Sucrose Gradient Centrifugation Research Articles

Isolation of Phytochrome B Photobodies.

Phytochrome B (phyB), a plant photoreceptor, forms a membraneless organelle known as a photobody. Here, we present a protocol for the isolation of phyB photobodies through fluorescence-activated particle sorting from mature transgenic Arabidopsis leaves expressing phyB-GFP. This protocol involves the isolation of nuclei from frozen ground leaves using sucrose gradient centrifugation, the disruption of nuclear envelopes by sonication, and the subsequent isolation of phyB photobodies through fluorescence-activated particle sorting. We include experimental tips and notes for each step.

A blocking ELISA based on virus-like nanoparticles chimerized with an antigenic epitope of ASFV P54 for detecting ASFV antibodies

African swine fever virus (ASFV) is a highly lethal pathogen of domestic and wild pigs. Due to no vaccines or drugs available, early accurate diagnosis and eradication of infected animals are the most important measures for ASFV prevention and control. Bluetongue virus (BTV) core-like particles (CLPs) are non-infectious hollow nanoparticles assembled from the BTV VP3 and VP7 proteins, which could be used as a platform for presenting foreign epitopes. In this study, the secondary structure of BTV VP7 protein was analyzed and predicted using the IEDB Analysis resource. Based on the prediction results of the VP7 protein, the chimeric CLPs with an ASFV P54 epitope were successfully prepared through the BAC-to-BAC baculovirus expression system and sucrose gradient centrifugation. Based on the chimeric CLPs and mAb 2E4 against AFSV P54 epitope, a blocking ELISA for detecting AFSV antibodies was established, and its reaction conditions were optimized. Through comprehensive evaluation of the method, the results showed the chimeric CLPs-based blocking ELISA displayed the best detection performance, with an AUC of 0.9961, a sensitivity of 97.65%, and a specificity of 95.24% in ROC analysis. Compared with western blot and a commercial c-ELISA for detecting anti-ASFV antibodies, this method had an excellent agreement of 96.35% (kappa value = 0.911) and 97.76% (kappa value = 0.946) with the other tests, respectively. This ELISA also had high repeatability, with CV < 10%, and no cross-reaction with the serum antibodies against other swine viruses or Orbivirus. In brief, this was the first report on developing a blocking ELISA based on virus-like nanoparticles chimerized with an antigenic epitope of ASFV P54 for serological diagnosis of ASFV.

An improved method for extraction of soil fungal mycelium

Fungal mycelium is a major component of the soil microbiome. The soil hyphosphere represents a complex and dynamic niche for specific microorganisms, where multitrophic interactions occur, affecting ecosystem processes. However, extracting fungal mycelium from the soil to enable its taxonomical, chemical, and structural characterisation is challenging in the absence of a fast, efficient, and low-cost procedure. In this study, an old method (Bingle and Paul 1985), based on successive soil wet filtrations and density gradient centrifugation, was improved and tested in three different soil types (silty clay, silty clay loam, and loamy sand). The improved method reduced the number of filtrations by about five times and the centrifugation time from 40 min to 1 min. It avoided using any chemical substance which may impair further chemical analyses or DNA isolation and amplification. The method efficiency was about 50 % in the clay and 23 % in the sandy soils. However, a pre-step consisting of removing the fine-root fragments and other debris under the stereomicroscope may increase the method efficiency to more than 65 %, independent of the soil type.•A simple, efficient, and low-cost method suitable for extracting soil mycelium from a large number of samples.•The protocol includes successive soil wet filtrations and sucrose gradient centrifugation.•The method efficiency increases if the fine-root fragments and other debris are previously removed from the soil.

Virus-like particle vaccines with epitopes from porcine epidemic virus and transmissible gastroenteritis virus incorporated into self-assembling ADDomer platform provide clinical immune responses in piglets.

Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are major intestinal coronaviruses that cause vomiting, diarrhea, dehydration, and mortality in piglets. These viruses coexist and lead to significant economic losses in the swine industry. Virus-like particles (VLPs) have emerged as promising alternatives to conventional inactivated vaccines due to their exceptional safety, efficacy, and ability to provide multi-disease protection with a single dose. Our study focused on specific antigenic epitopes from the PEDV S protein (SS2 and 2C10 regions) and the TGEV S protein (A and D sites) as target candidates. These epitopes were integrated into the ADDomer framework, and we successfully generated recombinant proteins AD, AD-P, AD-T, and AD-PT using the baculovirus expression vector system (BEVS). By meticulously optimizing conditions in High Five cells, we successfully expressed and purified the recombinant proteins. Subsequently, we developed the recombinant ADDomer-VLP vaccine and conducted a comprehensive evaluation of its efficacy in piglets. Following ultrafiltration concentration and sucrose gradient centrifugation purification, the recombinant proteins self-assembled into VLPs as observed by transmission electron microscopy (TEM). Administration of the vaccine did not result in any adverse reactions in the immunized piglets. Additionally, no significant instances of fever were detected in any of the experimental groups, and there were no notable changes in average daily weight gain compared to the control group that received PBS. The recombinant ADDomer-VLP vaccines demonstrated strong immunogenicity, effectively stimulating the production of neutralizing antibodies against both PEDV and TGEV. Moreover, the recombinant ADDomer-VLP vaccine induced elevated levels of IFN-γ, IL-2, and IL-4, and enhanced cytotoxic T lymphocyte (CTL) activity in the peripheral blood of piglets. These recombinant VLPs have demonstrated the ability to induce strong cellular and humoral immune responses in piglets, making them an incredibly promising platform for the rapid and simplified development of epitope vaccines.

Isolation of Neuronal Synaptic Membranes by Sucrose Gradient Centrifugation.

Sucrose gradient centrifugation is a very useful technique for isolating specific membrane types based on their size and density. This is especially useful for detecting fatty acids and lipid molecules that are targeted to specialized membranes. Without fractionation, these types of molecules could be below the levels of detection after being diluted out by the more abundant lipid molecules with a more ubiquitous distribution throughout the various cell membranes. Isolation of specific membrane types where these lipids are concentrated allows for their detection and analysis. We describe herein our synaptic membrane isolation protocol that produces excellent yield and clear resolution of five major membrane fractions from a starting neural tissue homogenate: P1 (nuclear), P2 (cytoskeletal), P3 (neurosynaptosomal), PSD (post-synaptic densities), and SV (synaptic vesicle).

Polysome Profiling in Adult Mouse Testes.

Polysome profiling is widely used to isolate and analyze polysome fractions, which consist of actively translating mRNAs and ribosomes. Compared to ribosome profiling and translating ribosome affinity purification, polysome profiling is simpler and less time consuming in sample preparation and library constructions. Spermiogenesis, i.e., the post-meiotic phase of male germ cell development, is a highly coordinated developmental process in which transcription and translation are decoupled because of nuclear condensation, resulting in translation regulation as the major mode for the regulation of gene expression in post-meiotic spermatids. To understand the translation regulation during spermiogenesis, an overview of translational state of spermiogenic mRNAs is required. Here, we describe a protocol to identify translating mRNAs using polysome profiling. Briefly, mouse testes are gently homogenized to release polysomes containing translating mRNAs, following polysome-bound mRNAs isolated by sucrose density gradient purification and characterized by RNA-seq. This protocol allows to quickly isolate translating mRNAs from testes and analyze the discrepancy of translational efficiency in mouse testes from different mouse lines. Key features Quickly obtain polysome RNAs from testes. Omit RNase digestion and RNA recovery from gel. High efficiency and robustness compared to ribo-seq. Graphical overview Schematic illustrating the experimental design for polysome profiling in mouse testes. Mouse testes are homogenized and lysed in Sample preparation, and polysome RNAs are enriched by sucrose gradient centrifugation and used to calculate translation efficiency in Sample analysis.

Systematic Analysis of Assembly Intermediates in Yeast to Decipher the Mitoribosome Assembly Pathway.

Studies of yeast mitoribosome assembly have been historically hampered by the difficulty of generating mitoribosome protein-coding gene deletion strains with a stable mitochondrial genome. The identification of mitochondrial DNA-stabilizing approaches allows for the generation of a complete set of yeast deletion strains covering all mitoribosome proteins and known assembly factors. These strains can be used to analyze the integrity and assembly state of mitoribosomes by determining the sedimentation profile of these structures by sucrose gradient centrifugation of mitochondrial extracts, coupled to mass spectrometry analysis of mitoribosome composition. Subsequent hierarchical cluster analysis of mitoribosome subassemblies accumulated in mutant strains reveals details regarding the order of protein association during the mitoribosome biogenetic process. These strains also allow the expression of truncated protein variants to probe the role of mitochondrion-specific protein extensions, the relevance of protein cofactors, or the importance of RNA-protein interactions in functional sites of the mitoribosome. In this chapter, we will detail the methodology involved in these studies.

Separating Inner and Outer Membranes of Escherichia coli by EDTA-free Sucrose Gradient Centrifugation.

The envelope of Gram-negative bacteria consists of an outer membrane (OM), a peptidoglycan cell wall, and an inner membrane (IM). The OM and IM have different components of proteins and lipids. Separating the IM and OM is a basic biochemical procedure to further study lipids and membrane proteins in different locations. Sucrose gradient ultracentrifugation of lysozyme/EDTA-treated total membrane is the most widely used method to separate the IM and OM of Gram-negative bacteria. However, EDTA is often harmful to protein structure and function. Here, we describe a relatively simple sucrose gradient ultracentrifugation method to separate the IM and OM of Escherichia coli. In this method, the cells are broken by a high-pressure microfluidizer, and the total cell membrane is collected by ultracentrifugation. The IM and OM are then separated on a sucrose gradient. Because EDTA is not used, this method is beneficial for subsequent membrane protein purification and functional study.

Expression of miRNA-Targeted and Not-Targeted Reporter Genes Shows Mutual Influence and Intercellular Specificity

The regulation of translation by RNA-induced silencing complexes (RISCs) composed of Argonaute proteins and micro-RNAs is well established; however, the mechanisms underlying specific cellular responses to miRNAs and how specific complexes arise are not completely clear. To explore these questions, we performed experiments with Renilla and firefly luciferase reporter genes transfected in a psiCHECK-2 plasmid into human HCT116 or Me45 cells, where only the Renilla gene contained sequences targeted by microRNAs (miRNAs) in the 3'UTR. The effects of targeting were miRNA-specific; miRNA-21-5p caused strong inhibition of translation, whereas miRNA-24-3p or Let-7 family caused no change or an increase in reporter Renilla luciferase synthesis. The mRNA-protein complexes formed by transcripts regulated by different miRNAs differed from each other and were different in different cell types, as shown by sucrose gradient centrifugation. Unexpectedly, the presence of miRNA targets on Renilla transcripts also affected the expression of the co-transfected but non-targeted firefly luciferase gene in both cell types. Renilla and firefly transcripts were found in the same sucrose gradient fractions and specific anti-miRNA oligoribonucleotides, which influenced the expression of the Renilla gene, and also influenced that of firefly gene. These results suggest that, in addition to targeted transcripts, miRNAs may also modulate the expression of non-targeted transcripts, and using the latter to normalize the results may cause bias. We discuss some hypothetical mechanisms which could explain the observed miRNA-induced effects.

Combining BN-PAGE and microscopy techniques to investigate pigment-protein complexes and plastid transitions in citrus fruit

BackgroundChlorophyll and carotenoids, the most widely distributed lipophilic pigments in plants, contribute to fruit coloration during development and ripening. These pigments are assembled with pigment-protein complexes localized at plastid membrane. Pigment-protein complexes are essential for multiple cellular processes, however, their identity and composition in fruit have yet to be characterized.ResultsBy using BN-PAGE technique in combination with microscopy, we studied pigment-protein complexes and plastid transformation in the purified plastids from the exocarp of citrus fruit. The discontinuous sucrose gradient centrifugation was used to isolate total plastids from kumquat fruit, and the purity of isolated plastids was assessed by microscopy observation and western blot analysis. The isolated plastids at different coloring stages were subjected to pigment autofluorescence observation, western blot, two-dimensional electrophoresis analysis and BN-PAGE assessment. Our results demonstrated that (i) chloroplasts differentiate into chromoplasts during fruit coloring, and this differentiation is accompanied with a decrease in the chlorophyll/carotenoid ratio; (ii) BN-PAGE analysis reveals the profiles of macromolecular protein complexes among different types of plastids in citrus fruit; and (iii) the degradation rate of chlorophyll-protein complexes varies during the transition from chloroplasts to chromoplasts, with the stability generally following the order of LHCII > PS II core > LHC I > PS I core.ConclusionsOur optimized methods for both plastid separation and BN-PAGE assessment provide an opportunity for developing a better understanding of pigment-protein complexes and plastid transitions in plant fruit. These attempts also have the potential for expanding our knowledge on the sub-cellular level synchronism of protein changes and pigment metabolism during the transition from chloroplasts to chromoplasts.

Avian Reovirus σB Interacts with Caveolin-1 in Lipid Rafts during Dynamin-Dependent Caveolae-Mediated Endocytosis.

Caveolin-1 (Cav-1) is the basic component of caveolae, a specialized form of lipid raft that plays an essential role in endocytic viral entry. However, the evidence of direct involvement of caveolae and Cav-1 in avian reovirus (ARV) entry remains insufficient. In this study, the membrane lipid rafts were isolated as detergent-resistant microdomains (DRMs) by sucrose gradient centrifugation, and the capsid protein σB of ARV was found to associate with Cav-1 in DRMs fractions. Additionally, the interaction between ARV σB protein and Cav-1 was demonstrated by immunofluorescence co-localization and co-immunoprecipitation assays. Furthermore, we found that the internalization of ARV is sensitive to caveolae and dynamin inhibitors, while it is insensitive to clathrin inhibitors. In conclusion, these results indicate that the ARV σB protein interacts with Cav-1 during dynamin-dependent caveolae-mediated endocytosis for the entry of ARV.

Efficient Isolation and Purification of High-Quality Arabidopsis thaliana Trichomes.

Trichomes are fine outgrowths on the surface of aerial plant organs which play a role in protecting plants against water loss, UV radiation, and herbivore feeding. Throughout the years, trichomes have become a popular paradigm in biological research. For example, trichomes on rosette leaves of the reference plant Arabidopsis thaliana have been used as a model to investigate cell development, cell differentiation, and, more recently, cell wall biogenesis. State of the art -omics studies on specific cell types or tissues often require physical separation, enrichment, and purification. This, of course, also applies to leaf trichomes, and various methods have thus been proposed to separate trichomes and leaf tissue. Though most of these methods are indeed suitable for trichome isolation, they suffer in part from tedious operating procedures, low yield, poor sample purity, and reduced trichome integrity. We have thus revised a previously reported method for trichome isolation, and report here an efficient and scalable procedure for the isolation and gradient centrifugation-based purification of high-quality A. thaliana trichomes. We describe the preparation of plant material and trichome release, which is based on prolonged gentle agitation of plant seedlings in the presence of a cation-chelating agent that weakens trichome-leaf interactions. We also outline the steps for the subsequent recovery and purification of the isolated crude trichome fraction, which is based on the use of discontinuous sucrose gradient centrifugation. In addition to A. thaliana, we have found that this procedure can be applied to release and enrich glandular and non-glandular trichomes from various species, including Solanum lycopersicum and Nicotiana benthamiana. The resulting purified leaf trichomes can be subjected to different types of bioassays, including histochemistry, biochemical quantification of cell wall monosaccharides, and transcriptomics, as well as proteomic profiling. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of plant material for release and enrichment of A. thaliana trichomes Basic Protocol 2: Purification of A. thaliana trichomes by density gradient centrifugation.

Comparison of the extraction methods of plasma exosomes from human umbilical cord blood

We compared ultracentrifugation, sucrose gradient centrifugation, improved ultracentrifugation, and polyethylene glycol (PEG) precipitation in the extraction of plasma exosomes from human umbilical cord blood, aiming at screening out a stable and efficient method. The morphology, structure, and size of exosomes were observed based on transmission electron microscopy and dynamic light scattering. Total protein content of exosomes was determined by bicinchoninic acid (BCA) assay, and the expression of exosome markers CD63 and HSP70 and exosome negative marker GM130 (Golgi marker) by Western blotting. Results showed that sucrose gradient centrifugation was more stable and yielded exosomes of uniform particle size compared with ultracentrifugation which had been considered as the "gold standard" for exosome extraction. However, sucrose gradient centrifugation had the limitations of complex operation and time-intensiveness. The improved ultracentrifugation featured ease of implementation and the extracted exosomes were of high purity. PEG precipitation extracted the most exosomes in a shorter timeframe, but the purity of the exosomes was low. In conclusion, all the four methods can separate exosomes from human umbilical cord blood plasma, but they are different in operation time, product purity, and product content. Therefore, the method for extracting plasma exosomes from human umbilical cord blood should be selected based on the experimental purpose and specific requirements.

Investigation of Avian Influenza H5N6 Virus-like Particles as a Broad-Spectrum Vaccine Candidate against H5Nx Viruses.

Highly pathogenic avian influenza (HPAI) clade 2.3.4.4 viruses have been reported to be the source of infections in several outbreaks in the past decades. In a previous study, we screened out a broad-spectrum virus strain, H5N6-Sichuan subtype, by using a lentiviral pseudovirus system. In this project, we aimed to investigate the potential of H5N6 virus-like particles (VLPs) serving as a broad-spectrum vaccine candidate against H5Nx viruses. We cloned the full-length M1 gene and H5, N6 genes derived from the H5N6-Sichuan into pFASTBac vector and generated the VLPs using the baculovirus-insect cell system. H5N6 VLPs were purified by sucrose gradient centrifugation, and the presence of H5, N6 and M1 proteins was verified by Western blot and SDS-PAGE. The hemagglutination titer of H5N6 VLPs after purification reached 5120 and the particle structure remained as viewed by electron microscopy. The H5N6 VLPs and 293T mammalian cell-expressed H5+N6 proteins were sent for mice immunization. Antisera against the H5+N6 protein showed 80 to 320 neutralizing antibody titers to various H5Nx pseudoviruses. In contrast, H5N6 VLPs not only elicited higher neutralizing antibody titers, ranging from 640 to 1280, but also induced higher IL-2, IL-4, IL-5, IFN-γ and TNF production, thus indicating that H5N6 VLPs may be a potential vaccine candidate for broad-spectrum H5Nx avian influenza vaccines.

LSD1 deletion decreases exosomal PD-L1 and restores T-cell response in gastric cancer

BackgroundHistone lysine-specific demethylase 1 (LSD1) expression has been shown to be significantly elevated in gastric cancer (GC) and may be associated with the proliferation and metastasis of GC. It has been reported that LSD1 repressed tumor immunity through programmed cell death 1 ligand 1 (PD-L1) in melanoma and breast cancer. The role of LSD1 in the immune microenvironment of GC is unknown.MethodsExpression LSD1 and PD-L1 in GC patients was analyzed by immunohistochemical (IHC) and Western blotting. Exosomes were isolated from the culture medium of GC cells using an ultracentrifugation method and characterized by transmission electronic microscopy (TEM), nanoparticle tracking analysis (NTA), sucrose gradient centrifugation, and Western blotting. The role of exosomal PD-L1 in T-cell dysfunction was assessed by flow cytometry, T-cell killing and enzyme-linked immunosorbent assay (ELISA).ResultsThrough in vivo exploration, mouse forestomach carcinoma (MFC) cells with LSD1 knockout (KO) showed significantly slow growth in 615 mice than T-cell-deficient BALB/c nude mice. Meanwhile, in GC specimens, expression of LSD1 was negatively correlated with that of CD8 and positively correlated with that of PD-L1. Further study showed that LSD1 inhibited the response of T cells in the microenvironment of GC by inducing the accumulation of PD-L1 in exosomes, while the membrane PD-L1 stayed constant in GC cells. Using exosomes as vehicles, LSD1 also obstructed T-cell response of other cancer cells while LSD1 deletion rescued T-cell function. It was found that while relying on the existence of LSD1 in donor cells, exosomes can regulate MFC cells proliferation with distinct roles depending on exosomal PD-L1-mediated T-cell immunity in vivo.ConclusionLSD1 deletion decreases exosomal PD-L1 and restores T-cell response in GC; this finding indicates a new mechanism with which LSD1 may regulate cancer immunity in GC and provides a new target for immunotherapy against GC.

Active nNOS Is Required for Grp94-Induced Antioxidant Cytoprotection: A Lesson from Myogenic to Cancer Cells.

The endoplasmic reticulum (ER) chaperone Grp94/gp96 appears to be involved in cytoprotection without being required for cell survival. This study compared the effects of Grp94 protein levels on Ca2+ homeostasis, antioxidant cytoprotection and protein–protein interactions between two widely studied cell lines, the myogenic C2C12 and the epithelial HeLa, and two breast cancer cell lines, MDA-MB-231 and HS578T. In myogenic cells, but not in HeLa, Grp94 overexpression exerted cytoprotection by reducing ER Ca2+ storage, due to an inhibitory effect on SERCA2. In C2C12 cells, but not in HeLa, Grp94 co-immunoprecipitated with non-client proteins, such as nNOS, SERCA2 and PMCA, which co-fractionated by sucrose gradient centrifugation in a distinct, medium density, ER vesicular compartment. Active nNOS was also required for Grp94-induced cytoprotection, since its inhibition by L-NNA disrupted the co-immunoprecipitation and co-fractionation of Grp94 with nNOS and SERCA2, and increased apoptosis. Comparably, only the breast cancer cell line MDA-MB-231, which showed Grp94 co-immunoprecipitation with nNOS, SERCA2 and PMCA, increased oxidant-induced apoptosis after nNOS inhibition or Grp94 silencing. These results identify the Grp94-driven multiprotein complex, including active nNOS as mechanistically involved in antioxidant cytoprotection by means of nNOS activity and improved Ca2+ homeostasis.

Organic viticulture enhanced the activity of native entomopathogenic nematodes in DOCa Rioja soils (North of Spain)

Vineyards and their associated socio-economic activities are relevant sectors worldwide. Still, this agroecosystem is one of the most intensely managed crops and erosion-prone land areas. The conventional viticulture practices to control pests, diseases, and weeds, like tillage and agrochemical applications, accelerate the loss of soil biodiversity and compromise the presence of beneficial soil organisms such as the entomopathogenic nematodes (EPNs). Such human disturbances in the agroecosystems can strongly affect abiotic (e.g., soil texture and properties) and biotic factors (natural enemies and potential competitors) that modulate the EPN activity as biological control agents. For the first time in viticulture, this study aimed to investigate the impact of differentiating management on the EPN community and associated soil organisms and if their assemblage will provide indicators of better practices for sustainable farming. We hypothesized that organic pest management and alternative strategies to tillage might enhance the abundance and activity of the native EPN community in vineyard soils. In autumn 2019, we collected two composite soil samples from 80 vineyards distributed across the Guaranteed Designation of Origin (denominated DOCa) Rioja region. The sites belonged to one category of each of the two factors: pest management (integrated vs. organic, 40 plots each) and soil managing (tillage vs. cover cropping, 48 and 32 vineyards, respectively). Isolated through sucrose-gradient centrifugation and employing species-specific primers/probe qPCR sets, we investigated the presence of ten EPN species and associated soil organisms: four free-living nematodes (FLNs), six nematophagous fungi (NF), and two ectoparasitic bacteria (EcPB). Besides, we estimated the EPN activity using the traditional insect-bait method. We included in the analysis twenty soil variables to characterize the evaluated treatments and assess their impact on soil organism distribution. Our results provide evidence on the support of organic viticulture to beneficial soil organisms, notably the activity of native EPNs. We also reported a higher abundance of S. feltiae (the predominant steinernematid species in Europe) and FLNs for organic farming than IMP, while the presence of NF and EcPB resulted in unaffected. Contrarily, the soil management practices considered did not differ in their impact on EPNs or their natural enemies/competitors, even if contrasted for several soil properties. Future research may expand the screened soil-dwelling species using novel molecular technics to unravel their complex interactions and determine the best farming practices to preserve soil health.

Preparation of Asymmetric Vesicles with Trapped CsCl Avoids Osmotic Imbalance, Non-Physiological External Solutions, and Minimizes Leakage.

The natural asymmetry of cellular membranes influences their properties. In recent years, methodologies for preparing asymmetric vesicles have been developed that rely on cyclodextrin-catalyzed exchange of lipids between donor lipid multilamellar vesicles and acceptor lipid unilamellar vesicles, and the subsequent separation of the, now asymmetric, acceptor vesicles from the donors. Isolation is often accomplished by preloading acceptor vesicles with a high concentration of sucrose, typically 25% (w/w), and separating from donor and cyclodextrin by sucrose gradient centrifugation. We found that when the asymmetric vesicles prepared using methyl-α-cyclodextrin exchange were dispersed under hypotonic conditions using physiological salt solutions, there was enhanced leakage of an entrapped probe, 6-carboxyfluorescein. Studies with symmetric vesicles showed this was due to osmotic pressure and was specific to hypotonic solutions. Inclusion of cholesterol partly reduced leakage but did not completely eliminate it. To avoid having to use hypotonic conditions or to suspend vesicles at nonphysiological solute concentrations to minimize leakage, a method for preparing asymmetric vesicles using acceptor vesicle-entrapped CsCl at a physiological ion concentration (100 mM) was developed. Asymmetric vesicles prepared with the entrapped CsCl protocol were highly resistant to 6-carboxyfluorescein leakage out of the vesicles.

Changes in synaptic proteins of the complex PSD-95/NMDA receptor/nNOS and mitochondrial dysfunction after levocabastine treatment

Nitric oxide generation is related to the activity of certain proteins located at synaptic sites. Previous findings show that NOS activity, nNOS protein expression, respiratory parameters and mitochondrial complex activities are altered in rat cerebral cortex by administration of levocabastine, an antagonist of histamine H1 and neurotensin NTS2 receptors. ATP provision by mitochondria may play an important role in the functional interaction between synaptic proteins NMDA receptor and PSD-95 with NO synthesis. In this context, our purpose was to evaluate the effect of levocabastine administration on protein expression of PSD-95, GluN2B and iNOS, as well as on mitochondrial ATP production. Male Wistar rats received a single (i.p.) dose of levocabastine (50 μg/kg) or saline solution (controls) and were decapitated 18 h later. Mitochondrial and synaptosomal membrane fractions were isolated from cerebral cortex by differential and sucrose gradient centrifugation. Expression of synaptic proteins was evaluated by Western blot assays in synaptosomal membrane fractions. Oxygen consumption, mitochondrial membrane potential and ATP production rate were determined in fresh crude mitochondrial fractions. After levocabastine treatment, protein expression of PSD-95, GluN2B and β-actin decreased 97, 45 and 55%, respectively, whereas that of iNOS enhanced 3.5-fold versus controls. In crude mitochondrial fractions levocabastine administration reduced roughly 15% respiratory control rate as assayed with malate-glutamate or succinate as substrates, decreased mitochondrial membrane potential (21%), and ATP production rates (57%). Results suggested that levocabastine administration induces alterations in synaptic proteins of the protein complex PSD-95/NMDA receptor/nNOS and in neuron cytoskeleton. Mitochondrial bioenergetics impairment may play a role in the functional link between synaptic proteins and NO synthesis.

The ultrastructure of infectious L-type bovine spongiform encephalopathy prions constrains molecular models.

Bovine spongiform encephalopathy (BSE) is a prion disease of cattle that is caused by the misfolding of the cellular prion protein (PrPC) into an infectious conformation (PrPSc). PrPC is a predominantly α-helical membrane protein that misfolds into a β-sheet rich, infectious state, which has a high propensity to self-assemble into amyloid fibrils. Three strains of BSE prions can cause prion disease in cattle, including classical BSE (C-type) and two atypical strains, named L-type and H-type BSE. To date, there is no detailed information available about the structure of any of the infectious BSE prion strains. In this study, we purified L-type BSE prions from transgenic mouse brains and investigated their biochemical and ultrastructural characteristics using electron microscopy, image processing, and immunogold labeling techniques. By using phosphotungstate anions (PTA) to precipitate PrPSc combined with sucrose gradient centrifugation, a high yield of proteinase K-resistant BSE amyloid fibrils was obtained. A morphological examination using electron microscopy, two-dimensional class averages, and three-dimensional reconstructions revealed two structural classes of L-type BSE amyloid fibrils; fibrils that consisted of two protofilaments with a central gap and an average width of 22.5 nm and one-protofilament fibrils that were 10.6 nm wide. The one-protofilament fibrils were found to be more abundant compared to the thicker two-protofilament fibrils. Both fibrillar assemblies were successfully decorated with monoclonal antibodies against N- and C-terminal epitopes of PrP using immunogold-labeling techniques, confirming the presence of polypeptides that span residues 100-110 to 227-237. The fact that the one-protofilament fibrils contain both N- and C-terminal PrP epitopes constrains molecular models for the structure of the infectious conformer in favour of a compact four-rung β-solenoid fold.