Identification of a PRMT5-dependent repressor complex linked to silencing of human fetal globin gene expression.

Defining the molecular mechanisms underpinning fetal (gamma) globin gene silencing may provide strategies for reactivation of gamma-gene expression, a major therapeutic objective in patients with beta-thalassemia and sickle cell disease (SCD). We have previously demonstrated that symmetric methylation of histone H4 Arginine 3 (H4R3me2s) by the protein arginine methyltransferase PRMT5 is required for recruitment of the DNA methyltransferase DNMT3A to the gamma-promoter, and subsequent DNA methylation and gene silencing. Here we show in an erythroid cell line, and in primary adult erythroid progenitors that PRMT5 induces additional repressive epigenetic marks at the gamma-promoter through the assembly of a multiprotein repressor complex containing the histone modifying enzymes SUV4-20h1, casein kinase 2alpha (CK2alpha), and components of the nucleosome remodeling and histone deacetylation complex.

The Asia Oceania Human Proteome Organisation Membrane Proteomics Initiative. Preparation and characterisation of the carbonate-washed membrane standard.

The Asia Oceania Human Proteome Organisation (AOHUPO) has embarked on a Membrane Proteomics Initiative with goals of systematic comparison of strategies for analysis of membrane proteomes and discovery of membrane proteins. This multilaboratory project is based on the analysis of a subcellular fraction from mouse liver that contains endoplasmic reticulum and other organelles. In this study, we present the strategy used for the preparation and initial characterization of the membrane sample, including validation that the carbonate-washing step enriches for integral and lipid-anchored membrane proteins.

Stabilization of proteins for storage.

Following isolation and purification, it is often necessary to store proteins and peptides for extended periods of time before performing detailed biophysical, enzymatic, and structural proteomics. Therefore, it is essential that the pure target protein maintain its original biological (or functional) behavior over an extended period of storage which may range from weeks to years. Protein pharmaceuticals must remain viable following extensive shipping and storage, and they must remain devoid of all possible inactivation processes.

SYPRO Orange fluorescent staining of protein gels.

This method for staining protein gels uses a fluorescent dye, SYPRO Orange, which is very sensitive to protein (4-10 ng protein/band). SYPRO Orange cannot be visualized with the naked eye and thus a fluorescence scanner is required for detection of protein bands (the excitation maxima [Ex] are 300/472 nm and the emission maximum [Em] is 570 nm). SYPRO Red fluorescent protein stain uses the same binding principle as SYPRO Orange; hence, this protocol can also be used for SYPRO Red stain (Ex are 300/550 nm and Em is 630 nm).

Rapid coomassie blue staining of protein gels.

Coomassie brilliant blue R250 (CBR-250) and silver staining are the most widely used methods for the routine visualization of proteins separated by SDS-PAGE. CBR-250 is an organic dye that complexes with basic amino acids, such as arginine, lysine, and histidine, as well as tyrosine. Conventional CBR-250 staining is capable of detecting as little as 30-100 ng of protein, but sensitivity can be improved by performing the staining and destaining at elevated temperatures.

CTAB-PAGE.

Although SDS-PAGE is the method of choice for most denaturing gel electrophoresis procedures, the anionic detergent SDS still presents some drawbacks. For example, SDS forms crystals at low temperatures and, in some cases, causes proteins to aggregate or precipitate. In addition, some proteins are not well-resolved in SDS gels or may migrate anomalously.

Pouring linear gradient gels with a gradient former.

Linear gradient gels, which are polyacrylamide gels having a gradient of increasing acrylamide concentration (and linearly decreasing pore size), can have advantages over fixed-concentration SDS-PAGE gels. First, a much greater range of protein M(r) values can be separated on a linear gradient gel than on a fixed-concentration gel. Second, there is a greater likelihood of resolving proteins with very similar M(r) values on gradient gels than on fixed-concentration gels.

An aspartyl protease directs malaria effector proteins to the host cell.

Plasmodium falciparum causes the virulent form of malaria and disease manifestations are linked to growth inside infected erythrocytes. To survive and evade host responses the parasite remodels the erythrocyte by exporting several hundred effector proteins beyond the surrounding parasitophorous vacuole membrane. A feature of exported proteins is a pentameric motif (RxLxE/Q/D) that is a substrate for an unknown protease.

Longitudinal, Diet-induced Weight Gain is Associated with Increased Blood Monocytes and Reduced TLR4 Expression.

Excessive weight gain increases systemic inflammation resulting in increased disease risk. Toll-like receptor 4 (TLR4) reportedly mediates increases in inflammation; however, its role in obesity-induced inflammation has not been fully evaluated. The purpose of this study was to determine the longitudinal effect of diet-induced weight gain on blood monocyte concentration and cell-surface TLR4 expression.

Coupling aging immunity with a sedentary lifestyle: has the damage already been done?--a mini-review.

The elderly population is at an unprecedented risk of infectious diseases and malignancy due to apparently inevitable age-related declines in immunity. The 'immune risk profile' (IRP) is an array of biomarkers that has been used to predict morbidity and mortality in older adults. As it is generally accepted that middle-aged and elderly individuals who habitually participate in moderate-intensity exercise are less likely to incur an infection than their sedentary counterparts, this review addresses current knowledge on the effects of regular exercise on aspects of adaptive immunity as they relate to the IRP.

Extracellular remodelling during oncogenic Ras-induced epithelial-mesenchymal transition facilitates MDCK cell migration.

Epithelial-mesenchymal transition (EMT) describes a process whereby immotile epithelial cells escape structural constraints imposed by cellular architecture and acquire a phenotype characteristic of migratory mesenchymal cells. Implicated in carcinoma progression and metastasis, EMT has been the focus of several recent proteomics-based studies aimed at identifying new molecular players. To gain insights into extracellular mediators associated with EMT, we conducted an extensive proteomic analysis of the secretome from MDCK cells following oncogenic Ras-induced EMT (21D1 cells).

Proteomics analysis of A33 immunoaffinity-purified exosomes released from the human colon tumor cell line LIM1215 reveals a tissue-specific protein signature.

Exosomes are 40-100-nm-diameter nanovesicles of endocytic origin that are released from diverse cell types. To better understand the biological role of exosomes and to avoid confounding data arising from proteinaceous contaminants, it is important to work with highly purified material. Here, we describe an immunoaffinity capture method using the colon epithelial cell-specific A33 antibody to purify colorectal cancer cell (LIM1215)-derived exosomes.

ExoCarta: A compendium of exosomal proteins and RNA.

Exosomes, membrane microvesicles (40-100 nm) secreted by most cell types, can be isolated in several ways while characterizing them is heavily based on electron microscopy and, most importantly, the identification of exosome marker proteins. Researchers rely on the identification of certain exosomal marker proteins including Alix, CD9 and CD63 to confirm the presence of exosomes in their preparations. An evolutionary-conserved set of protein molecules have been identified in most exosomes studied to date.

A centrifugal ultrafiltration strategy for isolating the low-molecular weight (

The low-molecular weight fraction (LMF) of the human plasma proteome is an invaluable source of biological information, especially in the context of identifying plasma-based biomarkers of disease. In this study, a separation and enrichment strategy based on centrifugal ultrafiltration was developed for the LMF (i.e.

International blood collection and storage: clinical use of blood products.

Human blood transfusion is the process of transferring blood or blood-based products from an individual into the circulatory system of another. From the theory of circulation of blood to the early practice of blood transfusion, transfusion medicine has been an important concept for many centuries. The practicality of transfusion, however, only became a possibility during and shortly after the Second World War.

Transgenic barley (Hordeum vulgare L.) expressing the wheat aluminium resistance gene (TaALMT1) shows enhanced phosphorus nutrition and grain production when grown on an acid soil.

Barley (Hordeum vulgare L.), genetically modified with the Al(3+) resistance gene of wheat (TaALMT1), was compared with a non-transformed sibling line when grown on an acidic and highly phosphate-fixing ferrosol supplied with a range of phosphorus concentrations. In short-term pot trials (26 days), transgenic barley expressing TaALMT1 (GP-ALMT1) was more efficient than a non-transformed sibling line (GP) at taking up phosphorus on acid soil, but the genotypes did not differ when the soil was limed.

Exosomes: proteomic insights and diagnostic potential.

Exosomes are 40-100-nm diameter membrane vesicles of endocytic origin that are released by most cell types upon fusion of multivesicular bodies with the plasma membrane, presumably as a vehicle for cell-free intercellular communication. While early studies focused on their secretion from diverse cell types in vitro, exosomes have now been identified in body fluids such as urine, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluid, breast milk, saliva and blood. Exosomes have pleiotropic biological functions, including immune response, antigen presentation, intracellular communication and the transfer of RNA and proteins.

Towards understanding epithelial-mesenchymal transition: a proteomics perspective.

Epithelial-mesenchymal transition (EMT) is recognised as a crucial embryonic process that converts immotile epithelial cells into migratory mesenchymal cells. More recently, EMT has been implicated in key stages of tumour progression, whereby it facilitates epithelial cancer cell metastasis. Traditionally, molecular and cell biology-based approaches have been utilised to study EMT.

Secretome-based proteomics reveals sulindac-modulated proteins released from colon cancer cells.

Although experiments in rodents and human population-based studies have demonstrated the efficacy of nonsteroidal anti-inflammatory drugs (NSAIDs) such as sulindac in colorectal cancer (CRC) prevention, a detailed knowledge of the underlying mechanism of action of this drug is limited. To better understand the chemopreventitive effects of sulindac, especially early sulindac-induced apoptotic events, we used the CRC cell line LIM1215 as an experimental model, focusing on proteins secreted into the LIM1215 culture medium - i.e.

Secretome-based proteomic profiling of Ras-transformed MDCK cells reveals extracellular modulators of epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is a highly conserved morphogenetic process by which epithelial cells lose their basic morphological characteristics such as cell-cell contact and gain mesenchymal properties such as increased motility and invasiveness. To gain insights into proteins released from cells that modulate the EMT process, we compared secretome protein expression profiles of MDCK cells and Ras-transformed MDCK cells (21D1) that stably express oncogenic Ras using 2D-DIGE/LC-MS/MS. Differentially expressed secretome proteins were compared with their corresponding gene expression profiles using the Affymetrix GeneChip system.

PRMT5-mediated methylation of histone H4R3 recruits DNMT3A, coupling histone and DNA methylation in gene silencing.

Mammalian gene silencing is established through methylation of histones and DNA, although the order in which these modifications occur remains contentious. Using the human beta-globin locus as a model, we demonstrate that symmetric methylation of histone H4 arginine 3 (H4R3me2s) by the protein arginine methyltransferase PRMT5 is required for subsequent DNA methylation. H4R3me2s serves as a direct binding target for the DNA methyltransferase DNMT3A, which interacts through the ADD domain containing the PHD motif.

Enrichment of human platelet membranes for proteomic analysis.

Platelets (thrombocytes) are the smallest human blood cells and are pivotal in processes of hemostasis and thrombosis. Central to their function, the activation of platelets includes a complex interplay of adhesion and signalling molecules mediated via the plasma and inner membrane. Because platelets are enucleated, the analysis of the proteome is the best way to approach their biology.

Isolation of extracellular membranous vesicles for proteomic analysis.

Membranous vesicles are constitutively released by a multitude of cell types. Following fusion of multivesicular bodies with the plasma membrane, endocytic vesicles, 30-90 nm in size termed exosomes are released extracellularly. Whilst several groups have reported the presence of exosomes in cell-culture conditioned medium, their biological and physiological functions still remain unclear.

Purification of basolateral integral membrane proteins by cationic colloidal silica-based apical membrane subtraction.

Epithelial cell polarity mediates many essential biological functions and perturbation of the apical/basolateral divide is a hallmark of epithelial to mesenchymal transition in carcinoma. Therefore, correct targeting of proteins to the apical and basolateral surfaces is essential to proper epithelial cell function. However, proteomic characterisation of apical/basolateral sorting has been largely ignored, due to ineffectual separation techniques and contamination of plasma-membrane preparations with housekeeping proteins.

Olig2-induced neural stem cell differentiation involves downregulation of Wnt signaling and induction of Dickkopf-1 expression.

Understanding stem cell-differentiation at the molecular level is important for clinical applications of stem cells and for finding new therapeutic approaches in the context of cancer stem cells. To investigate genome-wide changes involved in differentiation, we have used immortalized neural stem cell (NSC) line (HB1.F3) and Olig2-induced NSC differentiation model (F3.