Incorporation of reconstituted acetylcholine receptors from Torpedo into the Xenopus oocyte membrane.

Xenopus oocytes are a valuable aid for studying the molecular structure and function of ionic channels and neurotransmitter receptors. Their use has recently been extended by the demonstration that oocytes can incorporate foreign membranes carrying preassembled receptors and channels. Here we show that when reconstituted in an artificial lipid matrix and injected into Xenopus oocytes, purified nicotinic acetylcholine receptors are efficiently inserted into the plasma membrane, where they form "clusters" of receptors that retain their native properties. This constitutes an innovative approach that, besides allowing the analyses of membrane fusion processes, is also a powerful technique for studying the characteristics and regulation of many membrane proteins (with their native stoichiometry and configuration) upon reinsertion into the membrane of a very convenient host cell system.

Lipid A biosynthesis in Rhizobium leguminosarum: role of a 2-keto-3-deoxyoctulosonate-activated 4' phosphatase.

Lipid A from several strains of the N2-fixing bacterium Rhizobium leguminosarum displays significant structural differences from Escherichia coli lipid A, one of which is the complete absence of phosphate groups. However, the first seven enzymes of E. coli lipid A biosynthesis, leading from UDP-GlcNAc to the phosphorylated intermediate, 2-keto-3-deoxyoctulosonate (Kdo2)-lipid IVA, are present in R. leguminosarum. We now describe a membrane-bound phosphatase in R. leguminosarum extracts that removes the 4' phosphate of Kdo2-lipid IVA. The 4' phosphatase is selective for substrates containing the Kdo domain. It is present in extracts of R. leguminosarum biovars phaseoli, viciae, and trifolii but is not detectable in E. coli and Rhizobium meliloti. A nodulation-defective strain (24AR) of R. leguminosarum biovar trifolii, known to contain a 4' phosphatase residue on its lipid A, also lacks measurable 4' phosphatase activity. The Kdo-dependent 4' phosphatase appears to be a key reaction in a pathway for generating phosphate-deficient lipid A.

Older plasma lipoproteins are more susceptible to oxidation: a linking mechanism for the lipid and oxidation theories of atherosclerotic cardiovascular disease.

Increases in plasma cholesterol are associated with progressive increases in the risk of atherosclerotic cardiovascular disease. In humans plasma cholesterol is contained primarily in apolipoprotein B-based low density lipoprotein (LDL). Cells stop making the high-affinity receptor responsible for LDL removal as they become cholesterol replete; this slows removal of LDL from plasma and elevates plasma LDL. As a result of this delayed uptake, hypercholesterolemic individuals not only have more LDL but have significantly older LDL. Oxidative modification of LDL enhances their atherogenicity. This study sought to determine whether increased time spent in circulation, or aging, by lipoprotein particles altered their susceptibility to oxidative modification. Controlled synchronous production of distinctive apolipoprotein B lipoproteins (yolk-specific very low density lipoproteins; VLDLy) with a single estrogen injection into young turkeys was used to model LDL aging in vivo. VLDLy remained in circulation for at least 10 days. Susceptibility to oxidation in vitro was highly dependent on lipoprotein age in vivo. Oxidation, measured as hexanal release from n-6 fatty acids in VLDLy, increased from 13.3 +/- 5.5 nmol of 2-day-old VLDLy per ml, to 108 +/- 17 nmol of 7-day-old VLDLy per ml. Oxidative instability was not due to tocopherol depletion or conversion to a more unsaturated fatty acid composition. These findings establish mathematically describable linkages between the variables of LDL concentration and LDL oxidation. The proposed mathematical models suggest a unified investigative approach to determine the mechanisms for acceleration of atherosclerotic cardiovascular disease risk as plasma cholesterol rises.

Disruption of cellular signaling pathways by daunomycin through destabilization of nonlamellar membrane structures.

Albeit anthracyclines are widely used in the treatment of solid tumors and leukemias, their mechanism of action has not been elucidated. The present study gives relevant information about the role of nonlamellar membrane structures in signaling pathways, which could explain how anthracyclines can exert their cytocidal action without entering the cell [Tritton, T. R. & Yee, G. (1982) Science 217, 248-250]. The anthracycline daunomycin reduced the formation of the nonlamellar hexagonal (HII) phase (i.e., the hexagonal phase propensity), stabilizing the bilayer structure of the plasma membrane by a direct interaction with membrane phospholipids. As a consequence, various cellular events involved in signal transduction, such as membrane fusion and membrane association of peripheral proteins [e.g., guanine nucleotide-binding regulatory proteins (G proteins and protein kinase C-alpha beta)], where nonlamellar structures (negative intrinsic monolayer curvature strain) are required, were altered by the presence of daunomycin. Functionally, daunomycin also impaired the expression of the high-affinity state of a G protein-coupled receptor (ternary complex for the alpha 2-adrenergic receptor) due to G-protein dissociation from the plasma membrane. In vivo, daunomycin also decreased the levels of membrane-associated G proteins and protein kinase C-alpha beta in the heart. The occurrence of such nonlamellar structures favors the association of these peripheral proteins with the plasma membrane and prevents daunomycin-induced dissociation. These results reveal an important role of the lipid component of the cell membrane in signal transduction and its alteration by anthracyclines.

Unsaturation of the membrane lipids of chloroplasts stabilizes the photosynthetic machinery against low-temperature photoinhibition in transgenic tobacco plants.

Using tobacco plants that had been transformed with the cDNA for glycerol-3-phosphate acyltransferase, we have demonstrated that chilling tolerance is affected by the levels of unsaturated membrane lipids. In the present study, we examined the effects of the transformation of tobacco plants with cDNA for glycerol-3-phosphate acyltransferase from squash on the unsaturation of fatty acids in thylakoid membrane lipids and the response of photosynthesis to various temperatures. Of the four major lipid classes isolated from the thylakoid membranes, phosphatidylglycerol showed the most conspicuous decrease in the level of unsaturation in the transformed plants. The isolated thylakoid membranes from wild-type and transgenic plants did not significantly differ from each other in terms of the sensitivity of photosystem II to high and low temperatures and also to photoinhibition. However, leaves of the transformed plants were more sensitive to photoinhibition than those of wild-type plants. Moreover, the recovery of photosynthesis from photoinhibition in leaves of wild-type plants was faster than that in leaves of the transgenic tobacco plants. These results suggest that unsaturation of fatty acids of phosphatidylglycerol in thylakoid membranes stabilizes the photosynthetic machinery against low-temperature photoinhibition by accelerating the recovery of the photosystem II protein complex.

Lipid metabolism in Chlamydia trachomatis-infected cells: directed trafficking of Golgi-derived sphingolipids to the chlamydial inclusion.

Chlamydia trachomatis undergoes its entire life cycle within an uncharacterized intracellular vesicle that does not fuse with lysosomes. We used a fluorescent Golgi-specific probe, (N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]) aminocaproylsphingosine (C6-NBD-Cer), in conjunction with conventional fluorescence or confocal microscopy to identify interactions between the Golgi apparatus and the chlamydial inclusion. We observed not only a close physical association between the Golgi apparatus and the chlamydial inclusion but the eventual presence of a metabolite of this fluorescent probe associated with the chlamydiae themselves. Sphingomyelin, endogenously synthesized from C6-NBD-Cer, was specifically transported to the inclusion and incorporated into the cell wall of the intracellular chlamydiae. Incorporation of the fluorescent sphingolipid by chlamydiae was inhibited by brefeldin A. Chlamydiae therefore occupy a vesicle distal to the Golgi apparatus that receives anterograde vesicular traffic from the Golgi normally bound for the plasma membrane. Collectively, the data suggest that the chlamydial inclusion may represent a unique compartment within the trans-Golgi network.

Interações moleculares no mecanismo de ação da galectina-4 humana

A galectina-4 humana (HGal-4), pertencente à família das galectinas, possui dois domínios de reconhecimento de carboidratos (CRDs) com alta afinidade para β-galactosídeos e se encontra amplamente distribuída em células normais e neoplásicas de diferentes organismos. Suas funções snglobam uma grande variedade de eventos celulares, tais como processos inflamatórios, neoplásicos, progressão tumoral e metástase. Entretanto, muitas perguntas sobre suas interações com diferentes carboidratos, a especificidade destas interações e o papel específico das galectinas permanecem ainda sem resposta. No presente trabalho, propomos a investigação das interações galectina-glicano da galectina-4 humana e de seus domínios CRDs independentes (CRD-I e CRD-II) através de um conjunto de métodos biofísicos. Através do método de dicroísmo circular (CD), usando várias regiões espectrais, e fluorescência fomos capazes de entender mudanças ocorrentes na estrutura secundária e terciária das protéinas quando da interação com lactose/sacarose. Estes dados, juntamente com testes de hemaglutinação, mostraram que a glectina-4 e os CRDs respondem de forma distinta à ligação com açúcar. Por diferentes técnicas (fluorescência, ITC e MST) determinamos as constantes de dissociação para os domínios CRDs (Kd ~0,5 mM) e para HGal-4 e, de forma qualitativa, os valores obtidos indicaram possíveis estados oligoméricos dessas proteínas. A investigação da interação proteína-membrana da HGal-4 foi feita, primeiramente, com miméticos de membranas e monitorada pela técnica de RPE em crescente complexidade de composição de tais miméticos, indo desde composições mais simples, passando por lipid rafts na presença de diferentes glicolipídeos (GM1, LPS) e chegando-se à interação com células tumorais (U87MG, T98G e HT-29). Tais experimentos mostraram que galectina-4 reconhece e se liga naqueles modelos onde existem glicanos complexos na superfície. Investigamos também a participação de HGal-4 endógena e exógena no tratamento quimioterápico de células tumorais e verificamos um papel importante de HGal-4 para células HT-29. Finalizando esta tese, apresentamos o trabalho realizado em um ano de estágio na University of Oxford, durante o qual, investigamos a estrutura da região C-terminal de um receptor da família GPCR, qual seja o receptor de neurotensina NTS1. Aqui, mais uma vez, foi empregada a técnica de RPE que aliada à produção/marcação de mutantes do receptor, permitiu determinar que a hélice H8 se estabiliza quando em proteolipossomos.

Chronopotentiometric study of a Nafion membrane in presence of glucose

Chronopotentiometric and swelling experiments have been conducted to characterize the behavior of a Nafion membrane in NaCl and KCl aqueous solutions without and with glucose. A mixture solution with similar composition to the cerebrospinal fluid and blood plasma has also been studied. From the chronotentiograms, current-voltage curves have been obtained, and the values of the limiting current density, diffusion boundary layer thickness, difference between counter-ion transport number in membrane and free solution, and transition times have been determined for the investigated membrane systems. The obtained results indicate that the presence of glucose affects the ion transport through the membrane depending on the electrolyte and glucose concentrations. At low electrolyte concentration, experimental transition times are found to be smaller in presence of glucose, which has been related to an effective membrane area reduction in presence of glucose. The membrane system corresponding to the mixture solution shows a behavior similar to the single high concentration NaCl membrane system, indicating that the observed behavior is mainly associated to the Na^+ ions transport in higher proportion. In this case, the glucose presence does not affect significantly the investigated properties of the membrane, which is interesting for its utilization in a glucose fuel cell.

Thermogravimetry and Py–GC/MS techniques as fast qualitative methods for comparing the biochemical composition of Nannochloropsis oculata samples obtained under different culture conditions

Microalgae have many applications, such as biodiesel production or food supplement. Depending on the application, the optimization of certain fractions of the biochemical composition (proteins, carbohydrates and lipids) is required. Therefore, samples obtained in different culture conditions must be analyzed in order to compare the content of such fractions. Nevertheless, traditional methods necessitate lengthy analytical procedures with prolonged sample turn-around times. Results of the biochemical composition of Nannochloropsis oculata samples with different protein, carbohydrate and lipid contents obtained by conventional analytical methods have been compared to those obtained by thermogravimetry (TGA) and a Pyroprobe device connected to a gas chromatograph with mass spectrometer detector (Py–GC/MS), showing a clear correlation. These results suggest a potential applicability of these techniques as fast and easy methods to qualitatively compare the biochemical composition of microalgal samples.

Evolution of extracellular polymeric substances produced in two submerged membrane bioreactors working at high sludge age

This research paper deals with the evolution of the extracellular polymeric substances (EPS) produced in the mixed liquor of two 25 L bench-scale membrane bioreactors (MBRs), with micro (MF-MBR) and ultrafiltration (UF-MBR) submerged membranes. The conclusion focuses on the relationship between the operation and how EPS respond, demonstrating that significant changes in EPS concentration were commonly observed when abrupt changes in the operational conditions took place. Bound EPS (EPSb) showed moderate positive statistical correlations with sludge age and MLSS for the two MBRs. Soluble EPS (EPSs), on the other hand, showed a moderate negative statistical correlation between EPSs with the two parameters analyzed for MF-MBR and no correlation with the UF-MBR was found. With respect to the composition of EPS, EPSb were mostly made up of proteins (44–46%) whereas in EPSs, the three components (proteins, carbohydrates, and humic substances) appeared in approximately the same proportion. The statistical analysis exhibited strong positive correlations between EPSb and their constituents, however for EPSs, the correlation was strong only with carbohydrates and moderate with humic substances.

Competing Lipid-Protein and Protein-Protein Interactions Determine Clustering and Gating Patterns in the Potassium Channel from Streptomyces lividans (KcsA)

There is increasing evidence to support the notion that membrane proteins, instead of being isolated components floating in a fluid lipid environment, can be assembled into supramolecular complexes that take part in a variety of cooperative cellular functions. The interplay between lipid-protein and protein-protein interactions is expected to be a determinant factor in the assembly and dynamics of such membrane complexes. Here we report on a role of anionic phospholipids in determining the extent of clustering of KcsA, a model potassium channel. Assembly/disassembly of channel clusters occurs, at least partly, as a consequence of competing lipid-protein and protein-protein interactions at nonannular lipid binding sites on the channel surface and brings about profound changes in the gating properties of the channel. Our results suggest that these latter effects of anionic lipids are mediated via the Trp67–Glu71–Asp80 inactivation triad within the channel structure and its bearing on the selectivity filter.

Neurospora crassa transcriptomics reveals oxidative stress and plasma membrane homeostasis biology genes as key targets in response to chitosan

Chitosan is a natural polymer with antimicrobial activity. Chitosan causes plasma membrane permeabilization and induction of intracellular reactive oxygen species (ROS) in Neurospora crassa. We have determined the transcriptional profile of N. crassa to chitosan and identified the main gene targets involved in the cellular response to this compound. Global network analyses showed membrane, transport and oxidoreductase activity as key nodes affected by chitosan. Activation of oxidative metabolism indicates the importance of ROS and cell energy together with plasma membrane homeostasis in N. crassa response to chitosan. Deletion strain analysis of chitosan susceptibility pointed NCU03639 encoding a class 3 lipase, involved in plasma membrane repair by lipid replacement, and NCU04537 a MFS monosaccharide transporter related to assimilation of simple sugars, as main gene targets of chitosan. NCU10521, a glutathione S-transferase-4 involved in the generation of reducing power for scavenging intracellular ROS is also a determinant chitosan gene target. Ca2+ increased tolerance to chitosan in N. crassa. Growth of NCU10610 (fig 1 domain) and SYT1 (a synaptotagmin) deletion strains was significantly increased by Ca2+ in the presence of chitosan. Both genes play a determinant role in N. crassa membrane homeostasis. Our results are of paramount importance for developing chitosan as an antifungal.

Performance Assessment of a Polymer Electrolyte Membrane Electrochemical Reactor under Alkaline Conditions – A Case Study with the Electrooxidation of Alcohols

A novel polymer electrolyte membrane electrochemical reactor (PEMER) configuration has been employed for the direct electrooxidation of propargyl alcohol (PGA), a model primary alcohol, towards its carboxylic acid derivatives in alkaline medium. The PEMER configuration comprised of an anode and cathode based on nanoparticulate Ni and Pt electrocatalysts, respectively, supported on carbonaceous substrates. The electrooxidation of PGA was performed in 1.0 M NaOH, where a cathode based on a gas diffusion electrode was manufactured for the reduction of oxygen in alkaline conditions. The performance of a novel alkaline anion-exchange membrane based on Chitosan (CS) and Poly(vinyl) alcohol (PVA) in a 50:50 composition ratio doped with a 5 wt.% of poly (4-vinylpyridine) organic ionomer cross-linked, methyl chloride quaternary salt resin (4VP) was assessed as solid polymer electrolyte. The influence of 4VP anionic ionomer loading of 7, 12 and 20 wt.% incorporated into the electrocatalytic layers was examined by SEM and cyclic voltammetry (CV) upon the optimisation of the electroactive area, the mechanical stability and cohesion of the catalytic ink onto the carbonaceous substrate for both electrodes. The performance of the 4VP/CS:PVA membrane was compared with the commercial alkaline anion-exchange membrane FAA −a membrane generally used in direct alcohol alkaline fuel cells- in terms of polarisation plots in alkaline conditions. Furthermore, preparative electrolyses of the electrooxidation of PGA was performed under alkaline conditions of 1 M NaOH at constant current density of 20 mA cm−2 using a PEMER configuration to provide proof of the principle of the feasibility of the electrooxidation of other alcohols in alkaline media. PGA conversion to Z isomers of 3-(2-propynoxy)-2-propenoic acid (Z-PPA) was circa 0.77, with average current efficiency of 0.32. Alkaline stability of the membranes within the PEMER configuration was finally evaluated after the electrooxidation of PGA.

Rapid Communication: Cholesterol deficiency-associated APOB mutation impacts lipid metabolism in Holstein calves and breeding bulls.

During the last months, the number of reports on Holstein calves suffering from incurable idiopathic diarrhea dramatically increased. Affected calves showed severe hypocholesterolemia and mostly died within days up to a few months after birth. This new autosomal monogenic recessive inherited fat metabolism disorder, termed cholesterol deficiency (CD), is caused by a loss of function mutation of the bovine gene. The objective of the present study was to investigate specific components of lipid metabolism in 6 homozygous for the mutation (CDS) and 6 normal Holstein calves with different genotypes. Independent of sex, CDS had significantly lower plasma concentrations of total cholesterol (TC), free cholesterol (FC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), very-low-density lipoprotein cholesterol (VLDL-C), triacylglycerides (TAG), and phospholipids (PL) compared with homozygous wild-type calves ( < 0.05). Furthermore, we studied the effect of the genotype on cholesterol metabolism in adult Holstein breeding bulls of Swissgenetics. Among a total of 254 adult males, the homozygous mutant genotype was absent, 36 bulls were heterozygous carriers (CDC), and 218 bulls were homozygous wild-type (CDF). In CDC bulls, plasma concentrations of TC, FC, HDL-C, LDL-C, VLDL-C, TAG, and PL were lower compared with CDF bulls ( < 0.05). The ratios of FC:cholesteryl esters (CE) and FC:TC were higher in CDC bulls compared with CDF bulls, whereas the ratio of CE:TC was lower in CDC bulls compared with CDF bulls ( < 0.01). In conclusion, the CD-associated mutation was shown to affect lipid metabolism in affected Holstein calves and adult breeding bulls. Besides cholesterol, the concentrations of PL, TAG, and lipoproteins also were distinctly reduced in homozygous and heterozygous carriers of the mutation. Beyond malabsorption of dietary lipids, deleterious effects of apolipoprotein B deficiency on hepatic lipid metabolism, steroid biosynthesis, and cell membrane function can be expected, which may result in unspecific symptoms of reduced fertility, growth, and health.

Diatom abundance and fatty acid composition of ice algae and of Calanus glacialis in samples obtained in 2008 in the eastern Beaufort Sea

The copepod Calanus glacialis plays a key role in the lipid-based energy flux in Arctic shelf seas. By utilizing both ice algae and phytoplankton, this species is able to extend its growth season considerably in these seasonally ice-covered seas. This study investigated the impacts of the variability in timing and extent of the ice algal bloom on the reproduction and population success of C. glacialis. The vertical distribution, reproduction, amount of storage lipids, stable isotopes, fatty acid and fatty alcohol composition of C. glacialis were assessed during the Circumpolar Flaw Lead System Study. Data were collected in the Amundsen Gulf, south-eastern Beaufort Sea, from January to July 2008 with the core-sampling from March to April. The reduction in sea ice thickness and coverage observed in the Amundsen Gulf in 2007 and 2008 affected the life strategy and reproduction of C. glacialis. Developmental stages CIII and CIV dominated the overwintering population, which resulted in the presence of very few CV and females during spring 2008. Spawning began at the peak of the ice algal bloom that preceded the precocious May ice break-up. Although the main recruitment may have occurred later in the season, low abundance of females combined with a potential mismatch between egg production/development to the first feeding stage and phytoplankton bloom resulted in low recruitment of C. glacialis in the early summer of 2008.