Binding of ferritin-lectin conjugates to C-type virus in intact cells.

Ferritin-Ricin II and Ferritin-Concanavalin A bound to budding as well as mature C-type viral particles. No differencies in binding between the viral coat and adjacent plasma membrane were detected with either lectin conjugate. Aggregation of viral particles by lectin conjugates was observed, and linking of virus to the plasma membrane resulted in phagocytosis of viral particles.     

Capping of concanavalin A- or ricin-binding sites does not influence phagocytosis in polymorphonuclear leukocytes

Summary Rabbit polymorphonuclear leukocytes (PMNs) were capped with territin-conjugated concanavalin A or ricin, and then allowed to phagocytose yeast cells. Phagocytic activity and lectin distribution were determined by ultrastructural morphometry. Capped PMNs were found to phagocytose as efficiently as control PMNs, and always to ingest the particles with a lectin-free portion of their plasma membrane. This clearly indicates that concanavalin A- and ricin-binding sites of the PMN membrane are not involved in the recognition and phagocytosis of yeast particles.     

Visualisation of lectin binding sites on the surface of human platelets using lectins adsorbed to gold granules

Summary Washed human platelets have been incubated with the lectins WGA, ConA and RCA₁, adsorbed to differentsized gold particles. Plasma membrane receptors for each lectin were then located by scanning and transmission electron microscopy.Acknowledgments. We would like to thank Mlle Dominique Dupuis for technical assistance. This work was supported in part by grant No. CRL 78.5.128.1 from INSERM.     

Cell penetrating peptides: overview and applications to the delivery of oligonucleotides

Crossing biological barriers represents a major limitation for clinical applications of biomolecules such as nucleic acids, peptides or proteins. Cell penetrating peptides (CPP), also named protein transduction domains, comprise short and usually basic amino acids-rich peptides originating from proteins able to cross biological barriers, such as the viral Tat protein, or are rationally designed. They have emerged as a new class of non-viral vectors allowing the delivery of various biomolecules across biological barriers from low molecular weight drugs to nanosized particles. Encouraging data with CPP-conjugated oligonucleotides have been obtained both in vitro and in vivo in animal models of diseases such as Duchenne muscular dystrophy. Whether CPP-cargo conjugates enter cells by direct translocation across the plasma membrane or by endocytosis remains controversial. In many instances, however, endosomal escape appears as a major limitation of this new delivery strategy.     

Patterns of membrane organization in toad bladder epithelium: A freeze-fracture study

Summary Two cell-types of toad bladder epithelium show uncommon plasma membrane organization in freeze-fractured specimens. One type, the granular cell, contains a plasma membrane in which the A-face is poorly particulate luminally while the B-face discloses multiple large particles at this site. In contrast, the lateral and basal portions of the granular-cell membrane are typical in that more particles occupy the A-face than the B-face. In the other cell-type, which is mitochondriarich, the plasma membrane, luminally, laterally, and basally, contains rod-shaped and a few glubular particles in the A-face. We suggest that these two peculiar membrane organizations by considered in the localization of both vasopressin and aldosterone action in toad bladder.Some of these results were presented in abstract form at the 6th annual meeting of the Swiss Societies for Experimental Biology, Lausanne, 11–12, May 1974²¹ The authors are indebted to Mrs.Marthe Sidler-Ansermet, Mrs.Gorana Perrelet, MissMariella Ravazzola and Mr.Michel Bernard for valuable assistance.This work was supported in part by grants No. 3.0311.73, 3.8081.72, 3.553.75 and 3.1300.73 from Fonds National Suisse de la Recherche Scientifique, Bern, Switzerland, and by a grant-in-aid from Hoechst-Pharmaceuticals (Frankfurt-Hoechst, W. Germany).     

Presence of oncornavirus-like particles in the P388 murine leukemic cell line

A culture of P388 murine lymphoblastoid cells has been shown to contain type C oncornavirus-like particles budding at the plasma membrane. Occasionally intracytoplasmic type A and immature type B particles were also observed by electron microscope techniques. The discovery of oncornavirus-like particles in the P388 cell line increases the utility of this neoplastic system for detecting potential antineoplastic agents.     

Disruption of clc-5 leads to a redistribution of annexin A2 and promotes calcium crystal agglomeration in collecting duct epithelial cells

Mutations in CLCN5, which encodes the voltage-dependent Cl⁻/H⁺antiporter, CLC-5, cause Dent’s disease. This disorder is characterized by low molecularweight proteinuria, hypercalciuria, nephrocalcinosis and nephrolithiasis. Using a collecting duct cell model (mIMCD-3) in which endogenous clc-5 is disrupted by antisense clc-5 or overexpression of truncated clc-5, we demonstrate altered expression of the crystal adhesion molecule, annexin A2. Endogenously expressed annexin A2 is intracellular with limited plasma membrane localization. Following clc-5 disruption, there is both a marked increase in plasma membrane annexin A2 and an increase in cell surface crystal retention and agglomeration, which may be attenuated using pretreatment with anti-annexin A2 antibodies or wheat germ agglutinin lectin but not by concanavalin A. We hypothesize that in Dent’s disease, endocytic failure leads to an accumulation at the plasma membrane of crystal-binding molecules that include annexin A2 leading to retention of calcium crystals and ultimately nephrocalcinosis and nephrolithiasis. Received 22 October 2005; received after revision 26 November 2005; accepted 2 December 2005     

Ultrastructure of the particles reconstituted by complementation of extracts from chl-r mutants of Escherichia coli K 12]

By freeze-fracturing it is shown that the vesicles reconstituted by complementation of the chlA and chlB mutants of E. coli K 12 extracts are characterized by an asymmetric membrane bilayer. In a feature quite similar to the original intact plasma membranes, the membrane splits in two halves and the intramembranous particles are asymmetrically distributed on the two facture faces. It is proposed that the process of membrane reconstitution, which is also associated with the restoration of nitrate-reductase activity, relies on a sequence of increasing complexity of the molecular organisation.     

Detailed analysis of the plasma extracellular vesicle proteome after separation from lipoproteins

The isolation of extracellular vesicles (EVs) from blood is of great importance to understand the biological role of circulating EVs and to develop EVs as biomarkers of disease. Due to the concurrent presence of lipoprotein particles, however, blood is one of the most difficult body fluids to isolate EVs from. The aim of this study was to develop a robust method to isolate and characterise EVs from blood with minimal contamination by plasma proteins and lipoprotein particles. Plasma and serum were collected from healthy subjects, and EVs were isolated by size-exclusion chromatography (SEC), with most particles being present in fractions 8–12, while the bulk of the plasma proteins was present in fractions 11–28. Vesicle markers peaked in fractions 7–11; however, the same fractions also contained lipoprotein particles. The purity of EVs was improved by combining a density cushion with SEC to further separate lipoprotein particles from the vesicles, which reduced the contamination of lipoprotein particles by 100-fold. Using this novel isolation procedure, a total of 1187 proteins were identified in plasma EVs by mass spectrometry, of which several proteins are known as EV-associated proteins but have hitherto not been identified in the previous proteomic studies of plasma EVs. This study shows that SEC alone is unable to completely separate plasma EVs from lipoprotein particles. However, combining SEC with a density cushion significantly improved the separation of EVs from lipoproteins and allowed for a detailed analysis of the proteome of plasma EVs, thus making blood a viable source for EV biomarker discovery.     

Presence of oncornavirus-like particles in the P388 murine leukemic cell line

Summary A culture of P388 murine lymphoblastoid cells has been shown to contain type C oncornavirus-like particles budding at the plasma membrane. Occasionally intracytoplasmic type A and immature type B particles were also observed by electron microscope techniques. The discovery of oncornavirus-like particles in the P388 cell line increases the utility of this neoplastic system for detecting potential antineoplastic agents.Supported by Contract NO1-CM-67048 from the Division of Cancer Treatment (DCT), National Cancer Institute (NCI), National Institutes of Health, Department of Health, Education, and Welfare. The fannie E. Rippel Foundation, Talley Industries, and the Phoenix Coca Cola Bottling Co. We are also grateful to Miss Linda M. Lange for technical assistance.     

Biomedicine and Diseases: Review¶Assembling the human immunodeficiency virus type 1

Retroviral assembly proceeds through a series of concerted events that lead to the formation and release of infectious virion particles from the infected cell. Upon translation, structural proteins are targeted to the plasma membrane where they accumulate. There, the nascent particle forces the plasma membrane to form a bud, which pinches off releasing the virion particle from the cell. In this review we describe the molecular mechanisms now known to be behind the process of virion assembly. In particular, we focus on the human immunodeficiency virus type 1, the prototype member of the lentivirus subfamily of the Retroviridae.     

Compared expression of murine plasmacytoma associated virus in tumor cells and in mouse embryo cells]

A DNA complementary to the viral genome of C-type particles produced by a Mouse myeloma derived cell line (MF2 cell line) was synthesized. This cDNA was used as a probe to study the viral genome expression among the total RNA and the poly (A)-rich RNA extracted from the MF2 and Balb/c embryonic cells. As evidenced by molecular hybridization experiments, the presence of at least one endogenous Balb/c virus in the MF2 virus stocks is suggested. In the productive cells, the viral RNA sequences are expressed in the poly (A)-rich RNA fraction.     

Application of immunoelectron microscopy to the detection of viruses in a water medium]

Adenoviruses were immersed in demineralyzed and deionized water for 5 days. The water was subsequently analyzed by Immuno-Electron-Microscopy for detection of viral particles. An attempt of quantation was also made on control, untreated particles. Viruses dispersed in water can be detected by the technique employed but quantitation is limited by the heterogeneous dispersion of the particles on the grids.     

Dynamics of ESCRT proteins

Proteins of the ESCRT (endosomal sorting complex required for transport) complex function in membrane fission processes, such as multivesicular body (MVBs) formation, the terminal stages of cytokinesis, and separation of enveloped viruses from the plasma membrane. In mammalian cells, the machinery consists of a network of more than 20?proteins, organized into three complexes (ESCRT-I, -II, and -III), and other associated proteins such as the ATPase vacuolar protein sorting 4 (Vps4). Early biochemical studies of MVBs biogenesis in yeast support a model of sequential recruitment of ESCRT complexes on membranes. Live-cell imaging of ESCRT protein dynamics during viral budding and cytokinesis now reveal that this long-standing model of sequential assembly and disassembly holds true in mammalian cells.     

Degradation by phytohemagglutinin of the antiviral state induced by interferon]

Phytohaemagglutinin (PHA M and P forms), when added to L 929 cells previously treated by murine interferon, degrades the antiviral state and restores virus sensitivity in the cells. This degradation depends on the amount of the lectin, the contact period with the cell and the presence of PHA membrane receptors. The importance of membrane configuration not only in the induction but also in the maintenance of the antiviral state is discussed.     

Thiamine-binding activity of Saccharomyces cerevisiae plasma membrane

The specific binding activity to [14C]thiamine was found to be located in hte plasma membrane of Saccharomyces cerevisiae. The activity was inhibited by several thiamine analogs and it was hardly detectable in the plasma membrane from a thiamine transport mutant of Saccharomyces cerevisiae. Some properties of the thiamine-binding activity of yeast plasma membrane are discussed in connection with those of the thiamine transport system.     

Increased activity of dipeptidyl peptidase IV in serum of hepatoma-bearing rats coincides with the loss of the enzyme from the hepatoma plasma membrane

The specific activity of dipeptidyl peptidase IV (DPP IV E.C. 3.4.14.-) in the plasma membrane of Morris hepatoma 9121 or hepatoma 7777 was 3.5% and 2.9%, respectively, of that in the plasma membrane of rat liver. The enzyme activity in the serum of hepatoma-bearing rats was 141% (hepatoma 91219) and 162% (hepatoma 7777) of the normal value. Cytochemical investigation showed that the DPP IV activity was almost completely absent from the hepatoma cell plasma membrane and was not sequestered within these cells. Indirect immunofluorescence staining with a polyclonal antibody directed against DPP IV indicated that the loss of activity was due to the absence of DPP IV molecules in the plasma membrane. The possibility that the enzyme is transferred from the membrane into the serum as a result of structural alterations is discussed.     

Signal perception in plant pathogen defense

Highly sensitive and specific recognition systems for microbial pathogens are essential for disease resistance in plants. Structurally diverse elicitors from various pathogens have been identified and shown to trigger plant defense mechanisms. Elicitor recognition by the plant is assumed to be mediated by receptors. Plant receptors for fungus-derived elicitors appear to reside preferentially in the plasma membrane, whereas viral and bacterial elicitors may enter the plant cell and are perceived intracellularly. Receptor activation initiates an intracellular signal transduction cascade leading to stimulation of a characteristic set of plant defense responses. Isolation of plant elicitor receptors and their encoding genes is expected to provide significant information on the molecular basis of signal perception and intracellular signal generation in plant-pathogen interactions.     

Increased activity of dipeptidyl peptidase IV in serum of hepatoma-bearing rats coincides with the loss of the enzyme from the hepatoma plasma membrane

Summary The specific activity of dipeptidyl peptidase IV (DPPIV E.C. 3.4.14.-) in the plasma membrane of Morris hepatoma 9121 or hepatoma 7777 was 3.5% and 2.9%, respectively, of that in the plasma membrane of rat liver. The enzyme activity in the serum of hepatoma-bearing rats was 141% (hepatoma 91219) and 162% (hepatoma 7777) of the normal value. cytochemical investigation showed that the DPP IV activity was almost completely absent from the hepatoma cell plasma membrane and was not sequestered within these cells. Indirect immunofluorescence staining with a polyclonal antibody directed against DPP IV indicated that the loss of activity was due to the absence of DPP IV molecules in the plasma membrane. The possibility that the enzyme is transferred from the membrane into the serum as a result of structural alterations is discussed.     

Thiamine-binding activity ofSaccharomyces cerevisiae plasma membrane

Summary The specific binding activity to [¹⁴C]thiamine was found to be located in the plasma membrane ofSaccharomyces cerevisiae. The activity was inhibited by several thiamine analogs and it was hardly detectable in the plasma membrane from a thiamine transport mutant ofSaccharomyces cerevisiae. Some properties of the thiamine-binding activity of yeast plasma membrane are discussed in connection with those of the thiamine transport system.