C. albicans were pulsed with C. albicans in the presence of DAmb or MBCD for 2 hours, and then autologous purified memory CD4+ T cells were added. As shown in Fig 6A memory CD4+ T cells stimulated by untreated monocytes exhibited, as expected, a robust proliferative response and cytokine production in response to the fungus. On the contrary, the same popula- tion of T cells barely proliferated and produced IFN-γ and IL-17 in response to drugs treated C. albicans-pulsed monocytes. The impairment of T cell response to C. albicans following lipidraft disruption might be ascribed to several mechanisms other than the decreased fungus uptake. First, to evaluate whether the decreased T cell response might result from a direct drugs effect on T cells, treated monocytes were extensively washed after antigen pulse and then co-cultured with T cells (Fig 6A). Drugs removal only partially restored T cell response, reinforcing the hypothesis that drug's effects on monocytes are the main cause for the reduced T cell activation. Moreover the observed partial rescue of T cell proliferation could also be due to the phagocytosis of residual C. albicans cells, which cannot be totally removed from the culture by washings. According with these results, the memory CD4+ T cell response to MP65, a purified, antigenic Candida albicans protein (Fig 6B), which does not require phagocytosis for its uptake and processing, was left unmodified by the presence of drugs. These results also excluded that drugs could generically blunt antigen processing and/or presentation by monocytes.
Detergent resistant membranes (‘‘DRMs’’) are thought to be the biochemical equivalent of lipidraft microdomains [36,37]. As shown in Figure 5A, none of the FM that associated with total membranes from mucosal explants, labeled with the dye for 1 h, was detected in the corresponding DRM fraction, using Triton X- 100 as detergent. Since at this time point most of the FM was seen in TWEEs (Figure 1), the experiment implies that apical FM- positive early endosomes are mainly composed of detergent sensitive membranes, i.e., non-raft- or liquid disordered mem- branes . Figure 5B shows a similar DRM analysis performed with a microvillar membrane vesicle fraction directly exposed to FM. In this experiment the dye was likewise clearly seen in the total membranes, but in addition it was also detectable in the DRM fraction, indicating that FM spontaneously inserts into both raft- and non-raft microdomains of the brush border. Together, the results of the above experiments imply that FM is partially redistributed from raft- to non-raft microdomains before its internalization into TWEEs. As a control experiment to validate this conclusion, the distribution of the GPI-linked lipidraft marker AP in the membrane fractions is shown in Figure 6C. For both total mucosal- and microvillar membranes, the 67 kDa band of AP was equally prominent in both TM- and DRM fractions, indicating that lipid rafts were efficiently recovered in the latter. In contrast, the 160 kDa lactase/phlorizin hydrolase (LPH), the only major microvillar hydrolase predominantly residing in the non-raft part of the membrane [21,39], was absent from the DRM fractions. In addition, staining with Coomassie brilliant blue revealed that only a minor part of the total protein was present in the DRM fractons.
The use of membrane model systems, such as Giant unilamellar vesicles (GUVs), is a good approach to study in detail a limited number of molecular interactions, to determine, amongst the overwhelming complexity surrounding biomembranes, which of those interactions are involved in a given biological process, and if they are altered in pathologies such as TLE. As stated above, cellular membranes can have different domains with different biophysical properties, such as fluidity. These properties vary according to the environment and composition of the membrane itself and are reminiscent of the lipid phases found in membrane model systems 100 . The existing lamellar phases, i.e., those in which the lipids
similar results in hypergravity and in control samples. Such difference could be due to the upregulation of the TSHR in hypergravity. Microgravity also induced the release of total SM measured by thin layer chromatography. Here with a more sophisticated technique we did not notice any significant variation of individual SM species. On the other hand we had already shown that the enzymes responsible for SM metabolism behaved similarly under hypo- and hypergravity conditions . In hypergravity, no variation of SMase and SM- synthase1 expression was found as in hypogravity but the SMase translocated from the nucleus to the cytoplasm and had similar values of enzyme activity to those in hypogravity . Comparing the results from hypo-and hypergravity, it was evident that the plasma membrane was significantly altered either increasing or decreasing the mechanical load of the gravitational force. CHO removal perturbed lipid rafts that act as platform for TSHR. Therefore membrane CHO and not SM was critical for TSH-TSHR interaction. Depletion of membrane CHO by methyl-beta-cyclodextrin resulted in a disruption of lipid rafts in plasma membrane  whereas extensive sphingolipid depletion did not affect lipidraft integrity . Rapid changes of the cytoskeleton as reaction to gravitational changes were reported in diverse cell types [28,29] and they could be responsible for changes in surface receptors . Here we reported the first observation of altered receptor by perturbation of membrane CHO.
enous or exogenous BDNF on LTP (Chen et al., 1999; Krama´r et al., 2004; Fontinha et al., 2008). To evaluate the influence of lipidraft disruption on the facilitatory effect of BDNF on LTP, M␤CD was used at a low concentration to avoid marked changes in synaptic integrity that could cause alterations in the synaptic plasticity phenomena. Hippocampal slices were incubated with M␤CD (1 m M ) for 30 min before and during the entire LTP experiment. At this concentration, M␤CD had a very mild effect on basal synaptic transmission ( ⫺4.6 ⫾ 0.4%), as shown in Fig- ure 10 B. Furthermore, in M ␤CD-treated slices, the magnitude of LTP was similar to what was observed in other slices in the ab- sence of M␤CD (Fig. 10H, open bars). Most importantly, the magnitude of LTP in two consecutive pathways on the same slice (see Materials and Methods) was similar in the presence of M ␤CD throughout the entire LTP-inducing protocol ( p ⬎ 0.05, n ⫽ 3) (Fig. 10C). This allowed us to study the modulatory role of BDNF on LTP in slices that were perfused with M ␤CD through- out the entire recording period. To evaluate the effect of BDNF on LTP, we compared the magnitude of LTP in the first pathway (in the absence of BDNF) with that in the second pathway (in the presence of BDNF), in the same slice. The effect of BDNF on LTP in the absence or presence of M ␤CD was then compared.
The cytostome/ cytopharynx of T. cruzi is a plasma membrane invagination that penetrates deeply into the cytoplasm towards the nucleus (Milder & Deane 1969). Fluid-phase pinocytosis of peroxidase and receptor- mediated endocytosis of transferrin occur through this structure, resulting in accumulation of ingested proteins in organelles called reservosomes (Soares & De Souza 1988, Soares et al. 1992, Porto-Carreiro et al. 2000). Our biochemical analysis of a DRM fraction allowed the detection of flotillin-1 and a singular T. cruzi glycolipid that positively reacts with cholera B toxin, both labels considered as lipidraft markers (Nguyen & Hildreth 2000, Seveau et al. 2004). Furthermore, we have also obtained immunofluorescence co-localization for flotillin-1 and TRITC-labeled transferrin in a region cor- responding to the cytostome/ cytopharynx. Although no flotillin-1 gene has been found in the T. cruzi genome database (El-Sayed et al. 2005), the punctual positive reaction for flotillin-1 at the cytostome/ cytopharynx suggests that a similar protein might be present at this site, although the possibility of cross-reactivity with a non-related protein can not be excluded. Recent data from Hela and MEF cells have shown that coassembly of flotillin-1 and flotillin-2 into micro-domains induced membrane curvature, formation of plasma-membrane invaginations morphologically similar to caveolae, and the accumulation of intracellular vesicles. These microdomains are distinct from caveolin1-positive caveolae, are dynamic, and bud into the cell. It has been proposed that flotillin proteins are defining structural components of the machinery that mediates a clathrin- independent endocytic pathway (Frick et al. 2007).
The mechanism or combination of mechanisms proposed here does not exclude any other; namely, other mechanisms may act independently of or in conjunction with the ones presented here. Moreover, MD limitations are clear and restrict the conclusions to processes/mechanisms developing at very short time and length scales. Other mechanisms may act at longer times and/or larger lengths scales, and different numerical techniques should therefore be used for their investigation. For example, the high affinity of chlf for ld lipid phases indicates that this compound may alter the thermodynamics of lo/ld (liquid-liquid) coexistence in lipid mem- branes. This idea is particularly attractive in the context of the lipidraft hypothesis , which proposes the formation of lo nanodo- mains (rafts) as a fundamental organizing principle of the components of the cell membrane (lipids and proteins). Chloroform may alter the thermodynamics of lo/ld coexistence, providing a mechanism for anesthesia at a global scale involving lipid-mediated protein reorganization in the cell membrane. Large-scale coarse- grained simulations of multicomponent bilayers should be used to numerically explore this possibility. Additionally, experiments on giant unilamellar vesicles as those used in  can be specifically designed to explore the results presented in this paper.
As MVs e microvillar vesículas exibem a mesma composição lipídica, com mais alto conteúdo de colesterol, esfingomielina e fosfatidilserina quando comparado à membrana plasmática (Thouverey et al., 2009). A composição de fosfolipídios das MVs foi estudada por Thouverey e colaboradores (2009) encontrando valores aproximados de 36% de fosfatidiletanolamina (PE), 26,5% de fosfatidilcolina (PC), 3,5% de ácido fosfatídico (PA), 7% de fosfatidilinositol (PI), 16,5% de fosfatidilserina (PS) e 11% de esfingomielina (SM). Deve ser lembrado que estes autores não descreveram a presença de GM1 na composição lipídica das vesículas da matrix, provavelmente devido à técnica emprega para a extração, detecção dos diferentes lipídios e complexidade para separação de glicolipídios. Porém, a incorporação de GM1 em monocamadas de DPPC/colesterol fornece evidências de heterogeneidade, com a observação de pequenos micro domínios ricos em GM1. Acredita-se que estes micro domínios são relevantes para o entendimento dos mecanismos de formação de lipid rafts em membranas (Yuan e Johnston, 2000).
Oxidative stress has been considered as one of the factors responsible for hepatic diseases, which sometimes require new ways of treatment. The present study aimed to evaluate the in vitro antioxidant capacity of the tea of Echinodorus grandiforus ("leather hat" plant) in rat liver. Different preparations of tea were evaluated for phenolic composition, antioxidant activity by DPPH assay and ability to inhibit lipid peroxidation induced by copper sulfate. The antioxidant activity was assessed in liver tissue treated with sodium azide in the presence or absence of tea by assays for lipid peroxidation (TBARS), protein oxidation (carbonyl) and the antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD). The results show that different preparations of tea are important sources of polyphenols and contain theobromine, catechin and vitexin. Furthermore, the results indicate that this tea exhibits an antioxidant activity by its ability to scavenge DPPH radical. Different preparations of tea prevented damage to lipids and proteins induced by sodium azide, as well as assisting in restoring CAT and SOD activities. Thus, it can be seen that E. grandiforus tea had antioxidant activity in serum and liver being able to prevent oxidative damages generated by sodium azide. Key words: oxidative stress, phenolic compounds, reactive species, sodium azide.
quantification of the results in Figure 3. We applied Triton X- 100 to cells at 37 uC rather than 4uC to maintain cell viability and preliminary experiments showed that this was associated with more effects on fenestrations (Figure S1). Fenestrations were quantified by measuring their diameter or their porosity (which is the percentage of the surface area of the cell membrane containing fenestrations). The effects of 7KC were initially studied at four concentrations. At the lowest concen- tration (9 mM) there was an increase in fenestrations while at the highest concentration (73 mM) typically used to study rafts in lymphocytes, cell membrane retraction and damage were apparent, however remaining fragments of cell membrane were highly fenestrated (Figure S2). Therefore the 9 mM concentra- tion was used in all subsequent experiments. LAURDAN (6- lauroyl-2-dimethylaminonaphthalene) was used to visualize rafts with ratiometric two-photon fluorescence microscopy. LAUR- DAN undergoes a spectral shift when in lipid-disordered non- raft regions versus lipid-ordered raft domains, thus can identify both raft and non-raft regions simultaneously and is a widely accepted method for identifying lipid-disordered and lipid- ordered regions of cell membranes [18,19]. This is quantified with Generalized Polarization values (GP) which range from 21 to 1, where 21 represents most lipid disordered and 1 represents the most lipid ordered membranes . LAUR- DAN-stained LSECs confirmed that 7KC increased lipid disordered, non-raft regions in the cell membrane. The GP values decreased from 20.259 in controls to 20.320 in 7KC- treated cells. These results were confirmed using NBD- cholesterol where it was found that Triton X-100 increased raft staining while 7KC reduced staining (Figure S3). Scanning electron microscopy (SEM) revealed that 7KC caused a marked increase in both the diameter of fenestrations and total porosity in isolated LSECs (Figure 2 and 3). 7KC was associated with the development of some gaps which presumably represent deficits in the cell membrane where the rafts have been depleted. On the other hand, Triton X-100 was associated with a marked decrease in fenestration porosity as assessed by SEM (Figure 2 and 3). GP values did not statistically significantly change following treatment with Triton X-100 however the distribution was more homogeneous and fewer sieve plates were apparent.
solid lipid nanoparticles stabilized by a blend of saturated long-chain phospholipids and the bile salt sodium glycolate. The structures of the dispersions were investigated by DSC, WAXS and TEM. A platelet-like, layered structure was confirmed for particles in the β-modification, whereas a spheroidal shape was revealed for particles in the α-form. In our study, the morphological evaluation as well as the WAXS and DSC analyses indicated the formation of β-TS in SLNs. This result may be attributed to the preparation method. By using the hot solvent diffusion method to obtain the lipid nanocarriers, hot nanosized emulsions were generated by mixing the organic and aqueous phases at 10 °C, the melting temperature of the solid lipid and, subsequently, magnetically stirring the mixture until it cooled to room temperature. This procedure could have favored the formation of the β allomorph because the recrystallization step was slow.
treatment. In principle the monolayer vs. bilayer membrane situation was outlined also in a scheme shown by Fujimoto et al. . These authors questioned how LDs can fuse with endocytosed caveolin-vesicles. By double stimulation with AIM and OA treatment of preadipocytes, we received a variety of diverse LDs which had quite different sets of PLIN proteins binding to LD surfaces. Single-color-stained LDs, mosaic, heterogeneously mixed and merged LDs positive for PLIN proteins in various sizes were detectable. Many different types of LDs in single cells could be generated by combining specific ‘‘endogenous’’ adipogenic stimulation and ‘‘exogenous’’ OA-treatment. We noticed, after AIM conversion and upon the uptake of OA, a rapid increase in expression of adipophilin, TIP47 and S3-12. Obviously these latter proteins were independently involved in the endocytotic uptake of hydrophobic substances, in contrast to the endogenously derived perilipin expression at the ER. These results are in conflict with current models of LD maturation in adipocytes, where TIP47, S3- 12 and adipophilin are thought to be replaced continuously by newly expressed perilipin (; for ‘‘EPAT exchangeable model’’ see ; for TIP47 (PLIN3) at ER see ). Preliminary experiments of in vitro binding using recombinant PLIN proteins to ‘‘Membrane Lipid Strips’’ (Echelon Biosciences), testing spotted lipid substances, revealed different sets of binding pattern for perilipin (PLIN1), adipophilin (PLIN2), TIP47 (PLIN3) and MLDP (PLIN5) respectively, suggesting that each PLIN protein is specialized to different sets of lipid material (H.Heid, unpub- lished). This confirms the work of Hsieh  on LD sorting and on specific binding with preferences of individual PLIN proteins to diverse hydrophobic components, mainly either TAG- or mainly CE-containing droplets. Heterogeneities and diversities of LD- binding proteins at the surface of LDs in single cells were also demonstrated e.g. by Ozaki et al
Dyslipidemia in pregnancy and the parameters for lipid transfer from the mother to the fetus are still not fully understood. However, studies have shown that gestational dyslipidemia is influenced by the placental hormones that affect both the metabolism of glucose and that of lipids, to ensure that the fetus is supplied with nutrients essential for its develop- ment. There is, however, less flexibility in terms of metabolic adaptation in obese pregnant women compared to those of normal weight. 5,18 In obese
Student’s t test was used to compare the means of two groups. Linear association between lipid fractions and categories of nutritional status was tested with ANCOVA. The chi-squared test was used to compare proportions. A test for linear trends (Jonckheere- Terpstra test) was performed to test the linear association between the age range as the independent variable and lipid fractions as the dependent variable. Normal distribution curves of total cholesterol and triglycerides were drawn separately for gender and median (P 50th )
Fig. 2 – Scheme of a mussel raft with symbols as described for the model (see text). Fig. 3 – Ingestion rate (a), mean scope for growth (b) and mussel production (c) for a raft with “seed” mussels solely (0.05 g meat DW) as a function of mussel abundance and current speed, assuming a concentration of POM of 0.5 mg L -1 and ranges in mussel abundance and current speed within those observed (see text).
bovinos mestiços holandês-zebu, fistulados no rúmen. As fezes foram coletadas nos tempos zero (imediatamente após a administração da fibra complexada com cromo), 1, 2, 4, 6, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 56, 64, 72, 80, 88, 96, 120, 132, 144 e 192 horas. As estimativas dos parâmetros dos perfis de taxa de passagem foram ajustadas de acordo com procedimentos de regressão robusta. O tempo médio de retenção de partículas no raft ( ); o tempo médio de retenção de partículas na fase líquida ruminal ( ) e o tempo médio de retenção ruminal total ( ) foram ajustados para a cinética de passagem e estimados com o procedimento NLIN do SAS. A partir dos resultados observados, pode-se sugerir que a taxa de transferência possui comportamento inverso à taxa de escape ; os valores médios de , os tempos médios de retenção e os tempos médios de retenção de partículas na fase líquida ruminal ( ) não diferiram estatisticamente. As características cinéticas dos alimentos, após escaparem do raft e chegarem à fase líquida, foram equivalentes. Os das fibras das diferentes silagens não diferiram entre si ( ). Observaram-se pequenas diferenças numéricas dos nos diferentes alimentos. As fibras das silagens em estudo tiveram taxas semelhantes de λ, k, TMR1,TMR2 e TMRR. Portanto, a cinética da taxa de passagem ruminal de partículas das silagens dos consórcios é semelhante. Assim, a tomada de decisão pelo tipo de consórcio a ser utilizado deve ser baseada em outros fatores, tais como viabilidade do manejo de culturas e custo da silagem.
The aim of this work is to prepare Laponite RD-based nanocomposite latexes by aqueous emulsion polymerization, using the reversible addition-fragmentation chain transfer (RAFT) polymerization. Laponite platelets were selected as the inorganic filler due, especially, to their anisotropic shape, which allows the production of nanostructured films, but also for their thermal and mechanical properties, their high chemical purity and the uniform dispersity of the platelets. Hydrophilic polymers (macroRAFT) composed of poly(ethylene glycol) (PEG), acrylic acid (AA) or N,N-dimethylaminoethyl methacrylate (DMAEMA) and comprising hydrophobic n-butyl acrylate (BA) units (in some cases) and trithiocarbonate terminal group were initially synthesized. Then, the interaction between the macroRAFTs and the clay was studied through the plot of adsorption isotherms. By acting as coupling agents and stabilizers, the macroRAFT agents were used in the emulsion copolymerization of methyl (meth)acrylate and BA by semi-continuous process in the presence of the clay. Hybrid latex particles with different morphologies were obtained and the results were associated to the nature and concentration of the RAFT (co)polymers, to the pH of the macroRAFT/Laponite dispersion, the glass transition temperature of the final copolymer (function of the composition of the hydrophobic monomers mixture) and to the polymerization conditions. The cryo-TEM images indicate the formation of polymer- decorated Laponite platelets (several latex particles located at the surface of the platelets), dumbbell-like, janus, Laponite-decorated (armored) latex particles, and multiple encapsulated particles (several platelets inside each latex particle). The mechanical properties of polymer/Laponite films were studied by dynamic mechanical analysis and correlated with the particles morphology and the films microstructure.
Interestingly, the pretreatment of leukocytes with pro- tein synthesis inhibitors acting on transcription (actino- mycin D) and translation (cycloheximide) significantly, although partially, inhibited lipid body formation induced by PAF and fatty acids in vitro but not by eotaxin and RANTES, thus indicating that according to the stimulated conditions induction of lipid bodies depends on new pro- tein synthesis and its likely that specific and trans- criptionaly regulated early response genes are activated during the process of lipid body formation (Bozza et al. 1996a, b, 1997, Bandeira-Melo et al. 2001a, Pacheco et al. 2002). A role for peroxisome proliferator-activated recep- tors (PPARs), members of the nuclear receptor supergene family that function in ligand-activated transcription, in leukocyte lipid body formation has recently been sug- gested. Indeed, we observed that treatment of mouse mac- rophages with BRL 49653, a preferential PPARγ ligand, failed to induce lipid body formation. However, BRL 49653 treatment significantly potentiated lipid body formation induced by ox-LDL or PAF-like agonists, suggesting that PPAR have a role in regulating leukocyte lipid body for- mation (de Assis et al. 2003). However, we anticipate that differences in PPAR expression and role in lipid body for- mation may vary according to the cell type and stimuli studied and further studies are needed to characterize the roles of PPAR in lipid body formation and the involve- ment of PPAR targeted genes in this phenomenon.
Higher photosynthetic efficiency and biomass production with rapid growth makes microalgae as potential candidates over other energy crops in many applications. Heavy metals influence the production of secondary metabolites and lipd content of microalgae in particular. A study was conducted using six Chlorella species under heavy metal exposure to evaluate the copper stress on biomass, cellular and lipid contents. Preliminary growth studies indicated the growth tolerance levels of Chlorella in the presence of copper at 4.0 mg L −1 concentration. The total chlorophyll, protein and lipid content of the isolates were 1.7-3.45%, 0.43-0.70 mg g −1 and 0.02-0.11 mg g −1 respectively. Gas Chromatography-Mass Spectroscopy analysis revealed that the percent composition of fatty acids varied among the species studied and the major group of fatty acids were C16:0, C18:1 and C18:2. Highest percent of fatty acids were found in C. vulgaris, C. protothecoides and C. pyrenoidosa. Copper have an impact on Chlorella species where biomass content was directly proportional to the lipid productivity. The results reflects the fact that copper stress on Chlorella species as the evidence of lipid production in both qualitative and quantitative manner. In conclusion, Chlorella species can be used for the sustainable producion of renewable energy through copper stress and removal of copper from aqueous solutions.
As a corollary from all the data, we conclude that the plasma membrane physicochemical properties undoubtedly play a key role in the mechanism of action of ACT, and very likely, other RTX toxins. In this regard, many questions remain to be explored in the near future: Does ACT exploit or depend on the inherent properties of cholesterol-enriched domains to exert its cytotoxic activities? Is the toxin itself able to locally alter the physical properties of the lipid bilayer? And could ACT even indirectly induce local changes in the membrane lipid composition, i.e., activating calcium-dependent sphingomyelinases? Experiments are currently ongoing in our laboratory to provide answers.