Discontinuous Sucrose Density Gradient Research Articles

Separation of Single Core and Multicore Lytic Granules by Subcellular Fractionation and Immunoisolation.

Subcellular fractionation is an important tool used to separate intracellular organelles, structures or proteins. Here, we describe a stepwise protocol to isolate two types of lytic granules, multicore (MCG), and single core (SCG), from primary murine CTLs. We used cell disruption by nitrogen cavitation followed by separation of organelles via discontinuous sucrose density gradient centrifugation. Immunoisolation with a Synaptobrevin 2 antibody attached to magnetic beads was then used to harvest Synaptobrevin 2 positive granules for immunoblotting, mass spectrometry, electron, and light microscopy.

A2E Distribution in RPE Granules in Human Eyes.

A2E (N-retinylidene-N-retinylethanolamine) is a major fluorophore in the RPE (retinal pigment epithelium). To identify and characterize A2E-rich RPE lipofuscin, we fractionated RPE granules from human donor eyes into five fractions (F1–F5 in ascending order of density) by discontinuous sucrose density gradient centrifugation. The dry weight of each fraction was measured and A2E was quantified by liquid chromatography/mass spectrometry (LC/MS) using a synthetic A2E homolog as a standard. Autofluorescence emission was characterized by a customer-built spectro-fluorometer system. A significant A2E level was detected in every fraction, and the highest level was found in F1, a low-density fraction that makes up half of the total weight of all RPE granules, contains 67% of all A2E, and emits 75% of projected autofluorescence by all RPE granules. This group of RPE granules, not described previously, is therefore the most abundant RPE lipofuscin granule population. A progressive decrease in autofluorescence was observed from F2 to F4, whereas no autofluorescence emission was detected from the heavily pigmented F5. The identification of a novel and major RPE lipofuscin population could have significant implications in our understanding of A2E and lipofuscin in human RPE.

CaMKII Modulates the Cardiac Transient Outward K+ Current through its Association with Kv4 Channels in Non-Caveolar Membrane Rafts.

To test whether the physiological regulation of the cardiac Kv4 channels by the Ca2+/calmodulin-dependent protein kinase II (CaMKII) is restricted to lipid rafts and whether the interactions observed in rat cardiomyocytes also occur in the human ventricle. Ventricular myocytes were freshly isolated from Sprague-Dawley rats. Ito was recorded by the whole-cell Patch-Clamp technique. Membrane rafts were isolated by centrifugation in a discontinuous sucrose density gradient. The presence of the proteins of interest was analysed by western blot. Immunogold staining and electron microscopy of heart vibrosections was performed to localize Kv4.2/Kv4.3 and CaMKII proteins. Protein-protein interactions were determined by co-immunoprecipitation experiments in rat and human ventricular mycoytes. Patch-Clamp recordings in control conditions and after lipid raft or caveolae disruption show that the CaMKII-Kv4 channel complex must associate in non-caveolar lipid rafts to be functional. Separation in density gradients, co-immunoprecipitation and electron microscopy show that there are two Kv4 channel populations: one located in caveolae, that is CaMKII independent, and another one located in planar membrane rafts, which is bound to CaMKII. CaMKII regulates only the Kv4 channel population located in non-caveolar lipid rafts. Thus, the regulation of cardiac Kv4 channels in rat and human ventricle depends on their subcellular localization.

Loss of Caveolin‐3 (Cav‐3) Promotes G‐protein‐regulated Matrix Metalloprotease 14 (MMP14) Activation in the Aged Heart

Age‐associated cardiovascular disease significantly correlates with morbidity and mortality worldwide; therefore, investigation of cellular signaling pathways in the heart that change with age may help identify novel therapeutic strategies for mitigating the progression of cardiovascular pathologies. Our lab has uncovered a membrane‐delimited receptor transactivation pathway in the heart, wherein G‐protein coupled receptors (GPCRs) signal via heterotrimeric G‐proteins to activate membrane‐tethered matrix metalloprotease 14 (MMP14/MT1‐MMP) and consequently induce “shedding” of membrane‐bound receptor ligands (J Biol Chem 290: 9941‐7). Specifically, G‐protein‐regulated MMP14 releases heparin‐binding EGF‐like growth factor (HB‐EGF), which then triggers activation of epidermal growth factor receptor (EGFR) signaling. We hypothesized that the components of this mechanism are compartmentalized within caveolar lipid raft domains (i.e. cholesterol‐rich membranes containing caveolin‐1 (Cav‐1) and caveolin‐3 (Cav‐3) isoforms), wherein co‐localization with caveolin proteins serves to repress MMP14 enzymatic activity. Furthermore, we hypothesized that age‐dependent loss of Cav‐3 in the heart increases MMP14 activity and thereby alters the signaling pathway to contribute to deleterious cardiac aging phenotypes. We thus sought to compare changes in G‐protein‐regulated MMP14 activity and subsequent activation of HB‐EGF/EGFR signaling in membrane fractions isolated from cardiac myocytes (CMs) and cardiac fibroblasts (CFs) of young (2–3 month) and aged (18–21 month) mice, with or without transgenic CM‐specific overexpression of Cav‐3 (i.e., Cav‐3 OE), to determine if preserved Cav‐3 expression prevents or lessens harmful age‐associated membrane signaling. We observed approximately a two‐fold increase of MMP14 activity in membranes from CMs of aged wild‐type (WT) mice compared to young controls (p< 0.05). Immunoblot analysis of buoyant fractions isolated by differential centrifugation of cardiac tissue in a discontinuous sucrose density gradient confirmed that MMP14, G‐proteins, HB‐EGF, and EGFR co‐localize with caveolin protein isoforms in lipid rafts. Work in progress will test if co‐localization of these proteins is reduced in aged wild‐type (WT) animals but not in Cav‐3 OE animals, and if MMP14 and Cav‐3 co‐immunoprecipitate (co‐IP) within lipid raft domains and thus form a functional signaling module that is disrupted in aged WT subjects but remains intact in Cav‐3 OE mice. Taken together, the results suggest that GPCR transactivation of EGFR and regulation by signaling components in caveolar lipid rafts may be a feature of membrane signaling that is altered in the aged heart.Support or Funding InformationSupported by NIH grants R21AG053568 and R21AG52914.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Proteomic Analysis of Rice Golgi Membranes Isolated by Floating Through Discontinuous Sucrose Density Gradient.

The Golgi apparatus is an endomembrane system organelle and has roles in glycosylation, sorting, and secretion of proteins in the secretory pathway. It has a central function in living organism and is also essential for plant growth. Proteomic approaches to identify the Golgi membrane proteins have been performed in cell suspension cultures and many Golgi membrane-associated proteins were found, whereas it has well established in rice seedling yet. In this chapter, our recent improving published methods for isolated rice Golgi membranes by floating through a discontinuous sucrose density gradient are provided in detail with proteomic analyses.

Isolation of F. novicida-Containing Phagosome from Infected Human Monocyte Derived Macrophages.

Francisella is a gram-negative bacterial pathogen, which causes tularemia in humans and animals. A crucial step of Francisella infection is its invasion of macrophage cells. Biogenesis of the Francisella-containing phagosome (FCP) is arrested for ~15 min at the endosomal stage, followed by gradual bacterial escape into the cytosol, where the microbe proliferates. The crucial step in pathogenesis of tularemia is short and transient presence of the bacterium within phagosome. Isolation of FCPs for further studies has been challenging due to the short period of time of bacterial residence in it and the characteristics of the FCP. Here, we will for the first time present the method for isolation of the FCPs from infected human monocytes-derived macrophages (hMDMs). For elimination of lysosomal compartment these organelles were pre-loaded with dextran coated colloidal iron particles prior infection and eliminated by magnetic separation of the post-nuclear supernatant (PNS). We encountered the challenge that mitochondria has similar density to the FCP. To separate the FCP in the PNS from mitochondria, we utilized iodophenylnitrophenyltetrazolium, which is converted by the mitochondrial succinate dehydrogenase into formazan, leading to increased density of the mitochondria and allowing separation by the discontinuous sucrose density gradient ultracentrifugation. The purity of the FCP preparation and its acquisition of early endosomal markers was confirmed by Western blots, confocal and transmission electron microscopy. Our strategy to isolate highly pure FCPs from macrophages should facilitate studies on the FCP and its biogenesis.

Development of a Japanese encephalitis virus-like particle vaccine in silkworms using codon-optimised prM and envelope genes.

We have successfully prepared a Japanese encephalitis virus (JEV) − Nakayama virus like particle (NVLP) vaccine using synthetic codon-optimized prM and E genes. The expression of the recombinant JEV Nakayama-BmNPV (JEV-NNPV) virus was determined in infected silkworm Bm-N cells by fluorescence and Western blot analysis. The recombinant was inoculated into silkworm pupae and the yield of Nakayama VLP (NVLP) reached a peak in the homogenates after 3 days. Additionally, in the peptide analysis of infected pupae homogenate, it appeared approximately 300–500 μg E protein/pupa were produced. When purified the above eluates on the discontinuous sucrose density gradient centrifugation, NVLP showed a strong hemagglutination (HA) activity by using chicken red blood cell in phosphate-buffered saline (PBS) free from Mg++ and Ca++ ions. The immune antisera against NVLP strain could efficiently neutralize the plaque formation of Nakayama, Beijing-1 and Muar strains, showing tendency of much higher reaction with heterologous Muar strain than homologous Nakayama strain. Our findings suggest that the JEV-NVLP may be useful for JEV epidemic control in many endemic areas of Asian countries as a widely effective and less expensive JE vaccine.

Entire nucleotide sequences of Gossypium raimondii and G.arboreum mitochondrial genomes revealed A-genome species as cytoplasmic donor of the allotetraploid species.

Cotton (Gossypium spp.) is commonly grouped into eight diploid genomic groups, designated A-G and K, and an allotetraploid genomic group, AD. Gossypium raimondii (D5 ) and G.arboreum (A2 ) are the putative contributors to the progenitor of G.hirsutum (AD1 ), the economically important fibre-producing cotton species. Mitochondrial DNA from week-old etiolated seedlings was extracted from isolated organelles using discontinuous sucrose density gradient method. Mitochondrial genomes were sequenced, assembled, annotated and analysed in orderly. Gossypiumraimondii (D5 ) and G.arboreum (A2 ) mitochondrial genomes were provided in this study. The mitochondrial genomes of two diploid species harboured circular genome of 643,914bp (D5 ) and 687,482bp (A2 ), respectively. They differ in size and number of repeat sequences, both contain illuminating triplicate sequences with 7317 and 10,246bp, respectively, demonstrating dynamic difference and rearranged genome organisations. Comparing the D5 and A2 mitogenomes with mitogenomes of tetraploid Gossypium species (AD1 , G.hirsutum; AD2 , G.barbadense), a shared 11kbp fragment loss was detected in allotetraploid species, three regions shared by G.arboreum (A2 ), G.hirsutum (AD1 ) and G.barbadense (AD2 ), while eight regions were specific to G.raimondii (D5 ). The presence/absence variations and gene-based phylogeny supported that A-genome is a cytoplasmic donor to the progenitor of allotetraploid species G.hirsutum and G.barbadense. The results present structure variations and phylogeny of Gossypium mitochondrial genome evolution.

Rapid evolutionary divergence of Gossypium barbadense and G. hirsutum mitochondrial genomes.

BackgroundThe mitochondrial genome from upland cotton, G. hirsutum, was previously sequenced. To elucidate the evolution of mitochondrial genomic diversity within a single genus, we sequenced the mitochondrial genome from Sea Island cotton (Gossypium barbadense L.).MethodsMitochondrial DNA from week-old etiolated seedlings was extracted from isolated organelles using discontinuous sucrose density gradient method. Mitochondrial genome was sequenced with Solexa using paired-end, 90 bp read. The clean reads were assembled into contigs using ABySS and finished via additional fosmid and BAC sequencing. Finally, the genome was annotated and analyzed using different softwares.ResultsThe G. barbadense (Sea Island cotton) mitochondrial genome was fully sequenced (677,434-bp) and compared to the mitogenome of upland cotton. The G. barbadense mitochondrial DNA contains seven more genes than that of upland cotton, with a total of 40 protein coding genes (excluding possible pseudogenes), 6 rRNA genes, and 29 tRNA genes. Of these 75 genes, atp1, mttB, nad4, nad9, rrn5, rrn18, and trnD(GTC)-cp were each represented by two identical copies. A single 64 kb repeat was largely responsible for the 9 % difference in genome size between the two mtDNAs. Comparison of genome structures between the two mitochondrial genomes revealed 8 rearranged syntenic regions and several large repeats. The largest repeat was missing from the master chromosome in G. hirsutum. Both mitochondrial genomes contain a duplicated copy of rps3 (rps3-2) in conjunction with a duplication of repeated sequences. Phylogenetic and divergence considerations suggest that a 544-bp fragment of rps3 was transferred to the nuclear genome shortly after divergence of the A- and D- genome diploid cottons.ConclusionThese results highlight the insights to the evolution of structural variation between Sea Island and upland cotton mitochondrial genomes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1988-0) contains supplementary material, which is available to authorized users.

Primary research on abnormal hippocampal synaptic transmission induced in a chronic mild stressed rat model

Objective The differential proteins of hippocampal synapses in depression model rat was analyzed by two-dimensional gel electrophoresis.To provide clues for studies on the synaptic pathogenesis of depression. Methods According to the baseline of sucrose consumption, 58 male Sprague-Dawley rats were divided into Chronic Mild Stress (CMS) and control groups randomly (n=29 per group). After establishment of rat depression model via CMS experimental mode, rat hippocampal synapses from the CMS and control groups were prepared by discontinuous sucrose density gradient centrifugation.Subsequently, the differential proteomics analysis were performed by conventional two-dimensional gel electrophoresis (2DE) and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). Results The results from the sucrose consumption test showed consistent decreases of sucrose intake and preference in the CMS group (P<0.05), suggesting that the depression model rat had been successfully established. Furthermore, by 2DE combined with mass spectrometry analysis (2DE-MS), 16 differentially expressed proteins were obtained, of which 12 were up-regulated and 4 were down-regulated in the chronic mild stressed group as compared with the control group. These proteins were mainly classified into four functional categories including energy metabolism, substance metabolism, cytoskeleton and vesicular transport. Conclusion Differential proteins found in this study may participate in an important mechanism that underlies the abnormal transmission of hippocampal synapses found in the chronic mild stressed rat, thereby providing valuable clues for studies on the pathogenesis of depression. Key words: Depressive disorder; Chronic mild hippocampus; Hippocampus; Synapses; Two-dimensional gel electrophoresis; Proteomics

Annexin A2 mediates apical trafficking of renal Na‐K‐2Cl cotransporter (892.22)

Transport activity of the thick ascending limb (TAL) Na‐K‐2Cl‐cotransporter (NKCC2) critically depends on its association with lipid raft membrane microdomains (LR). The mechanisms involved in the recruitment of NKCC2 into LR have not been elucidated so far. In the present study we hypothesized that the lipid raft scaffolding protein annexin A2 serves as an essential mediator in this process. Detergent‐resistant LR were isolated from rat renal outer medullary tissue samples using discontinuous sucrose density gradient and ultracentrifugation (floating assay). Interaction between NKCC2 and Anxa2 was analyzed by immunoprecipitation, GST pull down, and peptide spot assays. Distribution of the products in the apical TAL membrane was studied by immunofluorescence and immunogold electron microscopy. Trafficking was studied in AVP‐deficient rats and cultured TAL cells using the AVP analogue desmopressin (dDAVP) or low chloride hypotonic stress. Functional relevance of Anxa2 for NKCC2 trafficking was studied using RNAi‐mediated knockdown strategies. Floating assay, immunoprecipitation and colocalization studies confirmed the parallel presence of NKCC2 and Anxa2 in apical membrane LR of the TAL. Binding assays approved physical interaction of Anxa2 and the non‐phosphorylated N‐terminus of NKCC2. Moreover, dDAVP and low chloride hypotonic stress caused parallel trafficking of NKCC2 and Anxa2 in rats and in cultured TAL cells and this response was inhibited by RNAi‐mediated knockdown of Anxa2. In conclusion our data demonstrate that Anxa2 is an essential component of the NKCC2 trafficking protein complex and mediates its recruitment into LR microdomains.

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.

A novel approach to the Langendorff technique: preparation of isolated cardiomyocytes and myocardial samples from the same rat heart

Researchers in biomedical sciences must continually re-evaluate their investment in experiments using laboratory animals. Our group is interested in various signalling pathways that underlie physiological and pathophysiological functioning of the mammalian heart. Two important resources for this type of work are isolated cardiomyocytes and homogenized or preserved sections of whole myocardium. In order to improve our experimental approach ethically, we devised an adaptation of the Langendorff whole-heart retrograde perfusion technique that allows the isolation of adult rat ventricular cardiomyocytes and processing/storage of myocardium from the same heart. This could result in a 50% reduction in the number of animals required for certain experiments. We believe that a generalized adoption of this method would represent a meaningful reduction of animal use in our field of research and, furthermore, strengthen data sets by permitting correlation between myocyte function and various parameters of myocardial biochemistry/structure in individual hearts. This approach is of particular relevance for studies of cardiac pathology, given the cost and time involved in generating experimental disease models.

Caveolin‐1 Supports the P2Y2 Receptor Signaling

Damage to the CNS cells’ can cause a differential spatiotemporal release of multiple factors into the extracellular space. Among such factors, nucleotides, and their interaction with the P2Y2 nucleotide receptors (P2Y2Rs) have gained prominence as putative modulators of gliotic responses after CNS injury. Yet, the molecular mechanisms underlying these interactions and responses remain to be explored. Discontinuous sucrose density gradient separation of 1321N1 cells (expressing recombinant hP2Y2R) homogenates revealed co‐fractionation of P2Y2Rs in cav‐1 (+) membrane‐raft fractions. Likewise, confocal microscopy revealed that a significant percent of P2Y2Rs co‐localized with cav‐1 in its sub‐cellular distribution. Blocking cav‐1 expression in hP2Y2‐1321N1 cells elicited abnormal intracellular Ca2+ mobilization responses and uncharacteristic patterns of AKT, ERK1/2 and p38 phosphorylation levels when stimulated with nucleotide agonists, as determined by microfluorometric calcium imaging analyses and immunoblotting, respectively. Our findings suggest that P2Y2Rs reside in membrane caveolae of non‐stimulated 1321N1 cells and that this sub‐cellular compartment may couple its downstream signaling machinery. We hypothesize that P2Y2Rs’ signaling cascades are linked to their expression in cav‐1 (+) micro‐domains. NAM is supported by the NIGMS‐NIH award number R25GM061838 at the UPR‐MSC.

Phycobilisome composition in Chondrus crispus (Gigartinales, Rhodophyta) from a wild type strain and its vegetatively derived green mutant

Intact phycobilisomes from a wild-type red Chondrus crispus and its vegetatively derived green mutant were isolated by centrifugation through a discontinuous sucrose density gradient. Pigment composition was subsequently characterized by spectrophotometry. Vegetative thalli of the two strains grown together for six months in the laboratory resulted in different pigment profiles. Two pigmented phycobilisome bands appeared in the sucrose gradient of the wild-type alga, a purple coloured one, and a pink one, whereas only a single blue band appeared in the gradient of the green mutant. Spectrophotometric and fluorescence analyses identified the phycobiliprotein composition of the purple band as the typical phycoerythrin-phycocyanin-allophycocyanin complement in the wild-type, but there was no detectable phycoerythrin present in the blue band of the green mutant. Sodium dodecyl sulphate, preparative polyacrylamide gel electrophoresis analysis confirmed the presence of allophycocyanin subunits in all extracts, but firm evidence of an R-phycoerythrin linker polypeptide in the blue band was missing. These results highlight the ability of C. crispus to adapt to a phycoerythrin deficiency by adjusting light harvesting pigment ratios.

αA-Crystallin and αB-Crystallin Reside in Separate Subcellular Compartments in the Developing Ocular Lens

αA-Crystallin (αA) and αB-crystallin (αB), the two prominent members of the small heat shock family of proteins are considered to be two subunits of one multimeric protein, α-crystallin, within the ocular lens. Outside of the ocular lens, however, αA and αB are known to be two independent proteins, with mutually exclusive expression in many tissues. This dichotomous view is buoyed by the high expression of αA and αB in the lens and their co-fractionation from lens extracts as one multimeric entity, α-crystallin. To understand the biological function(s) of each of these two proteins, it is important to investigate the biological basis of this perceived dichotomy; in this report, we address the question whether αA and αB exist as independent proteins in the ocular lens. Discontinuous sucrose density gradient fractionation and immunoconfocal localization reveal that in early developing rat lens αA is a membrane-associated small heat shock protein similar to αB but with remarkable differences. Employing an established protocol, we demonstrate that αB predominantly sediments with rough endoplasmic reticulum, whereas αA fractionates with smooth membranes. These biochemical observations were corroborated with immunogold labeling and transmission electron microscopy. Importantly, in the rat heart also, which does not contain αA, αB fractionates with rough endoplasmic reticulum, suggesting that αA has no influence on the distribution of αB. These data demonstrate presence of αA and αB in two separate subcellular membrane compartments, pointing to their independent existence in the developing ocular lens.

Isolation, Purification and Verification of Peripheral Nerve Myelin Derived from Bovine Cauda Equina

Background: Animal models of peripheral neuroinflammation are essential to understanding the pathogenesis of, and deducing new treatments for demyelinating polyneuropathies. This paper updates and describes adapted methods for the isolation, purification and verification of bovine peripheral nerve myelin used to reliably induce peripheral neuritis in susceptible mice. Methods: Myelin was isolated via discontinuous sucrose and continuous cesium chloride density gradient ultracentrifugation from frozen adult bovine cauda equina. Negatively stained electron microscopy was employed to confirm myelin structure. Phase contrast and routine light microscopy, gravimetric analyses, gel electrophoresis and lens culinaris fluorescent western blotting, analytical colorimetric assays and indirect immunohistochemistry were performed to analyze whole myelin or its protein and lipid subcomponents. Myelin isolates were used to induce experimental autoimmune neuritis in 8-12 Weeks old female Swedish Jim Lambert/ Jackson mice. Results: Negatively-stained electron microscopy demonstrated the two-dimensional repetitive lamella structure of myelin and a lack of organelle contamination. Lipids accounted for ~72% of the dry mass of bovine peripheral nerve myelin, while ~25% was protein/ proteolipid and <4% insoluble residue based on gravimetric analyses. Glycoproteins accounted for the majority of isolated myelin proteins, with myelin protein zero (~28 KDa) being the most prevalent protein. Phospholipids accounted for ~60% of the total dry weight of bovine peripheral nerve myelin. Surface expression of galactocerebroside and sulfatide, two major lipid subcomponents present in myelin, was detected by indirect fluorescent immunohistochemistry. Bovine peripheral nerve myelin reliably induced a severe macrophagepredominant demyelinating polyneuropathy with associated axonal loss in female mice, consistent with experimental autoimmune neuritis. Conclusions: These updated and adapted methods using readily available resources can be applied to rapidly isolate, purify and verify peripheral nerve myelin from animal sources, and determine its protein and lipid composition prior to use in peripheral neuritis animal models.

Comparison of Chloroplast Proteomes Extracted from Florets of Physiological and Genic Male Sterile Lines and Their Maintainer Line in Wheat

The objective of this study was to explain the male sterile mechanism of wheat (Triticum aestivum L.) based on chloroplast proteome. A method for isolating intact chloroplast proteome from wheat floret was established. Using this method, the chloroplast proteomes were extracted from florets of the genic and physiological male sterile lines and their maintainer line, and separated in 2-dimensional electrophoresis (2-DE) gels. The cytoplasmic-nuclear sterile line, ms(S)-1376, had identical nuclear background with the maintainer line, (A)-1376, and the physiological male sterile line, ms(A)-1376, was derived from (A)-1376 after induction of chemical hybridizing agent SQ-1. The extraction method was effective to obtain high purity of intact chloroplast using discontinuous sucrose density gradient centrifugation with 3-step gradient densities of 30, 45, and 60%. The 2-DE result showed that the floret chloroplast protein profiles were different among the 3 lines at uninucleate anther stage, and 239 protein spots were visible on each gel (pH 4-7, molecular weight 14.4-66.2 kD). Six differentially expressed proteins were analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) and indexed in bioinformation database. They were identified as acyl-CoA dehydrogenase domain protein, calmodulin-binding protein phosphatase, multiple catalytic peptidase, heat-shock protein 60, light receptor protein 2, and a protein of unknown function. These proteins are involved in series of physiological reactions such as metabolism of energy substances, chloroplast defendance, chloroplasts signal transduction, and plant growth. The differential expressions of these proteins among the 3 lines are likely related to the male sterility in wheat.

Detergent-free isolation and characterization of cholesterol-rich membrane domains from trans-Golgi network vesicles

Cholesterol is an abundant lipid of the trans-Golgi network (TGN) and of certain endosomal membranes where cholesterol-rich microdomains are important in the organization and compartmentalization of vesicular trafficking. Here we describe the development of a rapid method to isolate a cholesterol-rich endomembrane fraction. We show that widely used subcellular fractionation techniques incompletely separate cholesterol-rich membranes, such as the TGN, from organelles, such as late endosomes and lysosomes. To address this issue, we devised a new subcellular fractionation scheme involving two rounds of velocity centrifugation, membrane sonication, and discontinuous sucrose density gradient centrifugation. This strategy resulted in the isolation of a cholesterol and GM1 glycosphingolipid-enriched membrane fraction that was completely cleared of plasma membrane, endoplasmic reticulum, and mitochondria. This buoyant fraction was enriched for the TGN and recycling endosome proteins Rab11 and syntaxin-6, and it was well resolved from cis-Golgi and early and late endosomal membranes. We demonstrate that this technique can give useful insights into the compartmentation of phosphoinositide synthesis, and it facilitates the isolation of cholesterol-rich membranes from a population of TGN-trafficking vesicles.

Distinct subcellular localization of three isoforms of insulinoma-associated protein 2β in neuroendocrine tissues

Aims Insulinoma-associated protein 2β (IA-2β) is considered to play a significant role in regulated secretion. Recent studies have shown that the mouse brain expresses three major isoforms of IA-2β, named IA-2β60, IA-2β64, and IA-2β71. In this study, we analyzed the tissue-, cell- and organelle-specific distributions of IA-2β isoforms in mice. Main methods To localize IA-2β expression in mouse tissues and cells, western blot and immunohistochemical analyses were carried out. The subcellular distribution of IA-2β isoforms was assessed by sedimentation of mouse brain homogenates in a discontinuous sucrose density gradient. Key findings IA-2β60 was abundant in the cerebrum, cerebellum, medulla oblongata, pancreas, adrenal gland, and pituitary, and in the muscular and mucosal layers of the digestive organs. In contrast, the expression of IA-2β64 and IA-2β71 was restricted to the cerebrum, cerebellum, medulla oblongata, and pituitary, and the muscular layers of the digestive organs. Immunohistochemical analysis of mouse pancreatic islets revealed that pancreatic beta cells expressed IA-2β60 exclusively, whereas alpha and delta cells expressed all three isoforms. By the sedimentation of mouse brain homogenates, it was shown that IA-2β64 and IA-2β71 were co-localized with IA-2 on secretory granules, but were absent from synaptic vesicles (SVs). On the other hand, IA-2β60 was co-localized with synaptophisin on SVs, but was absent from secretory granules. Significance The tissue-, cell- and organelle-specific distributions of IA-2β isoforms suggest that IA-2β60 has a role in secretion from SVs, whereas IA-2β64 and IA-2β71 are involved in secretion from secretory granules.