Sucrose Gradient Ultracentrifugation Research Articles

Selective mRNA translation by eIF2 phosphorylation during ER stress

When the protein flux into the endoplasmic reticulum exceeds its folding capacity, a condition referred to as ER stress, there is an induction of translational and transcriptional gene expression designed to alleviate the underlying stress. This Unfolded Protein Response (UPR) features a global decrease in translation, which is facilitated by PERK phosphorylation of eIF2 (eIF2~P). Coincident with global translational control, eIF2~P directs the preferential translation of mRNAs induced by the UPR. In this study, we use sucrose gradient ultracentrifugation and a genome‐wide microarray approach to measure changes in translational efficiencies for individual mRNAs during ER stress. We find that while many transcripts are repressed during eIF2~P, some appear resistant to stress, while a subset is preferentially translated. ATF5 mRNA showed the greatest shift towards higher polysomes during ER stress. Translation of ATF5 is controlled by a mechanism of delayed ribosome reinitiation. Additionally, CHOP mRNA was shifted substantially towards the higher polysome fractions via a ribosomal bypass of an inhibitory uORF situated in the 5′‐leader of the mRNA. Lastly, we report novel mRNAs involved in protein turnover and synthesis and mRNA processing, which are preferentially translated. Identification of novel preferentially translated genes identifies new control schemes for key regulatory steps in the UPR.

Alternative polyadenylation mediated shortening of the Hsp70.3 3′UTR increases polyribosome loading of the mRNA transcript

IntroductionHeat shock protein 70.3 (Hsp70.3) expression is increased in response to cellular stress and plays a cytoprotective role, including a cardioprotective role in the heart against ischemia/reperfusion (I/R) injury. Recent work from our lab shows that Hsp70.3 expression following I/R is controlled through alternative polyadenylation (APA). Truncation of the 3′UTR removes potential regulatory sequences and is observed concomitant with upregulation of Hsp70.3 expression. Herein, we investigated the hypothesis that APA truncation of the 3′UTR enhances polysome loading of the Hsp70.3 transcript.MethodsSucrose gradient ultracentrifugation was used to isolate polysome bound mRNA from HL‐1 cardiac myocyte cells subjected to control (long 3′UTR) or a 1 hr heat shock (HS; short 3′UTR).ResultsCells subjected to HS for one hour showed a decrease in the total amount of polysome loaded mRNA. The Hsp70.3 transcript found in the polysome dense fractions existed as a higher ratio of the APA truncated short 3′UTR, thus supporting our hypothesis that APA truncation of the Hsp70.3 3′UTR increases polysome loading.Our results provide an enhanced understanding of APA on the regulation of Hsp70.3 and will have a significant impact in understating the regulation of cardiac gene regulation after I/R injury.This work was partially funded by a University of Cincinnati Provost Pilot Research Project grant (MT).

Characterization of the cytosolic pool of perilipin 5

The PAT family of lipid storage droplet (LD) associated proteins consists of five members, each an established regulator of neutral lipid metabolism. The most recently discovered family member, perilipin 5, has been shown to localize to two distinct intracellular pools: the lipid storage droplet and a cytosolic fraction, the nature of which is less understood. Characterization of the cytosolic fraction by way of sucrose gradient ultracentrifugation and non‐denaturing gel electrophoresis has indicated a density of 1.15 g/mL and a mass of 575 kDa for the particles on which LD‐dissociated perilipin 5 resides. The observed density and susceptibility of these structures to lipolytic degradation suggest that their core is rich in neutral lipid, much like the larger LD's. Based on the current understanding of these complexes, our lab is using a combination of immunoprecipitation and crosslinking techniques to identify other proteins that localize to these particles and interact with perilipin 5. Characterization of these candidate proteins holds the promise of yielding new insights into the regulation and function of the perilipin 5 protein, and to expand our understanding of lipid metabolism as a whole.

Human Sperm Tail Proteome Suggests New Endogenous Metabolic Pathways

Proteomic studies are contributing greatly to our understanding of the sperm cell, and more detailed descriptions are expected to clarify additional cellular and molecular sperm attributes. The aim of this study was to characterize the subcellular proteome of the human sperm tail and, hopefully, identify less concentrated proteins (not found in whole cell proteome studies). Specifically, we were interested in characterizing the sperm metabolic proteome and gaining new insights into the sperm metabolism issue. Sperm were isolated from normozoospermic semen samples and depleted of any contaminating leukocytes. Tail fractions were obtained by means of sonication followed by sucrose-gradient ultracentrifugation, and their purity was confirmed via various techniques. Liquid chromatography and tandem mass spectrometry of isolated sperm tail peptides resulted in the identification of 1049 proteins, more than half of which had not been previously described in human sperm. The categorization of proteins according to their function revealed two main groups: proteins related to metabolism and energy production (26%), and proteins related to sperm tail structure and motility (11%). Interestingly, a great proportion of the metabolic proteome (24%) comprised enzymes involved in lipid metabolism, including enzymes for mitochondrial beta-oxidation. Unexpectedly, we also identified various peroxisomal proteins, some of which are known to be involved in the oxidation of very long chain fatty acids. Analysis of our data using Reactome suggests that both mitochondrial and peroxisomal pathways might indeed be active in sperm, and that the use of fatty acids as fuel might be more preponderant than previously thought. In addition, incubation of sperm with the fatty acid oxidation inhibitor etomoxir resulted in a significant decrease in sperm motility. Contradicting a common concept in the literature, we suggest that the male gamete might have the capacity to obtain energy from endogenous pools, and thus to adapt to putative exogenous fluctuations.

Enrichment of Golgi membranes from HeLa cells by sucrose gradient ultracentrifugation

This is a protocol to extract intact Golgi Membranes from HeLa cells using sucrose gradient centrifugation. This extraction is very useful for several applications including pull-down of Golgi membrane proteins, electron microscopy and reconstitution of protein transport into an isolated system. Protocol adapted from Balch et al. (1984).

Gas6-Induced Tissue Factor Expression in Endothelial Cells Is Mediated Through Caveolin-1-Enriched Domains

AbstractAbstract 3308Gas6 is the ligand for the TAM family of receptors, which are composed of three members namely Tyro3, Axl and Mer. These receptors belong to the large family of type I transmembrane receptor tyrosine kinases. Our laboratory has identified important intracellular signaling pathways important in gas6-Axl mediated protection of endothelial cells from apoptosis. However, as both gas6 and Axl null mice are protected from lethal thromboembolism, we sought to explore novel gas6-Axl intracellular signaling pathways regulating thrombosis. Caveolae have been shown to play a crucial role in the activation of signaling cascades following ligand binding to receptor tyrosine kinases. Caveolae are formed from lipid rafts by polymerization of caveolins. Caveolin-1 is the most abundant protein found in caveolae. Caveolin-1-enriched microdomains are well known to play a role as a docking platform for receptor tyrosine kinases and intracellular adaptor signaling proteins. Axl association with these highly specialized domains of the plasma membrane has not been previously elucidated. In the present study, we investigated the role of caveolin-1-enriched microdomains in gas6/Axl signaling in endothelial cells. First, we demonstrated that gas6-induced Akt and Erk1/2 phosphorylation required the presence of a functional Axl receptor as shown by Axl siRNA knockdown experiments in human umbilical vein endothelial cells. Then, caveolin-1 fractions, enriched by a detergent-free cell lysis followed by sucrose gradient ultra centrifugation, were studied by western blot analysis. After 5 and 10 min of gas6 treatment, Axl colocalized with caveolin-1 suggesting Axl recruitment into caveolin-1-enriched cell fractions. We found that c-Src, a signaling molecule known to behave as a transient docking platform in lipid rafts, also moved in caveolin-1-enriched cell fractions after gas6 stimulation. Caveolin-1 siRNA abolished gas6-induced Akt, Erk1/2 and c-Src phosphorylation suggesting that caveolin-1 enriched fractions are required for gas6-Axl signaling. Interestingly, we have shown that gas6-induced Akt phosphorylation required c-Src activation using c-Src siRNA and the pharmacological inhibitor (PP2). However, gas6-induced Erk1/2 phosphorylation was independent of c-Src. Finally, we found that gas6 increased tissue factor expression through the Axl-c-Src-Akt signaling cascade. Taken together, our results demonstrate that caveolin-1-enriched domains are required for gas6-Axl signaling and lead to the upregulation of tissue factor expression by gas6 in endothelial cells. These results highlight new insights of gas6-Axl signaling and function in endothelial cells. Disclosures:No relevant conflicts of interest to declare.

Focal adhesion kinase regulates intestinal epithelial barrier function via redistribution of tight junction

Disruption of epithelial barrier function was identified as one of the pathologic mechanisms in inflammatory bowel diseases (IBD). Epithelial barrier consists of various intercellular junctions, in which the tight junction (TJ) is an important component. However, the regulatory mechanism of tight junction is still not clear. Here we examined the role of focal adhesion kinase (FAK) in the epithelial barrier function on Caco-2 monolayers using a specific FAK inhibitor, PF-573, 228 (PF-228). We found that the decrease of transepithelial resistance and the increase of paracellular permeability were accompanied with the inhibition of autophosphorylation of FAK by PF-228 treatment. In addition, PF-228 inhibited the FAK phosphorylation at Y576/577 on activation loop by Src, suggesting Src-dependent regulation of FAK in Caco-2 monolayers. In an ethanol-induced barrier injury model, PF-228 treatment also inhibited the recovery of transepithelial resistance as well as these phosphorylations of FAK. In a sucrose gradient ultracentrifugation, FAK co-localized with claudin-1, an element of the TJ complex, and they co-migrate after ethanol-induced barrier injury. Immunofluorescence imaging analysis revealed that PF-228 inhibited the FAK redistribution to the cell border and reassembly of TJ proteins in the recovery after ethanol-induced barrier injury. Finally, knockdown of FAK by siRNA resulted in the decrease of transepithelial resistance. These findings reveal that activation of FAK is necessary for maintaining and repairing epithelial barrier in Caco-2 cell monolayer via regulating TJ redistribution.

Pilot Scale Production of Highly Efficacious and Stable Enterovirus 71 Vaccine Candidates

BackgroundEnterovirus 71 (EV71) has caused several epidemics of hand, foot and mouth diseases (HFMD) in Asia and now is being recognized as an important neurotropic virus. Effective medications and prophylactic vaccine against EV71 infection are urgently needed. Based on the success of inactivated poliovirus vaccine, a prototype chemically inactivated EV71 vaccine candidate has been developed and currently in human phase 1 clinical trial.Principal FindingIn this report, we present the development of a serum-free cell-based EV71 vaccine. The optimization at each step of the manufacturing process was investigated, characterized and quantified. In the up-stream process development, different commercially available cell culture media either containing serum or serum-free was screened for cell growth and virus yield using the roller-bottle technology. VP-SFM serum-free medium was selected based on the Vero cell growth profile and EV71 virus production. After the up-stream processes (virus harvest, diafiltration and concentration), a combination of gel-filtration liquid chromatography and/or sucrose-gradient ultracentrifugation down-stream purification processes were investigated at a pilot scale of 40 liters each. Although the combination of chromatography and sucrose-gradient ultracentrifugation produced extremely pure EV71 infectious virus particles, the overall yield of vaccine was 7–10% as determined by a VP2-based quantitative ELISA. Using chromatography as the downstream purification, the virus yield was 30–43%. To retain the integrity of virus neutralization epitopes and the stability of the vaccine product, the best virus inactivation was found to be 0.025% formalin-treatment at 37°C for 3 to 6 days. Furthermore, the formalin-inactivated virion vaccine candidate was found to be stable for >18 months at 4°C and a microgram of viral proteins formulated with alum adjuvant could induce strong virus-neutralizing antibody responses in mice, rats, rabbits, and non-human primates.ConclusionThese results provide valuable information supporting the current cell-based serum-free EV71 vaccine candidate going into human Phase I clinical trials.

Histone deacetylase inhibitor MS-275 treatment alters immune molecule content and categories in hepatocarcinoma exosomes

To investigate the effects of the histone deacetylase inhibitor, MS-275, on the immune molecule content and categories in hepatocarcinoma exosomes. Exosomes were isolated from the human hepatocarcinoma cell lines, HepG2 and Hep3b, and purified by a combination technique of ultrafiltration centrifugation and sucrose gradient ultracentrifugation. The expressions of heat shock protein (HSP)70, human leukocyte antigen (HLA)-I, HLA-DR, cluster of differentiation (CD) 80 and NY-ESO-1 on exosomes were analyzed with immunoelectron microscopy and Western blotting before and after MS-275 treatment. Intergroup differences were statistically analyzed by the Student's paired t-test. MS-275 treatment of both HepG2 and Hep3b cell types significantly increased the numbers of exosomes, their total protein content, and expression of HSP70, HLA-I and CD80 (per 100 exosomes), as compared to non-treated cells (all, P less than 0.01). MS-275 was also found to induce de novo expression of HLA-DR, but had no significant effect on NY-ESO-1 expression (P more than 0.05). The findings from immunoelectron microscopy confirmed those from Western blotting. The histone deacetylase inhibitor, MS-275, can significantly alter the immune molecule content and categories in exosomes of hepatocarcinoma cells. The differential expression profile may reflect an anti-cancer immune response and represent molecular targets for novel anti-hepatoma therapeutic or preventative strategies.

Capture and Qualitative Analysis of the Activated Fc Receptor Complex from Live Cells

This unit describes the isolation of activated Fc receptor complexes from RAW 264.7 macrophages using live-cell affinity receptor chromatography (LARC). The Fc receptor complex is activated and captured by IgG-coated microbeads on the surface of live macrophages. After the cells are disrupted, the receptor complexes are isolated by washing and sucrose gradient ultracentrifugation. Soluble proteins associated with the receptor complex are then eluted from the beads using a stepwise series of salt buffers and aqueous acetonitrile. The eluted proteins and the residual insoluble proteins on the beads can then be digested with trypsin and subjected to liquid chromatography, electrospray ionization, and tandem mass spectrometry (LC-ESI-MS/MS). Controls include IgG-coated beads incubated with crude cell lysates or growth medium and beads coated with oxidized LDL or bovine serum albumin. Using this method, proteins present in IgG-FcR complexes can be distinguished from those in control scavenger receptor complexes (oxLDL or BSA). Thus, LARC is capable of detecting specific members of IgG receptor supramolecular complexes.

Vaccination with coxsackievirus B3 virus-like particles elicits humoral immune response and protects mice against myocarditis

Coxsackievirus B3 (CVB3), along with other enteroviruses, is involved in about 50% of myocarditis cases and in the pathogenesis of dilated cardiomyopathy. Prevention of CVB3 infection is therefore highly desirable. Virus-like particles (VLPs) are structurally similar to native virus particles and therefore are far better immunogens than any other subunit vaccines. Recombinant baculoviruses carrying either the intact, entire coding region of CVB3 or the four individual coding regions for virus proteins 1-4 (VP1-4) were constructed. Expression of CVB3 capsid proteins in insect cells infected with recombinant baculovirus was detected by immunofluorescence and Western blot analysis. Sucrose gradient ultracentrifugation fractions of the infected cell lysates contained peaks of CVB3 antigen with an approximate density of 1.14g/ml. Electron microscopy demonstrated the presence of VLP in these sucrose fractions. The CVB3 VLP was non-infectious in tissue culture. SWR (H-2(q)) mice vaccinated with CVB3 VLP developed antibodies to CVB3 capsid proteins after the first boost. Antibody titre was comparable to the level induced by an attenuated CVB3 vaccine. Vaccinated animals were protected from myocarditis when subsequently challenged with cardiovirulent CVB3 (chimera-2). Vaccination with VLP produced from the complete CVB3 coding region gave a greater immune response and afforded better protection than with VLP from the quadruple expression vector. These results demonstrate that CVB3 capsid proteins expressed in insect cells have the intrinsic capacity to assemble into non-infectious VLP, which afforded protection from CVB3 infection to mice when used as a vaccine.

An alternative import pathway of AIF to the mitochondria

In eukaryotic cells, transport of the newly synthesized proteins and phospholipids to the appropriate subcellular target compartments is essential for maintaining organelle morphology and cell survival. In animal cells, mitochondria are major organelles containing DNA genome that encodes only for a small fraction of their proteins, which are required for the organelle function. Most mitochondrial proteins are encoded by the nuclear genes and imported to the mitochondria following protein synthesis. Apoptosis-inducing factor (AIF), an essential FAD-dependent NADH oxidase for the oxidative phosphorylation, is located in the intermembranous space and contains mitochondrial localization signals. However, the import mechanism of AIF to the mitochondria is not yet studied. Using sucrose gradient ultracentrifugation and immunoblotting, AIF was detected in fractions of the endoplasmic reticulum, mitochondria-associated membranes (MAM) and mitochondria, and AIF from these fractions was resistant to trypsin in the absence of digitonin, suggesting that AIF could be protected by phospholipids. Knockdown of dynamin-related protein 1 (DRP1kd) expression reduced AIF levels in the mitochondria, but increased AIF concentrations in the MAM. Knockdown of mitofusin-2 (Mfn-2kd) or ATPase family AAA domain containing 3A (ATAD3Akd) expression, however, reduced AIF levels in the mitochondria and increased the number of transport vesicles that contained AIF in the cytosol, indicating that ATAD3A and Mfn-2 were respectively essential for the import and fusion of transport vesicles into the mitochondria. Here we show that AIF is imported from the endoplasmic reticulum to the mitochondria via mitochondria-associated membranes and transport vesicles.

Relationship between Localization on Cellular Membranes and Cytotoxicity of Vibrio vulnificus Hemolysin

Vibrio vulnificus secretes a hemolysin/cytolysin (VVH) that induces cytolysis in target cells. A detergent resistant membrane domain (DRM) fraction of the cells after sucrose gradient centrifugation includes cholesterol-rich membrane microdomains which have been called “lipid rafts”. It was reported that some pore-forming toxins require association with DRM and/or lipid rafts to exert their cytotoxicity. It has also been thought that cellular cholesterol is involved in VVH cytotoxicity because VVH cytotoxicity was inhibited by pre-incubation with cholesterol. However, both cellular localization and mode of action of VVH cytotoxicity remain unclear. In this study, we investigated the relationship between VVH localization on the cellular membrane and its cytotoxicity. Oligomers of VVH were detected from DRM fractions by sucrose gradient ultracentrifugation but all of these oligomers shifted from DRM fractions to non-DRM fractions after treatment with methyl-beta-cyclodextrin (MβCD), a cholesterol sequestering agent. On the other hand, immunofluorescence analysis showed that VVH did not co-localize with major lipid raft markers on cellular membrane of CHO cells. These data suggested that VVH localized at membrane regions which are relatively abundant in cholesterol but which are not identical with lipid rafts. To determine the linkage between localization and cytotoxicity of VVH, cytotoxicity was evaluated in MβCD-treated CHO cells. The cytotoxicity of VVH was not decreased by the MβCD treatment. In addition, the amount of VVH oligomer did not decrease in MβCD-treated CHO cells. Thus, we found that the amount of oligomer on cellular membrane is important for induction of cytotoxicity, whereas localization to lipid rafts on the cellular membrane was not essential to cytotoxicity.

Cholesterol Influences Voltage-Gated Calcium Channels and BK-Type Potassium Channels in Auditory Hair Cells

The influence of membrane cholesterol content on a variety of ion channel conductances in numerous cell models has been shown, but studies exploring its role in auditory hair cell physiology are scarce. Recent evidence shows that cholesterol depletion affects outer hair cell electromotility and the voltage-gated potassium currents underlying tall hair cell development, but the effects of cholesterol on the major ionic currents governing auditory hair cell excitabilityare unknown. We investigated the effects of a cholesterol-depleting agent (methyl beta cyclodextrin, MβCD) on ion channels necessary for the early stages of sound processing. Large-conductance BK-type potassium channels underlie temporal processing and open in a voltage- and calcium-dependent manner. Voltage-gated calcium channels (VGCCs) are responsible for calcium-dependent exocytosis and synaptic transmission to the auditory nerve. Our results demonstrate that cholesterol depletion reduced peak steady-state calcium-sensitive (BK-type) potassiumcurrent by 50% in chick cochlear hair cells. In contrast, MβCD treatment increased peak inward calcium current (∼30%), ruling out loss of calcium channel expression or function as a cause of reduced calcium-sensitive outward current. Changes in maximal conductance indicated a direct impact of cholesterol on channel number or unitary conductance. Immunoblotting following sucrose-gradient ultracentrifugation revealed BK expression in cholesterol-enriched microdomains. Both direct impacts of cholesterol on channel biophysics, as well as channel localization in the membrane, may contribute to the influence of cholesterol on hair cell physiology. Our results reveal a new role for cholesterol in the regulation of auditory calcium and calcium-activated potassium channels and add to the growing evidence that cholesterol is a key determinant in auditory physiology.

Purification of human respiratory syncytial virus fusion glycoprotein

Human respiratory syncytial virus (RSV) fusion glycoprotein (F) elicits neutralizing antibodies to RSV and has therefore attracted much attention as a suitable candidate antigen in the development of gene-based vaccines against RSV infections. However, a major obstacle in vaccine development has been the problem of antigen purification. To address this problem, we have developed a new method that combines sucrose gradient ultracentrifugation and a two-step chromatographic process, to purify RSV F from RSV particles propagated in HEp-2 cells. Analysis of the fractions produced using this method showed recovery of a functional homodimer with a molecular weight of 140kDa, and 54% preservation of the original F.

Release reaction of brain-derived neurotrophic factor (BDNF) through PAR1 activation and its two distinct pools in human platelets

Brain-derived neurotrophic factor (BDNF) is a cytokine that plays important roles in the survival, development, and plasticity of neurons. BDNF is also expressed in peripheral tissues and cells. In this article, we report the BDNF release reaction through thrombin stimulation and its localization in human platelets. Platelets from healthy volunteers were subjected to PAR1-AP or PAR4-AP stimulation. Release of BDNF was measured by ELISA. Localization of BDNF in resting and thrombin-activated platelets was examined by immunoelectron microscopy and sucrose gradient ultracentrifugation following western blotting. BDNF was released dose-dependently with PAR1-AP concentrations with drastic release at low PAR1-AP concentrations and gently release at high PAR1-AP concentrations. Maximum BDNF release was approximately 37% at 132 μM PAR1-AP. In contrast, 3.8% BDNF was released with 1.13 mM PAR4-AP stimulation. In immunoelectron microscopy and sucrose gradient ultracentrifugation analyses, BDNF was detected not only in α-granules but also cytoplasm in of the resting platelets, and it was distributed in the swollen open canalicular system fused to α-granules at 1 min and disappeared at 5 min after stimulation by thrombin. However, BDNF in cytoplasm remained throughout platelet activation. In conclusions, we demonstrate that BDNF is released from platelets through predominately PAR1 regulation. Furthermore, we identified two pools of BDNF in the α-granules and cytoplasm of human platelets, and only BDNF in α-granules is released through platelet activation.

Induction of a tumour-specific CTL response by exosomes isolated from heat-treated malignant ascites of gastric cancer patients

Purpose: Tumour cell-derived exosomes may represent a novel type of cancer vaccine. However, the immunogenicity of exosomes derived from tumour cells has been shown to be poor. Therefore, in this study, exosome immunogenicity following heat treatment of exosomes from malignant ascites obtained from gastric cancer patients was evaluated.Materials and methods: Tumour-derived exosomes were isolated from heat-treated and untreated malignant ascites of gastric cancer patients using serial centrifugation and sucrose gradient ultracentrifugation. Next, in vitro experiments were performed to investigate the influence of heat treatment on exosome immunogenicity.Results: Exosomes from heat-treated malignant ascites of gastric cancer patients (HS exosomes) were found to contain higher concentrations of heat shock proteins, Hsp70 and Hsp60, than exosomes derived from untreated malignant ascites obtained from gastric cancer patients. Additional in vitro studies suggest that exosomes derived from heat-treated malignant ascites are able to promote dendritic cell (DC) maturation and induce a tumour-specific cytotoxic T lymphocyte (CTL) response.Conclusions: Overall, these results demonstrate that exposure to heat stress can improve the immunogenicity of exosomes obtained from malignant ascites of gastric cancer patients.

Lipid bodies in coral–dinoflagellate endosymbiosis: Proteomic and ultrastructural studies

Gastrodermal lipid bodies (LBs) are organelles involved in the regulation of the mutualistic endosymbiosis between reef-building corals and their dinoflagellate endosymbionts (genus Symbiodinium). As their molecular composition remains poorly defined, we herein describe the first gastrodermal LB proteome and examine in situ morphology of LBs in order to provide insight into their structure and function. After tissue separation of the tentacles of the stony coral Euphyllia glabrescens, buoyant LBs of the gastroderm encompassing a variety of sizes (0.5-4 μm in diameter) were isolated after two cycles of subcellular fractionation via stepwise sucrose gradient ultracentrifugation and detergent washing. The purity of the isolated LBs was demonstrated by their high degree of lipid enrichment and as well as the absence of contaminating proteins of the host cell and Symbiodinium. LB-associated proteins were then purified, subjected to SDS-PAGE, and identified by MS using an LC-nano-ESI-MS/MS. A total of 42 proteins were identified within eight functional groups, including metabolism, intracellular trafficking, the stress response/molecular modification and development. Ultrastructural analyses of LBs in situ showed that they exhibit defined morphological characteristics, including a high-electron density resulting from a distinct lipid composition from that of the lipid droplets of mammalian cells. Coral LBs were also characterized by the presence of numerous electron-transparent inclusions of unknown origin and composition. Both proteomic and ultrastructural observations seem to suggest that both Symbiodinium and host organelles, such as the ER, are involved in LB biogenesis.

The G‐protein on cholesterol‐rich membrane microdomains mediates mucosal sensing of short‐ chain fatty acid and secretory response in rat colon

Short-chain fatty acids (SCFA) stimulate colonic contraction and secretion, which are mediated by an enteric reflex via a mucosal sensing and cholinergic mechanisms. The involvement of G-protein signal transduction was examined in the secretory response to luminal propionate sensing in rat distal colon. Mucosa-submucosa and mucosa preparations were used to measure short-circuit current (I(sc)) and acetylcholine (ACh) release respectively. Cholesterol-rich membrane microdomains, lipid rafts/caveolae, were fractionated using a sucrose gradient ultra-centrifugation after detergent-free extraction of the isolated colonic crypt. Luminal addition of methyl-β-cyclodextrin (10 mm) and mastoparan (30 μm), lipid rafts/caveolae disruptors, significantly inhibited luminal propionate-induced (0.5 mm) increases in I(sc) , but did not affect increases in I(sc) induced by serosal ACh (0.05 mm) or electrical field stimulation (EFS). Luminal addition of YM-254890 (10 μm), a Gα(q/11) -selective inhibitor, markedly inhibited propionate-induced increase in I(sc) , but did not affect I(sc) responses to ACh and EFS. Both methyl-β-cyclodextrin and YM-254890 significantly inhibited luminal propionate-induced non-neuronal release of ACh from colonocytes. Real-time PCR demonstrated that in mRNA expression of SCFA receptors, GPR 43 was far higher than that of GPR41 in the colon. Western blotting analysis revealed that the cholesterol-rich membrane microdomains that fractionated from colonic crypt cells were associated with caveolin-1, flotillin-1 and Gα(q/11) , but not GPR43. Uncoupling of Gα(q/11) from flotillin-1 in lipid rafts occurred under desensitization of the I(sc) response to propionate. These data demonstrate that the secretory response to luminal propionate in rat colon is mediated by G-protein on cholesterol-rich membrane microdomains, provably via Gα(q/11) .

Exosome-loaded dendritic cells elicit tumor-specific CD8+ cytotoxic T cells in patients with glioma

In this study, we demonstrate that tumor-derived exosome-loaded dendritic cells can elicit a specific CD8(+) cytotoxic T-lymphocyte (CTL) response against autologous tumor cells in patients with malignant glioma. Exosomes were purified by ultrafiltration centrifugation and sucrose gradient ultracentrifugation. Exosomes had antigen-presenting molecules (MHC-I, HSP70), tumor antigen (MAGE-1) and adherent molecule (ICAM-1). After incubation with exosomes, the dendritic cells (DCs) could activate the T lymphocytes to become glioma-specialized CTL. The CTL had vigorous cytotoxicity to glioma cells as opposed to autologous lymphoblast cells. These data demonstrate that tumor exosome-loaded DC can be an effective tool in inducing glioma-specific CD8(+) CTLs able to kill autologous glioma cells in vitro. In conclusion, exosomes are a natural and new source of tumor-rejection antigens, opening up new avenues for immunization against glioma.