary secretion, since the driving force for Cl- transport via basolateral Na+-K+-2Cl- cotransport is generated by the Na+-K+ pump. In addition, the continuous electrochemical gradient for Cl- flow during acinar cell stimulation is maintained by the basolateral K+ efflux. However, using a combination of single-cell electrophysiology and Ca2+-imaging, we demonstrate that photolysis of Ca2+ close to the apical membrane of parotid acinar cells triggered significant K+ current, indicating that a substantial amount of K+ is secreted into the lumen during stimulation. Nevertheless, the K+ content of the primary saliva is relatively low, suggesting that K+ might be reabsorbed through the apical membrane. Therefore, we investigated the localization of Na+-K+ pumps in acinar cells. We show that the pumps appear evenly distributed throughout the whole plasma membrane, including the apical pole of the cell. Based on these results, a new mathematical model of salivary fluid secretion is presented, where the pump reabsorbs K+ from and secretes Na+ to the lumen, which can partially supplement the paracellular Na+ pathway.

Remodelling of primary human CD4+ T cell plasma membrane order by n-3 PUFA.

Cell membrane fatty acids influence fundamental properties of the plasma membrane, including membrane fluidity, protein functionality, and lipid raft signalling. Evidence suggests that dietary n-3 PUFA may target the plasma membrane of immune cells by altering plasma membrane lipid dynamics, thereby regulating the attenuation of immune cell activation and suppression of inflammation. As lipid-based immunotherapy might be a promising new clinical strategy for the treatment of inflammatory disorders, we conducted in vitro and in vivo experiments to examine the effects of n-3 PUFA on CD4+ T cell membrane order, mitochondrial bioenergetics and lymphoproliferation. n-3 PUFA were incorporated into human primary CD4+ T cells phospholipids in vitro in a dose-dependent manner, resulting in a reduction in whole cell membrane order, oxidative phosphorylation and proliferation. At higher doses, n-3 PUFA induced unique phase separation in T cell-derived giant plasma membrane vesicles. Similarly, in a short-term human pilot study, supplementation of fish oil (4 g n-3 PUFA/d) for 6 weeks in healthy subjects significantly elevated EPA (20 : 5n-3) levels in CD4+ T cell membrane phospholipids, and reduced membrane lipid order. These results demonstrate that the dynamic reshaping of human CD4+ T cell plasma membrane organisation by n-3 PUFA may modulate down-stream clonal expansion.