Sucrose Density Gradient Ultracentrifugation Research Articles

7-Dehydrocholesterol (7-DHC), But Not Cholesterol, Causes Suppression of Canonical TGF-β Signaling and Is Likely Involved in the Development of Atherosclerotic Cardiovascular Disease (ASCVD).

For several decades, cholesterol has been thought to cause ASCVD. Limiting dietary cholesterol intake has been recommended to reduce the risk of the disease. However, several recent epidemiological studies do not support a relationship between dietary cholesterol and/or blood cholesterol and ASCVD. Consequently, the role of cholesterol in atherogenesis is now uncertain. Much evidence indicates that TGF-β, an anti-inflammatory cytokine, protects against ASCVD and that suppression of canonical TGF-β signaling (Smad2-dependent) is involved in atherogenesis. We had hypothesized that cholesterol causes ASCVD by suppressing canonical TGF-β signaling in vascular endothelium. To test this hypothesis, we determine the effects of cholesterol, 7-dehydrocholesterol (7-DHC; the biosynthetic precursor of cholesterol), and other sterols on canonical TGF-β signaling. We use Mv1Lu cells (a model cell system for studying TGF-β activity) stably expressing the Smad2-dependent luciferase reporter gene. We demonstrate that 7-DHC (but not cholesterol or other sterols) effectively suppresses the TGF-β-stimulated luciferase activity. We also demonstrate that 7-DHC suppresses TGF-β-stimulated luciferase activity by promoting lipid raft/caveolae formation and subsequently recruiting cell-surface TGF-β receptors from non-lipid raft microdomains to lipid rafts/caveolae where TGF-β receptors become inactive in transducing canonical signaling and undergo rapid degradation upon TGF-β binding. We determine this by cell-surface 125 I-TGF-β-cross-linking and sucrose density gradient ultracentrifugation. We further demonstrate that methyl-β-cyclodextrin (MβCD), a sterol-chelating agent, reverses 7-DHC-induced suppression of TGF-β-stimulated luciferase activity by extrusion of 7-DHC from resident lipid rafts/caveolae. These results suggest that 7-DHC, but not cholesterol, promotes lipid raft/caveolae formation, leading to suppression of canonical TGF-β signaling and atherogenesis. J. Cell. Biochem. 118: 1387-1400, 2017. © 2016 Wiley Periodicals, Inc.

Formation of mos RNA granules in the zebrafish oocyte that differ from cyclin B1 RNA granules in distribution, density and regulation

Many translationally repressed mRNAs are deposited in the oocyte cytoplasm for progression of the meiotic cell cycle and early development. mos and cyclin B1 mRNAs encode proteins promoting oocyte meiosis, and translational control of these mRNAs is important for normal progression of meiotic cell division. We previously demonstrated that cyclin B1 mRNA forms RNA granules in the zebrafish and mouse oocyte cytoplasm and that the formation of RNA granules is crucial for regulating the timing of translational activation of the mRNA. However, whether the granule formation is specific to cyclin B1 mRNA remains unknown. In this study, we found that zebrafish mos mRNA forms granules distinct from those of cyclin B1 mRNA. Fluorescent in situ hybridization analysis showed that cyclin B1 RNA granules were assembled in dense clusters, while mos RNA granules were distributed diffusely in the animal polar cytoplasm. Sucrose density gradient ultracentrifugation analysis showed that the density of mos RNA granules was partly lower than that of cyclin B1 mRNA. Similar to cyclin B1 RNA granules, mos RNA granules were disassembled after initiation of oocyte maturation at the timing at which the poly(A) tail was elongated. However, while almost all of the granules of cyclin B1 were disassembled simultaneously, a fraction of mos RNA granules firstly disappeared and then a large part of them was disassembled. In addition, while cyclin B1 RNA granules were disassembled in a manner dependent on actin filament depolymerization, certain fractions of mos RNA granules were disassembled independently of actin filaments. These results suggest that cytoplasmic regulation of translationally repressed mRNAs by formation of different RNA granules is a key mechanism for translational control of distinct mRNAs in the oocyte.

Simple One-step Purification of Hepatitis B Core Antigen in Escherichia coli

Hepatitis B core antigen (HBcAg) is one of the most important diagnostic reagents in hepatitis B virus (HBV) infection. The purifi- cation of HBcAg using conventional methods, such as sucrose density gradient ultracentrifugation, is time and cost consuming. To overcome this diculty , we aimed at developing an ecient method for producing a soluble form of HBcAg for diagnostic applica- tion. The HBcAg construct was secreted into periplasm space of E. coli, which facilitates purification by selective disruption of the outer membrane. The His-tagged HBcAg was purified from prepared periplasmic fraction using Ni-NTA anity chromatography with an average yield of 20 mg/L culture. The results of competitive ELISA indicated that the antigenicity of periplasmic HBcAg is comparable with commercial E. coli-derived antigen. These results demonstrate the periplasmic system is suitable for the rapid and simple purification of bioactive and soluble HBcAg.

Shiga toxin glycosphingolipid receptors and their lipid membrane ensemble in primary human blood-brain barrier endothelial cells.

Shiga toxin (Stx)-mediated injury to microvascular endothelial cells in the brain significantly contributes to the pathogenesis of the hemolytic-uremic syndrome caused by enterohemorrhagic Escherichia coli (EHEC). Stxs are AB5 toxins and the B-pentamers of the two major Stx subtypes Stx1a and Stx2a preferentially bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer) expressed by human endothelial cells. Here we report on comprehensive structural analysis of the different lipoforms of Gb3Cer (Galα4Galβ4Glcβ1Cer) and globotetraosylceramide (Gb4Cer, GalNAcβ3Galα4Galβ4Glcβ1Cer, the less effective Stx receptor) of primary human brain microvascular endothelial cells and their association with lipid rafts. Detergent-resistant membranes (DRMs), obtained by sucrose density gradient ultracentrifugation, were used as lipid raft-analogous microdomains of the liquid-ordered phase and nonDRM fractions were employed as equivalents for the liquid-disordered phase of cell membranes. Structures of the prevalent lipoforms of Gb3Cer and Gb4Cer were those with Cer (d18:1, C16:0), Cer (d18:1, C22:0) and Cer (d18:1, C24:1/C24:0) determined by electrospray ionization mass spectrometry that was combined with thin-layer chromatography immunodetection using anti-Gb3Cer and anti-Gb4Cer antibodies as well as Stx1a and Stx2a subtypes. Association of Stx receptor GSLs was determined by co-localization with lipid raft-specific membrane protein flotillin-2 and canonical lipid raft marker sphingomyelin with Cer (d18:1, C16:0) and Cer (d18:1, C24:1/C24:0) in the liquid-ordered phase, whereas lyso-phosphatidylcholine was detectable exclusively in the liquid-disordered phase. Defining the precise microdomain structures of primary endothelial cells may help to unravel the initial mechanisms by which Stxs interact with their target cells and will help to develop novel preventive and therapeutic measures for EHEC-mediated diseases.

Production of the virus-like particles of nipah virus matrix protein in Pichia pastoris as diagnostic reagents.

The matrix (M) protein of Nipah virus (NiV) is a peripheral protein that plays a vital role in the envelopment of nucleocapsid protein and acts as a bridge between the viral surface and the nucleocapsid proteins. The M protein is also proven to play an important role in production of virus-like particles (VLPs) and is essential for assembly and budding of NiV particles. The recombinant M protein produced in Escherichia coli assembled into VLPs in the absence of the viral surface proteins. However, the E. coli produced VLPs are smaller than the native virus particles. Therefore, the aims of this study were to produce NiV M protein in Pichia pastoris, to examine the structure of the VLPs formed, and to assess the potential of the VLPs as a diagnostic reagent. The M protein was successfully expressed in P. pastoris and was detected with anti-myc antibody using Western blotting. The VLPs formed by the recombinant M protein were purified with sucrose density gradient ultracentrifugation, high-performance liquid chromatography (HPLC), and Immobilized Metal Affinity Chromatography (IMAC). Immunogold staining and transmission electron microscopy confirmed that the M protein assembled into VLPs as large as 200 nm. ELISA revealed that the NiV M protein produced in P. pastoris reacted strongly with positive NiV sera demonstrating its potential as a diagnostic reagent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1038-1045, 2016.

Betulinic acid enhances TGF-β signaling by altering TGF-β receptors partitioning between lipid-raft/caveolae and non-caveolae membrane microdomains in mink lung epithelial cells.

BackgroundTGF-β is a key modulator in the regulation of cell proliferation and migration, and is also involved in the process of cancer development and progression. Previous studies have indicated that TGF-β responsiveness is determined by TGF-β receptor partitioning between lipid raft/caveolae-mediated and clathrin-mediated endocytosis. Lipid raft/caveolae-mediated endocytosis facilitates TGF-β degradation and thus suppressing TGF-β responsiveness. By contrast, clathrin-mediated endocytosis results in Smad2/3-dependent endosomal signaling, thereby promoting TGF-β responsiveness. Because betulinic acid shares a similar chemical structure with cholesterol and has been reported to insert into the plasma membrane, we speculate that betulinic acid changes the fluidity of the plasma membrane and modulates the signaling pathway associated with membrane microdomains. We propose that betulinic acid modulates TGF-β responsiveness by changing the partitioning of TGF-β receptor between lipid-raft/caveolae and non-caveolae microdomain on plasma membrane.MethodsWe employed sucrose-density gradient ultracentrifugation and confocal microscopy to determine membrane localization of TGF-β receptors and used a luciferase assay to examine the effects of betulinic acid in TGF-β-stimulated promoter activation. In addition, we perform western blotting to test TGF-β-induced Smad2 phosphorylation and fibronectin production.Results and conclusionsBetulinic acid induces translocation of TGF-β receptors from lipid raft/caveolae to non-caveolae microdomains without changing total level of TGF-β receptors. The betulinic acid-induced TGF-β receptors translocation is rapid and correlate with the TGF-β-induced PAI-1 reporter gene activation and growth inhibition in Mv1Lu cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12929-016-0229-4) contains supplementary material, which is available to authorized users.

Ethanol Enhances TGF‐β Activity by Recruiting TGF‐β Receptors From Intracellular Vesicles/Lipid Rafts/Caveolae to Non‐Lipid Raft Microdomains

Regular consumption of moderate amounts of ethanol has important health benefits on atherosclerotic cardiovascular disease (ASCVD). Overindulgence can cause many diseases, particularly alcoholic liver disease (ALD). The mechanisms by which ethanol causes both beneficial and harmful effects on human health are poorly understood. Here we demonstrate that ethanol enhances TGF-β-stimulated luciferase activity with a maximum of 0.5-1% (v/v) in Mv1Lu cells stably expressing a luciferase reporter gene containing Smad2-dependent elements. In Mv1Lu cells, 0.5% ethanol increases the level of P-Smad2, a canonical TGF-β signaling sensor, by ∼ 2-3-fold. Ethanol (0.5%) increases cell-surface expression of the type II TGF-β receptor (TβR-II) by ∼ 2-3-fold from its intracellular pool, as determined by I(125) -TGF-β-cross-linking/Western blot analysis. Sucrose density gradient ultracentrifugation and indirect immunofluorescence staining analyses reveal that ethanol (0.5% and 1%) also displaces cell-surface TβR-I and TβR-II from lipid rafts/caveolae and facilitates translocation of these receptors to non-lipid raft microdomains where canonical signaling occurs. These results suggest that ethanol enhances canonical TGF-β signaling by increasing non-lipid raft microdomain localization of the TGF-β receptors. Since TGF-β plays a protective role in ASCVD but can also cause ALD, the TGF-β enhancer activity of ethanol at low and high doses appears to be responsible for both beneficial and harmful effects. Ethanol also disrupts the location of lipid raft/caveolae of other membrane proteins (e.g., neurotransmitter, growth factor/cytokine, and G protein-coupled receptors) which utilize lipid rafts/caveolae as signaling platforms. Displacement of these membrane proteins induced by ethanol may result in a variety of pathologies in nerve, heart and other tissues.

Cellular Factor XIIIA Transglutaminase Localizes in Caveolae and Regulates Caveolin-1 Phosphorylation, Homo-oligomerization and c-Src Signaling in Osteoblasts.

Transglutaminases (TGs) are a family of widely distributed enzymes that catalyze protein crosslinking by forming a covalent isopeptide bond between the substrate proteins. We have shown that MC3T3-E1 osteoblasts express Factor XIII-A (FXIII-A), and that the extracellular crosslinking activity of FXIII-A is involved in regulating matrix secretion and deposition. In this study, we have investigated the localization and potential role of intracellular FXIII-A. Conventional immunofluorescence microscopy and TIRF microscopy analyses showed that FXIII-A co-localizes with caveolin-1 in specialized membrane structures, caveolae, in differentiating osteoblasts. The caveolae-disrupting agent methyl-β-cyclodextrin abolished FXIII-A staining and co-localization with caveolin-1 from the osteoblast plasma membrane. The presence of FXIII-A in caveolae was confirmed by preparing caveolae-enriched cellular fractions using sucrose density gradient ultracentrifugation followed by western blotting. Despite this association of FXIII-A with caveolae, there was no detectable transglutaminase activity in caveolae, as measured by monodansylcadaverine incorporation. TG inhibitor NC9--which can alter TG enzyme conformation--localized to caveolae and displaced FXIII-A from these structures when added to the osteoblast cultures. The decreased FXIII-A levels in caveolae after NC9 treatment increased c-Src activation, which resulted in caveolin-1 phosphorylation, homo-oligomerization and Akt phosphorylation, suggesting cellular FXIII-A has a role in regulating c-Src signaling in osteoblasts.

Role of PTEN in Hypoxic Pulmonary Vasoconstriction

RationaleHypoxic pulmonary vasoconstriciton optimizes ventilation/perfusion matching in the lung, and critically depends on transient receptor potential canonical (TRPC) 6 activation and trafficking to caveolae. However, underlying regulatory mechanisms are yet unclear. In endothelial cells, phosphatase and tensin homolog (PTEN) has been shown to serve as a scaffold for TRPC6, enabling cell surface expression of the channel and subsequent Ca2+ entry independent of its phosphatase activity. Based on these data we hypothesized that the interaction between PTEN and TRPC6 in PASMC may be required for TRPC6 transloaction to caveolae and an intact HPV response.MethodsInteraction between PTEN and TRPC6 was tested by co‐immunoprecipitation in whole lung lysates, and by proximity ligation assay in pulmonary artery smooth muscle cells (PASMC). Caveolar recruitment of proteins was assessed in PASMC by sucrose density gradient ultracentrifugation. HPV was analyzed in isolated perfused lungs as hypoxia‐induced incresae in perfusion pressure.ResultsHypoxia induced protein‐protein interaction between PTEN and TRPC6 in both whole lungs and PASMC. Hypoxia also translocated PTEN to caveolae (caveolin 1‐rich fraction) but inhibition of ROCK (Y27632; 5 µM) prevented this translocation. HPV response was significantly attenuated by Y27632, yet not by the PTEN phosphatase inhibitor VO‐OHpic (1‐50 µM).ConclusionHypoxia induces PTEN interaction with TRPC6 in PASMC, and PTEN translocation to caveolae in a ROCK‐dependent manner. PTEN may play an important role in TRPC6 recruitment and activation downstream of ROCK and thus, mediate HPV in a manner that is independent of its phosphatase activity.Funded by a CIHR operational grant to WMK.

Heteromers of amyloid precursor protein in cerebrospinal fluid.

BackgroundSoluble fragments of the amyloid precursor protein (APP) generated by α- and β-secretases, sAPPα and sAPPβ, have been postulated as promising new cerebrospinal fluid (CSF) biomarkers for the clinical diagnosis of Alzheimer’s disease (AD). However, the capacity of these soluble proteins to assemble has not been explored and could be relevant. Our aim is to characterize possible sAPP oligomers that could contribute to the quantification of sAPPα and sAPPβ in CSF by ELISA, as well as to characterize the possible presence of soluble full-length APP (sAPPf).ResultsWe employed co-immunoprecipitation, native polyacrylamide gel electrophoresis and ultracentrifugation in sucrose density gradients to characterize sAPP oligomers in CSF. We have characterized the presence of sAPPf in CSF from NDC and AD subjects and demonstrated that all forms, including sAPPα and sAPPβ, are capable of assembling into heteromers, which differ from brain APP membrane-dimers. We measured sAPPf, sAPPα and sAPPβ by ELISA in CSF samples from AD (n = 13) and non-disease subjects (NDC, n = 13) before and after immunoprecipitation with antibodies against the C-terminal APP or against sAPPβ. We demonstrated that these sAPP heteromers participate in the quantification of sAPPα and sAPPβ by ELISA. Immunoprecipitation with a C-terminal antibody to remove sAPPf reduced by ~30% the determinations of sAPPα and sAPPβ by ELISA, whereas immunoprecipitation with an APPβ antibody reduced by ~80% the determination of sAPPf and sAPPα.ConclusionsThe presence of sAPPf and sAPP heteromers should be taken into consideration when exploring the levels of sAPPα and sAPPβ as potential CSF biomarkers.Electronic supplementary materialThe online version of this article (doi:10.1186/1750-1326-10-2) contains supplementary material, which is available to authorized users.

Comparative membrane proteomics: a technical advancement in the search of renal cell carcinoma biomarkers.

Renal Cell Carcinoma (RCC) is the most common kidney cancer, accounting for 3% of adult malignancies, with high metastatic potential and radio-/chemo-resistance. To investigate the protein profile of membrane microdomains (MD), plasma membrane supramolecular structures involved in cell signaling, transport, and neoplastic transformation, we set up a proteomic bottom-up approach as a starting point for the identification of potential RCC biomarkers. We purified MD from RCC and adjacent normal kidney (ANK) tissues, through their resistance to non-ionic detergents followed by ultracentrifugation in sucrose density gradient. MD from 5 RCC/ANK tissues were then pooled and analysed by LC-ESI-MS/MS. In order to identify the highest number of proteins and increase the amount of membrane and hydrophobic ones, we first optimized an enzymatic digestion protocol based on Filter Aided Sample Preparation (FASP), coupled to MD delipidation. The MS analysis led to the identification of 742 ANK MD and 721 RCC MD proteins, of which, respectively, 53.1% and 52.6% were membrane- bound. Additionally, we evaluated RCC MD differential proteome by label-free quantification; 170 and 126 proteins were found to be, respectively, up-regulated and down-regulated in RCC MD. Some differential proteins, namely CA2, CD13, and ANXA2, were subjected to validation by immunodecoration. These results show the importance of setting up different protocols for the proteomic analysis of membrane proteins, specific to the different molecular features of the samples. Furthermore, the subcellular proteomic approach provided a list of differentially expressed proteins among which RCC biomarkers may be looked for.

Comparative Studies of the Potency of Foot and Mouth Disease Virus Trivalent Vaccine with Different Concentration of the Antigenic Content (146S).

Estimation of antigenic content (146S) of FMDV serotypes (A, O, SAT2) by sucrose density gradient (SDG) ultracentrifugation by determining the absorbance at 254 nm using ISCO520C density gradient system to produce a highly potent trivalent virus vaccine. The antigenic mass 146S (µg/ml) of serotypes (O Pan Asia2, A Iran O5 and SAT2/EGY/2012) were 6.5, 6.2 and 5.9, respectively. The vaccine was injected into three groups of calves (2individuals/each group) subcutaneously in lateral part of the neck for a dose 3 ml (6.2µg/serotype/ml), a dose 1.5 ml (4.1µg/serotype/ml) and a dose 1 ml (2µg/ml), the sera samples were collected at 7th day post vaccination (dpv), 14th dpv, 21th dpv, 28th dpv and every 2wks till 40 weeks to evaluate the immune response along that period. The antibody titers/40wpv for a 3 ml dose (6.2µg/ml) of serotypes (O Pan Asia-2, A Iran O5 and SAT-2/EGY/2012) were 2.08, 2 and 1.94, respectively (over the protective titer, PT=1.5 in SNT for cattle), a dose (4.1µg/ml) of the three serotypes were 1.56, 1.62 and 1.63 (over PT), respectively, But for (2µg/ml) dose of the three serotypes, the antibodies titer were 1.25, 1.19 and 1.2 (below PT), that show the antibodies titer depend on the concentration of the antigenic mass (146S) and with increase of the 146S concentration increase of the potency of the vaccine. The potency testing of that study depend upon the correlation between 146S and the neutralizing antibody titers were measured by SNT which are the perfect alternative of other potency tests which employ the challenge of the cattle with virulent virus. The immune response of the highly potent vaccine (4.1µg/serotype/ml and 6.2µg/serotype/ml) started early after 1st wpv and the protective titer remain for more than 38 wpv (especially in 6.2µg/ml injected calves) and that confer the potency of the vaccine of that dose.

Isolation and purification of proteasomes from primary cells.

Proteasomes play an important role in cell homeostasis and in orchestrating the immune response by systematically degrading foreign proteins and misfolded or damaged host cell proteins. We describe a protocol to purify functionally active proteasomes from human CD4(+) T cells and dendritic cells derived from peripheral blood mononuclear cells. The purification is a three-step process involving ion-exchange chromatography, ammonium sulfate precipitation, and sucrose density gradient ultracentrifugation. This method can be easily adapted to purify proteasomes from cell lines or from organs. Methods to characterize and visualize the purified proteasomes are also described.

Targeting of aquaporin 4 into lipid rafts and its biological significance

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system and is considered to be caused by the binding of NMO-IgG to aquaporin 4 (AQP4) on astrocytes, which initiates complement-dependent cytotoxicity. AQP4 has two isoforms, i.e., M1 and M23. AQP4 is considered to form heterotetramers containing both isoforms in vivo. Most of the previous studies were performed using either one of the isoforms expressing cell lines. In this study, we generated a fluorescent epitope-tagged AQP4 M1 and M23 co-expressing astrocyte cell line and examined the subcellular targeting of AQP4. In this cell line, AQP4 was targeted mostly to membrane lipid rafts fraction evidenced by sucrose density gradient ultracentrifugation followed by Western blotting with anti-AQP4 antibody. Cholesterol depletion with methyl-β-cyclodextrin or simvastatin resulted in the dislocation (relocation) of AQP4 from lipid rafts to non-rafts fraction of the membrane and AQP4 was not internalized intracellularly. This change in the localization of AQP4 on membrane significantly reduced complement-dependent cytotoxic effects of NMO-IgG obtained from patients with NMO without affecting AQP4 orthogonal arrays. Thus, these data strongly suggest that the targeting of AQP4 in the lipid rafts is closely related to the pathogenic effects of NMO-IgG.

Comparative biochemical analysis of the major yolk protein in the sea urchin egg and coelomic fluid.

The major yolk protein (MYP) is localized to the egg and coelomic fluid of the adult sea urchin. While the egg-localized MYP has been extensively studied, much less is known about the coelomic fluid-localized protein. Therefore, we have conducted a comparative biochemical analysis of these proteins. Sucrose density gradient ultracentrifugation revealed unique elution profiles for the MYP species present in the egg, 170- and 240 kDa, and the coelomic fluid, 180- and 250 kDa. Fractionation in polyacrylamide gels revealed that under reducing conditions both species were present in each location. However, in the absence of reducing agent only one species was present in each fraction: 240 kDa in the egg and 250 kDa in the coelomic fluid. In addition, V8 peptide mapping indicated that all four species have very similar primary structures. Circular dichroic spectral analysis and endogenous tryptophan measurements of the purified 170- and 180 kDa species revealed distinctive secondary and tertiary structural features with notable differences in their responses to calcium: apparent Kds of 245- and 475 μmol/L were measured for the 170- and 180 kDa species, respectively. Further analysis revealed that both species have differing calcium requirements for binding to membranes as well as protein-dependent, membrane-membrane interaction. We discuss the functional implications arising from the structural differences which exist between the egg and coelomic fluid resident MYPs.

Cationic lipid nanodisks as an siRNA delivery vehicle.

The term nanodisk (ND) describes reconstituted high-density lipoprotein particles that contain one or more exogenous bioactive agents. In the present study, ND were assembled from apolipoprotein A-I, the zwitterionic glycerophospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the synthetic cationic lipid 1,2-dimyristoyl-3-trimethylammonium-propane (DMTAP). ND formulated at a DMPC:DMTAP ratio of 70:30 (by weight) were soluble in aqueous media. The particles generated were polydisperse, with diameters ranging from ∼20 to <50 nm. In nucleic acid binding studies, agarose gel retardation assays revealed that a synthetic 23-mer double-stranded oligonucleotide (dsOligo) bound to DMTAP containing ND but not to ND formulated with DMPC alone. Sucrose density gradient ultracentrifugation studies provided additional evidence for stable dsOligo binding to DMTAP-ND. Incubation of cultured hepatoma cells with DMTAP-ND complexed with a siRNA directed against glyceraldehyde 3-phosphate dehydrogenase showed 60% knockdown efficiency. Thus, incorporation of synthetic cationic lipid (i.e., DMTAP) to ND confers an ability to bind siRNA and the resulting complexes possess target gene knockdown activity in a cultured cell model.

Reduction of spiked porcine circovirus during the manufacture of a Vero cell-derived vaccine

Porcine circovirus-1 (PCV1) was recently identified as a contaminant in live Rotavirus vaccines, which was likely caused by contaminated porcine trypsin. The event triggered the development of new regulatory guidance on the use of porcine trypsin which shall ensure that cell lines and porcine trypsin in use are free from PCV1. In addition, manufacturing processes of biologicals other than live vaccines include virus clearance steps that may prevent and mitigate any potential virus contamination of product. In this work, artificial spiking of down-scaled models for the manufacturing process of an inactivated pandemic influenza virus vaccine were used to investigate inactivation of PCV1 and the physico-chemically related porcine parvovirus (PPV) by formalin and ultraviolet-C (UV-C) treatment as well as removal by the purification step sucrose gradient ultracentrifugation. A PCV1 infectivity assay, using a real-time PCR infectivity readout was established. The formalin treatment (0.05% for 48h) showed substantial inactivation for both PCV1 and PPV with reduction factors of 3.0log10 and 6.8log10, respectively, whereas UV-C treatment resulted in complete PPV (≥5.9log10) inactivation already at a dose of 13mJ/cm but merely 1.7log10 at 24mJ/cm2 for PCV1. The UV-C inactivation results with PPV were confirmed using minute virus of mice (MVM), indicating that parvoviruses are far more sensitive to UV-C than PCV1. The sucrose density gradient ultracentrifugation also contributed to PCV1 clearance with a reduction factor of 2log10. The low pH treatment during the production of procine trypsin was investigated and showed effective inactivation for both PCV1 (4.5log10) and PPV (6.4log10). In conclusion, PCV1 in general appears to be more resistant to virus inactivation than PPV. Still, the inactivated pandemic influenza vaccine manufacturing process provides for robust virus reduction, in addition to the already implemented testing for PCV1 to avoid any contaminations.

HIV-1 Tat Interacts with and Regulates the Localization and Processing of Amyloid Precursor Protein

HIV-1 Tat protein plays various roles in virus proliferation and in the regulation of numerous host cell functions. Accumulating evidence suggests that HIV-1 Tat also plays an important role in HIV-associated neurocognitive disorders (HAND) by disrupting intracellular communication. Amyloid beta (Aβ) is generated from amyloid precursor protein (APP) and accumulates in the senile plaques of Alzheimer's disease patients. This study demonstrates that Tat interacts with APP both in vitro and in vivo, and increases the level of Aβ42 by recruiting APP into lipid rafts. Co-localization of Tat with APP in the cytosol was observed in U-87 MG cells that expressed high levels of Tat, and redistribution of APP into lipid rafts, a site of increased β- and γ-secretase activity, was demonstrated by discontinuous sucrose density gradient ultracentrifugation in the presence of Tat. Furthermore, Tat enhanced the cleavage of APP by β-secretase in vitro, resulting in 5.5-fold higher levels of Aβ42. This was consistent with increased levels of β-C-terminal fragment (β-CTF) and reduced levels of α-CTF. Moreover, stereotaxic injection of a lentiviral Tat expression construct into the hippocampus of APP/presenilin-1 (PS1) transgenic mice resulted in increased Tat-mediated production and processing of Aβ in vivo. Increased levels of Aβ42, as well as an increase in the number and size of Aβ plaques, were observed in the hippocampus following injection of Tat virus compared with mock virus. These results suggest that HIV-1 Tat may contribute to HAND by interacting with and modifying APP processing, thereby increasing Aβ production.

Raft-like membranes from the trans-Golgi network and endosomal compartments

This article describes a procedure to prepare a raft-like intracellular membrane fraction enriched for the trans-Golgi network (TGN) and endosomal compartments. The initial step in this technique involves cell disruption by homogenization, followed by clearance of the plasma membrane, late endosomes, mitochondria and the endoplasmic reticulum by differential sedimentation. Carbonate treatment, sonication and sucrose density-gradient ultracentrifugation are subsequently used to isolate the target membranes. The isolated subcellular fraction contains less than 1% of the total cellular proteins, but it is highly enriched for syntaxin-6 and Rab11. Typically, 40-60% of the cellular pool of GM1 glycosphingolipid and 10-20% of the total cellular cholesterol cofractionate with this buoyant membrane fraction. Given the role of GM1 as a cell-surface receptor for the cholera toxin and that levels of both GM1 and cholesterol in the TGN-endosomal compartment are upregulated in some inherited diseases, this protocol can potentially be applied to the analysis of disease-associated changes to GM1-enriched intracellular membranes. The isolated membranes are very well separated from caveolin-rich domains of the plasma membrane, the TGN and recycling endosomes. The entire protocol can be completed in as little as 1 d.

Isolation and Characterization of the Herpes Simplex Virus 1 Terminase Complex

During herpes simplex virus 1 (HSV-1) infection, empty procapsids are assembled and subsequently filled with the viral genome by means of a protein complex called the terminase, which is comprised of the HSV-1 UL15, UL28, and UL33 proteins. Biochemical studies of the terminase proteins have been hampered by the inability to purify the intact terminase complex. In this study, terminase complexes were isolated by tandem-affinity purification (TAP) using recombinant viruses expressing either a full-length NTAP-UL28 fusion protein (vFH476) or a C-terminally truncated NTAP-UL28 fusion protein (vFH499). TAP of the UL28 protein from vFH476-infected cells, followed by silver staining, Western blotting, and mass spectrometry, identified the UL15, UL28, and UL33 subunits, while TAP of vFH499-infected cells confirmed previous findings that the C terminus of UL28 is required for UL28 interaction with UL33 and UL15. Analysis of the oligomeric state of the purified complexes by sucrose density gradient ultracentrifugation revealed that the three proteins formed a complex with a molecular mass that is consistent with the formation of a UL15-UL28-UL33 heterotrimer. In order to assess the importance of conserved regions of the UL15 and UL28 proteins, recombinant NTAP-UL28 viruses with mutations of the putative UL28 metal-binding domain or within the UL15 nuclease domain were generated. TAP of UL28 complexes from cells infected with each domain mutant demonstrated that the conserved cysteine residues of the putative UL28 metal-binding domain and conserved amino acids within the UL15 nuclease domain are required for the cleavage and packaging functions of the viral terminase, but not for terminase complex assembly.