Raft Structure Research Articles

Anisotropic Stress Rupture Properties of a 3rd-Generation Nickel-Based Single-Crystal Superalloy at 1100 °C/150 MPa

The influence of orientation on the stress rupture behaviors of a 3rd-generation nickel-based single-crystal superalloy was investigated at 1100 °C/150 MPa. It is found that the stress rupture anisotropy is shown at 1100 °C, but not so obvious compared with that at intermediate temperatures. The [001] specimens display the longest rupture life, [111] specimens show the shortest rupture life, and [011] specimens exhibit the intermediate life. Detailed observations show that the final fracture is caused by crack initiation and propagation, and the anisotropy of three oriented specimens is related to the fracture modes, γ/γʹ microstructures, interfacial dislocation networks and cutting mechanisms in γʹ phase. For [001] specimens, N-type rafted structures are formed which can well hinder the slip and climb of dislocations. Besides, the regular interfacial dislocation networks can prevent dislocations from cutting into γʹ phase, leading to the improvement of the creep resistance. For [011] specimens, ± 45° rafted structures and irregular networks result in less strain hardening. For [111] specimens, a large number of crack propagation paths and inhomogeneous deformations caused by irregular rafted structures deteriorate the property and result in the shortest life. Furthermore, a[100] superdislocations with low mobility are widely formed in [001] and [011] specimens which suggests the low creep strain rate during steady creep stage, whereas superdislocations in [111] specimens possess high mobility, which indicates the high strain rate and corresponding poor stress rupture property.

Combined phase-field crystal plasticity simulation of P- and N-type rafting in Co-based superalloys

We combine a phase-field model with a crystal plasticity model to simulate the microstructural evolution during creep in the Co-based superalloy ERBOCo-2Ta. Three-dimensional simulations of tensile and compressive creep tests in [100] direction were performed to study the rafting behavior in Co-based superalloys. The loss of coherency between γ matrix and γ′ precipitate, which is essential for the understanding of rafted structures, is modeled in relation to the dislocation activity in the γ-channels. Special attention is given to the interplay between creep deformation and microstructure stability. Appropriate constitutive modeling is applied to simulate realistic microstructure evolution under creep conditions. Thus, with the removal of the misfit stress, γ′ precipitates lose their cuboidal shape and form rafts. During N-type rafting more γ′ precipitates coalesce than during P-type rafting. The γ′ volume fraction during rafting increases under tensile stress but decreases under compressive stress. The morphological evolution of γ′ precipitates under tensile and compressive stresses in Co-based superalloy is consistent with the rafting characteristics in experimental observations.

Human Peripheral Blood Mononuclear Cells Express High Levels of the Vitamin a Transport Protein, Stimulated by Retinoic Acid 6 (P19-004-19)

ObjectivesStimulated by retinoic acid 6 (STRA6) was the first to be identified in a novel category of proteins, cytokine signaling transporters, due to its ability to function as both a cell surface receptor and a membrane protein that binds to retinol binding protein facilitating cellular uptake of retinol. Retinol is metabolized to all-trans retinoic acid which functions as a steroid hormone transcription factor ligand to regulate genes generally involved in cellular differentiation. The role of vitamin A, as all-trans retinoic acid, in immunity is well characterized, but the role of STRA6 in mediating cellular uptake of vitamin A in immune cells is not understood. The objective of this research was to characterize the expression of STRA6 on human peripheral blood mononuclear cell (PBMC) subsets. We hypothesized STRA6 would be expressed by all PBMC subsets, but at different intensities. MethodsA convenience sample of volunteers were recruited and PBMC were isolated by density gradient centrifugation. Multicolor flow cytometry was used to identify PBMC subsets and expression of STRA6. Confocal microscopy was used to determine cellular location and lipid raft association of STRA6 on cell lines and isolated PBMC. ResultsAll T cell, natural killer cell, monocyte, and dendritic cell subsets analyzed expressed STRA6. Expression of STRA6 (median fluorescence intensity) was higher on CD303+ and CD141+ dendritic cells compared to CD1c+ dendritic cells, CD8+ T cells compared to CD4+ T cells, CD14+/CD16+ and CD14+/CD16- monocytes compared to CD14-/CD16+ monocytes, and CD56-/CD16+ natural killer cells compared to both CD56+/CD16 variable and CD56-/CD16 dim natural killer cells. Expression was not significantly correlated with age, sex, or body composition. Confocal microscopy showed STRA6 was not associated with GM1 cholera toxin subunit B staining and therefore not associated with lipid raft structures. ConclusionsThese results provide preliminary data in which to target vitamin A signaling, through cellular uptake of retinol, in immune cells to improve immune homeostasis in diverse populations. Funding SourcesFunding for this project was provided by internal university support.

Creep Damage of a Re/Ru-Containing Single Crystal Nickel-Based Superalloy at Elevated Temperature

By means of creep properties measurement, microstructure observation and contrast analysis of dislocation configuration, the creep behavior of a 4.5%Re/3.0%Ru-containing single crystal nickel-based superalloy at elevated temperature is investigated. Results show that the creep life of the alloy at 1040°C/160MPa is measured to be 725h to exhibit a better creep resistance at high temperature. In the primary stage of creep at high temperature, the γ phase in alloy has transformed into the N-type rafted structure along the direction vertical to the stress axis, the deformation mechanism of alloy during steady state creep is dislocations slipping in γ matrix and climbing over the rafted γ phase. In the latter period of creep, the deformation mechanism of alloy is dislocations slipping in γ matrix and shearing into the rafted γ phase. Wherein the dislocations shearing into the γ phase may cross-slip from {111} to {100} planes for forming the K-W locks to restrain the slipping and cross-slipping on {111} plane, which is thought to be the main reason of the alloy having a better creep resistance. As the creep goes on, the alternate slipping of dislocations results in the twisted of the rafted γ phase to promote the initiation and propagation of the cracks along the interfaces of γ/γ phase up to creep fracture, which is thought to be the damage and fracture mechanism of alloy during creep at high temperature.

Caveolar Kv1.3 Targeting Participates in the Adipocyte Physiology

The voltage-gated potassium channel Kv1.3 contributes to peripheral insulin sensitivity. KO mice for Kv1.3 are resistant to diet-induced weight gain. Therefore, this protein is being considered as a pharmacological target for obesity and associated type II diabetes. However, the expression of Kv1.3 in adipocytes generates controversy. Here, we found that Kv1.3 is present in white adipose tissue from humans and rodents. In addition, Kv1.1, Kv1.2, Kv1.4 and especially Kv1.5, from the same Shaker family are also present. Although elevated insulin levels and adipogenesis remodeled the Kv phenotype, which could trigger to numerous hybrid complexes, Kv1.3 noticeably participated in the insulin-dependent regulation of glucose transport in differentiated adipocytes. Adipogenesis increased the expression of Kv1.3 into caveolae by molecular interactions with caveolin 1. In a caveolin 1-deficient 3T3-L1 adipocyte cell line, we verified that the localization of Kv1.3 in caveolar lipid raft structures is necessary for proper insulin signaling. Insulin-dependent phosphorylation of the Kv1.3 occurs at the beginning of insulin-mediated signaling. However, when Kv1.3 localized out of these lipid microdomains, reduced phosphorylation was demonstrated. Our data contribute to the knowledge of the putative role of Kv1.3 in adipocyte physiology. Supported by the Ministerio de Ciencia, Innovación y Universidades, Spain (BFU2014-54928-R and BFU2017-87104-R) and Fondo Europeo de Desarrollo Regional (FEDER).

Effect of directional solidification process on microstructure and stress rupture property of a hot corrosion resistant single crystal superalloy

The influences of different directional solidification processes, i.e., the high rate solidification (HRS) and liquid metal cooling (LMC), on microstructure and stress rupture property of DD488 alloy were investigated. The DD488 alloy was directional solidified by both HRS and LMC processes. The microstructure and stress rupture properties at 980 oC/250 MPa were investigated by using optical microscopy (OM), scanning electron microscopy (SEM), electron microprobe analyzer (EPMA), transmission electron microscopy (TEM) and stress rupture testing. The results indicated that the LMC process refined the primary dendrite arm and decreased the microporosity volume fraction and solidification segregations of Cr and Co in as-cast DD488 alloy. After standard heat treatment of 1,260 oC/4 h, AC (air cooling) + 1,080 oC/4 h, AC + 870 oC/24 h, AC, the γ′ morphology in LMC alloy was more cuboidal than that in HRS alloy, and the γ′ volume fraction of LMC alloy was higher than that of HRS alloy. The stress rupture life at 980 oC/250 MPa of HRS alloy was 76.8 h, and it increased to 110.0 h in LMC alloy. The LMC process increased the stress rupture life due to the higher γ′ volume fraction, more perfect rafting structure and finer interfacial dislocation networks.

Piercing the lipid raft: the case of Vibrio cholerae cytolysin.

In a recent issue of Biochemical Journal, Kathuria et al. [Biochem. J. (2018) 475, 3039-3055] report that membrane binding of the pore-forming toxin Vibrio cholerae cytolysin (VCC) is facilitated by the presence of cholesterol, and the presence of this sterol within the lipid bilayer is key for the formation of a functional pore. Yet, in the presence of accessory non-lipid components, VCC retains its membrane-binding capability likely through membrane lipid raft structures. In light of their results, the authors provide new insights into the roles of cholesterol and of membrane microstructures in the binding, the oligomeric assembly and the cytolytic pore formation of VCC which all take place following infection by V. cholerae.

Determining the target of membrane sterols on voltage-gated potassium channels

Cholesterol, an essential lipid component of cellular plasma membranes, regulates fluidity, mechanical integrity, raft structure and may specifically interact with membrane proteins. Numerous effects on ion channels by cholesterol, including changes in current amplitude, voltage dependence and gating kinetics, have been reported. We have previously described such changes in the voltage-gated potassium channel Kv1.3 of lymphocytes by cholesterol and its analog 7-dehydrocholesterol (7DHC). In voltage-gated channels membrane depolarization induces movement of the voltage sensor domains (VSD), which is transmitted by a coupling mechanism to the pore domain (PD) to open the channel. Here, we investigated whether cholesterol effects were mediated by the VSD to the pore or the PD was the direct target. Specificity was tested by comparing Kv1.3 and Kv10.1 channels having different VSD-PD coupling mechanisms. Current recordings were performed with two-electrode voltage-clamp fluorometry, where movement of the VSDs was monitored by attaching fluorophores to external cysteine residues introduced in the channel sequence. Loading the membrane with cholesterol or 7DHC using methyl-β-cyclodextrin induced changes in the steady-state and kinetic parameters of the ionic currents while leaving fluorescence parameters mostly unaffected in both channels. Non-stationary noise analysis revealed that reduction of single channel conductance rather than that of open probability caused the observed current decrease. Furthermore, confocal laser scanning and stimulated emission depletion microscopy demonstrated significant changes in the distribution of these ion channels in response to sterol loading. Our results indicate that sterol-induced effects on ion channel gating directly target the pore and do not act via the VSD.

Effect of temperature on raft-dependent endocytic cluster formation during activation of Jurkat T cells by concanavalin A

Temperature plays an important role in the immune response. Acclimatization occurs when there are changes in ambient temperature over a long period. In this study, we used the human leukemic Jurkat T cell line to study the effect of temperature on the immune system using concanavalin A (ConA), a plant-derived immunostimulant, as a trigger for T-cell activation. Previously, we have reported endocytic intracellular cluster formation during T-cell activation by ConA with the aid of rafts and polymerization of the cytoskeleton (actin and microtubules). Here, we investigated the effect of temperature on cluster formation (with the aid of three-dimensional images of the cells) and on the stability of rafts, actin, and microtubules. When the temperature was changed between 23°C and 37°C (physiological temperature), clusters could be observed throughout this temperature range. Raft structure was stabilized at lower temperatures but destabilized at higher temperatures. Actin was stable when the temperature was higher than 27°C. When actin was depolymerized, clustering was not observed at 37°C but could be observed at 23°C. There were no changes in microtubules within this temperature range. Thus, raft clustering may be associated with raft stability at lower temperatures (<27°C) and with actin at higher temperatures (≥27°C). Hence, we provided insight into the associations between temperature, rafts, actin, and microtubules in the immune response.

Creep property and microstructural evolution of DD11 alloy under high temperature and low stress

In this paper, the interrupted and ruptured creep tests were carried out in a novel second generation single crystal superalloy named DD11 at 1100 °C/130 MPa. The alloy exhibited typical creep curve including primary, steady, and tertiary three creep stages. The microstructural evolution at different stages of the creep were analyzed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results show that the γ′ phases transform into rafted structure at the early stage of the steady creep and keep stable during the steady creep stage. As the creep goes on, the rafted structure further coarsens and the topological inversion occurs. In addition, at the primary creep, the dislocations mainly move in the γ matrix and pile up in the γ/γ′ interface since the matrix channels widen slightly. The formation of the regular interfacial dislocation networks occurs at the early stage of the steady creep. Under the low stress, the dominated deformation mechanism during steady creep stage is the climbing of the 〈010〉 type edge dislocation. Furthermore, the effect of the deformation mechanism on creep property was discussed in detail.

Caveolar targeting links Kv1.3 with the insulin-dependent adipocyte physiology.

The voltage-dependent potassium channel Kv1.3 participates in peripheral insulin sensitivity. Genetic ablation of Kv1.3 triggers resistance to diet-induced weight gain, thereby pointing to this protein as a pharmacological target for obesity and associated type II diabetes. However, this role is under intense debate because Kv1.3 expression in adipose tissue raises controversy. We demonstrated that Kv1.3 is expressed in white adipose tissue from humans and rodents. Moreover, other channels, such as Kv1.1, Kv1.2, Kv1.4 and especially Kv1.5, from the same Shaker family are also present. Although elevated insulin levels and adipogenesis remodel the Kv phenotype, which could lead to multiple heteromeric complexes, Kv1.3 markedly participates in the insulin-dependent regulation of glucose uptake in mature adipocytes. Adipocyte differentiation increased the expression of Kv1.3, which is targeted to caveolae by molecular interactions with caveolin 1. Using a caveolin 1-deficient 3T3-L1 adipocyte cell line, we demonstrated that the localization of Kv1.3 in caveolar raft structures is important for proper insulin signaling. Insulin-dependent phosphorylation of the channel occurs at the onset of insulin-mediated signaling. However, when Kv1.3 was spatially outside of these lipid microdomains, impaired phosphorylation was exhibited. Our data shed light on the putative role of Kv1.3 in weight gain and insulin-dependent responses contributing to knowledge about adipocyte physiology.

Lipid raft‑associated β‑adducin participates in neutrophil migration.

Previous studies have demonstrated that lipid rafts and β-adducin serve an important role in leukocyte rolling. In the present study the migratory ability and behavior of neutrophils was demonstrated to rely on the integrity of the lipid raft structure. β-adducin was demonstrated to have a critical role in neutrophil migration. Knockdown of β-adducin attenuated the migratory ability of dHL-60 cells and the distribution of β-adducin in lipid raft structures was changed by N-formylmethionyl-leucyl-phenyl-alanine treatment. Furthermore, the findings demonstrated that the tyrosine phosphorylation of β-adducin was required for its relocation. The results of the present study suggested that the lipid raft-associated protein β-adducin may be a novel control point for the excessive infiltration of neutrophils during inflammation.

Imaging of immunogold labeling in cells and tissues by helium ion microscopy.

Helium ion microscopy (HIM) scans samples with a fine ion beam exploiting the very short de Broglie wavelength of helium ions. Because the radiation induces only a small sample region to emit secondary electrons (SEs), very high resolution is expected. In order to explore the applications of SE-HIM in biology, COS7 kidney fibroblast cells and C2C12 myoblast cells cultured on a silicon (Si) nitride (SiN)/Si bilayer were dried and directly observed in high vacuum, without coating or staining. High contrast, high depth-of-field images were obtained revealing the nucleus, endoplasmic reticulum, cytoskeleton and putative mitochondria above a bright background from the support. Gold-tagged antibodies were employed to aid organelle identification. Signals from the gold tags were most clearly distinguishable by secondary electron (SE)-HIM when cells were grown on thin SiN film, and the minimum gap measured between gold particles showed the resolution to be 2 nm. Wheat germ agglutinin-gold labeling revealed clusters of gold particles ~50–200 nm in diameter on COS7 cells, which might represent assemblies of glycosylated proteins, suggesting the formation of membrane raft structures that include membrane proteins. SE-HIM also delivered high contrast images of unstained, uncoated, thin sections of Epon-embedded mouse kidney tissues mounted on a SiN/Si bilayer, revealing the details of sub-tissues and cell organelles. A charge-coupled mechanism explaining the observed SE-HIM contrast is proposed. Ionoluminescence-HIM was also performed targeting zinc oxide particles on cells. In conclusion, the high depth-of-field, high-resolution imaging achieved using HIM may have applications in various fields, including soft materials.

"Lipid raft aging" in the human frontal cortex during nonpathological aging: gender influences and potential implicationsin Alzheimer's disease.

Lipid rafts are highly dynamic membrane domains featured by distinctive biochemical composition and physicochemical properties compared with the surrounding plasma membrane. These microstructures are associated not only with cellular signaling and communication in normal nerve cells but also with pathological processing of amyloid precursor protein in Alzheimer's disease. Using lipid rafts isolated from human frontal cortex in nondemented subjects aging 24 to 85years, we demonstrate here that lipid structure of lipid rafts undergo significant alterations of specific lipid classes and phospholipid-bound fatty acids as brain cortex correlating with aging. Main changes affect levels of plasmalogens, polyunsaturated fatty acids (especially docosahexaenoic acid and arachidonic acid), total polar lipids (mainly phosphatidylinositol, sphingomyelin, sulfatides, and cerebrosides), and total neutral lipids (particularly cholesterol and sterol esters). Besides, relevant relationships between main fatty acids and/or lipid classes were altered in an age-related manner. This "lipid raft aging" exhibits clear gender differences and appear to be more pronounced in women than in men, especially in older (postmenopausal) women. The outcomes led us to conclude that human cortical lipid rafts are modified by aging in a gender-dependent fashion. Given the central role of bilayer lipid matrix in lipid rafts functionality and neuronal signaling, we hypothesize that these findings might underlie the higher prevalence of cognitive decline evolving toward Alzheimer's disease in postmenopausal women.

Estrogen Interactions With Lipid Rafts Related to Neuroprotection. Impact of Brain Ageing and Menopause.

Estrogens (E2) exert a plethora of neuroprotective actions against aged-associated brain diseases, including Alzheimer's disease (AD). Part of these actions takes place through binding to estrogen receptors (ER) embedded in signalosomes, where numerous signaling proteins are clustered. Signalosomes are preferentially located in lipid rafts which are dynamic membrane microstructures characterized by a peculiar lipid composition enriched in gangliosides, saturated fatty acids, cholesterol, and sphingolipids. Rapid E2 interactions with ER-related signalosomes appear to trigger intracellular signaling ultimately leading to the activation of molecular mechanisms against AD. We have previously observed that the reduction of E2 blood levels occurring during menopause induced disruption of ER-signalosomes at frontal cortical brain areas. These molecular changes may reduce neuronal protection activities, as similar ER signalosome derangements were observed in AD brains. The molecular impairments may be associated with changes in the lipid composition of lipid rafts observed in neurons during menopause and AD. These evidences indicate that the changes in lipid raft structure during aging may be at the basis of alterations in the activity of ER and other neuroprotective proteins integrated in these membrane microstructures. Moreover, E2 is a homeostatic modulator of lipid rafts. Recent work has pointed to this relevant aspect of E2 activity to preserve brain integrity, through mechanisms affecting lipid uptake and local biosynthesis in the brain. Some evidences have demonstrated that estrogens and the docosahexaenoic acid (DHA) exert synergistic effects to stabilize brain lipid matrix. DHA is essential to enhance molecular fluidity at the plasma membrane, promoting functional macromolecular interactions in signaling platforms. In support of this, DHA detriment in neuronal lipid rafts has been associated with the most common age-associated neuropathologies, namely AD and Parkinson disease. Altogether, these findings indicate that E2 may participate in brain preservation through a dual membrane-related mechanism. On the one hand, E2 interacting with ER related signalosomes may protect against neurotoxic insults. On the other hand, E2 may exert lipostatic actions to preserve lipid balance in neuronal membrane microdomains. The different aspects of the emerging multifunctional role of estrogens in membrane-related signalosomes will be discussed in this review.

Gangliosides and Lysolipids Regulate the Size of Membrane Rafts Depending on the Membrane Composition

Gangliosides are the group of glycolipids abundant in brain and thought to be involved in amyloid aggregation and development of Alzheimer's disease. Nevertheless, in some cases it can play even neuroprotective role. Here we show that these contradictive activities of gangliosides can be the consequence of their influence on membrane liquid-ordered lipid domains, also called rafts. Despite the existence of rafts in vivo is still an open question, they are widely observed in model systems and can be some dynamical structures in cell membranes. Rafts are assumed to be important players in membrane signal transduction and trafficking. Recently, we demonstrated both experimentally and theoretically that ganglioside GM1 regulates the raft size distribution in two different ways depending on the membrane cholesterol content. Therefore, it can both increase and decrease the line tension of the lipid domain boundary. The same observations for the effect of lysolipids produced in the cell membranes by enzymes phospholipase pointed us to the possible common mechanism of the lipid molecular geometry and overall membrane composition on the structure of membrane rafts and their influence on membrane processes. This work was supported in part by the Russian Foundation for Basic Research (project # 18-54-74001).

Effectiveness of domestic wastewater treatment using floating rafts a promising phyto-remedial approach: A review

Treatment of wastewater will lead to the problems again, if we will not use new more efficient alternative technologies/methods to avoid drawback of old technologies. Loss of water can be reduced through application of easy, inexpensive and eco-friendly technologies for wastewater treatment. Using Floating rafts to purify polluted wastewater is a process/method of ecological restoration at in-situ, as well as a complicated physical (attachment of pollutants to the root surface), chemical (degradation of metals into less toxic form) and biological process (microbial processes). Its core is utilizing aquatic plants such as Canna and Water lily and root attached microbes such as bac-teria, fungi and algae to absorb pollutants such as nitrogen and phosphorus, degrade organic matter and accumulates heavy metals in their biomass. Phytoremediation of polluted wastewater using the Floating rafts technique is an Eco-friendly method of wastewater treatment, which is economically effective to construct, requires little maintenance and increase the biodiversity as different types of plants are used. Floating rafts technique has been applied to some water pollution control projects at domestic and abroad. However, there are some factors such as plants, temperature, seasons, hydraulic retention time, coverage and initial concentration of pollutants etc. influenced to the pollutants removal efficiency of Floating rafts. In the future, the development orientation has been subjected to plant and its combinations, the transformation of Floating rafts structure and the utilization of aquatic plants, and probed the technology of Floating rafts building and management, to implement the win-win of landscape benefit and ecological function.

Neutrophil CD16b crosslinking induces lipid raft-mediated activation of SHP-2 and affects cytokine expression and retarded neutrophil apoptosis

Two different types of FcRs for IgG are constitutively expressed on the surface of human neutrophils, namely, FcγRIIA (CD32a) and FcγRIIIB (CD16b). Unlike FcγRIIA, FcγRIIIb is GPI anchored to the cell membrane and its signal transduction is still ambiguous. To further understand the signal transduction of CD16b, we compared neutrophil cytokine expression and apoptosis by the cross-linking of CD32a and CD16b respectively. We found that both CD32a and CD16b crosslinking can activate neutrophils, but did not exactly share cytokine expression profiles. On the other hand, CD16b cross-linking retarded neutrophil apoptosis while CD32a promoted it. By interrupting the lipid raft with methyl-β-cyclodextrin (MβCD) and inhibiting the ITAM-SYK pathway with an SYK inhibitor (piceatannol), we found reduced apoptosis was at least partially mediated by lipid raft structure, but not the ITAM-SYK pathway. Additionally, CD16b but not CD32a cross-linking triggered SHP-2 phosphorylation and led to its translocation into lipid rafts. SHP-2 phosphorylation and translocation were inhibited by MβCD. Moreover, pre-inhibition of SHP-2 by a specific inhibitor (SHP099) converted IL-10 and SOCS3 expression level and promoted neutrophil apoptosis after CD16b crosslinking. In conclusion, these results, for the first time, collectively indicate that SHP-2 is activated by CD16b crosslinking in neutrophils and functions as a component of the raft-mediated signaling pathway.

Cavin-1 regulates caveolae-mediated LDL transcytosis: crosstalk in an AMPK/eNOS/ NF-κB/Sp1 loop.

Caveolae are specialized lipid rafts structure in the cell membrane and critical for regulating endothelial functions, e.g. transcytosis of macromolecules like low density lipoprotein (LDL) etc. Specifically, the organization and functions of caveolae are mediated by structure protein (caveolin-1) and adapter protein (cavin-1). The pathogenic role of caveolin-1 is well studied; nevertheless, mechanisms whereby cavin-1 regulates signaling transduction remain poorly understood. The aim of this study was designed to explore the role of cavin-1 in caveolae-mediated LDL transcytosis across endothelial cells. We reported here that cavin-1 knockdown mediated by small interfering RNA (siRNA) caused a significant decrease of LDL transcytosis. Moreover, cavin-1 knockdown increased the activity of endothelial nitric oxide synthase (eNOS) and the production of nitric oxide (NO). Consequently, an eNOS inhibitor, N-Nitro-L-Arginine Methyl Ester (L-NAME), not only suppressed the activity of specificity protein (Sp1) and nuclear factor kappa B (NF-κB), but also inhibited both activities via activating adenosine 5‘-monophosphate- activated protein kinase (AMPK). In conclusion, we proposed an AMPK/eNOS/NF-κB/Sp1 circuit loop was formed to regulate caveolae residing proteins’ expression, e.g. LDL receptor (LDLR), caveolin-1, eNOS, thereby to regulate caveolae-mediated LDL transcytosis in endothelial cells.

Identification of sterol-containing domains in vacuolar membranes by confocal microscopy

Sterol-containing domains in the tonoplast of beet root cells were identified by confocal microscopy with a membrane probe filipin. The presence of sterol-containing domains in the tonoplast was confirmed using methyl-β-cyclodextrin. Sterol-containing domains with separate small intensely fluorescing sterol-enriched sites were found in the vacuolar membrane; these sites can be attributed to raft structures. It was shown that sterol content in the isolated rafts can be assessed by confocal microscopy. β-Sitosterol, stigmasterol, α-tocopherol, and cholesterol were identified among the sterols of the isolated rafts.