Annette Alfsen

Entry of viruses through the epithelial barrier: pathogenic trickery

Mucosal surfaces — such as the lining of the gut or the reproductive tract — are the main point of entry for viruses into the body. As such, almost all viruses interact with epithelial cells, and make use of the normal epithelial signalling and trafficking pathways of the host cell. In addition to protein receptors, carbohydrate chains of proteoglycans and epithelial-membrane glycosphingolipids have emerged as a new class of receptors for viral attachment to the host cell.

Study of Folch-Pi apoprotein. I. Isolation of two components, aggregation during delipidation.

Abstract The apoprotein of Folch-Pi proteolipid was isolated by delipidation in organic solvents and was transferred into water. At each step of the delipidation, the polypeptide chain composition of the proteolipid was characterized by polycrylamide gel electrophoresis in sodium dodecyl sulfate and simultaneously, the number of free sulfhydryl groups was determined. The electrophoretic pattern of the proteolipid showed two main components of respective molecular weights 25 100 and 20 700, which were isolated by preparative electrophoresis. The amino acid analysis, performed on each of the two components, did not reveal any significant difference. When the apoprotein is completely delipidated in organic solvents, a new component of higher apparent molecular weight is formed which is present in larger amounts in the water solution. This component can be related to other forms of this apoprotein described by different authors.

Study of the apoprotein of Folch-Pi bovine proteolipid. II. Characterization of the components isolated from sodium dodecyl sulfate solutions

Acrylamide gel electrophoresis in dodecyl sulfate solutions of Folch-Pi apoprotein shows several bands. The different components were separated by Biogel P-200 filtration and then reduced and carboxymethylated. A comparative study of the amino acid composition, N-terminal sequence and C-terminal amino acid of the different components led to the assumption that their primary sequences are similar. Evidence for a contamination of the protein by free amino acids might explain the difference in terminal groups found by us and by other groups. It has been shown that the purified components can polymerize independently of S-S bond formation or exchange. The polymerization products were found to resist dissociation by dodecyl sulfate. It has been suggested therefore that the differences in migration rates of the various components are related to their shape rather than to their molecular weight.

Separation of ricin A- and B-chains after dithiothreitol reduction

After complete cleavage of ricin interchain disulfide bridge by 0.05 M dithiothreitol in nondenaturing conditions at 37 degrees C during 1 h 30 min, A- and B-chains were separated on a lactosaminyl-aminoethyl Biogel P-150 column at 4 degrees C, in the presence of 0.01 M dithiothreitol and 0.5 M MgCl2. A- and B-chains have been characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunology. Their specific activities have been tested by protein synthesis inhibition in a cell-free assay (rabbit reticulocyte lysate) and on whole cells (Zajdela hepatoma cells) and by hemagglutination. From these tests, the apparent cross contamination of the chains was about 0.1%.

Dictyostelium discoideum cells shed vesicles with associated DNA and vital stain Hoechst 33342

Abstract.Dictyostelium discoideum cells are highly resis tant to xenobiotics. We previously observed that these primitive eukaryotic cells contain a 170-kDa P-glycoprotein, mediating multidrug resistance in mammalian cells, but nonfunctional in Dictyostelium cells. We show here that D. discoideum cells vitally stained with the DNA-specific dye, Hoechst 33342, release fluorescent material in their culture medium. Electron microscopy and lipid analysis demonstrate the vesicular nature of this material. Moreover, nucleic acids associate with these extracellular vesicles independently of Hoechst vital staining. The main vesicular DNA component exhibits a size >21 kb. Shedding of microvesicles during cell growth is not concomitant with programmed cell death. We propose that these extracellular vesicles are involved in a new cellular resistance mechanism against xenobiotics. Furthermore, since the association of DNA with vesicles occurs in physiological growth conditions and independently of vital staining, the new shedding process might be involved in a more general intercellular mechanism.

Structural studies of the apoprotein of the Folch-Pi bovine brain myelin proteolipid : characterization of the CNBr-fragments and of a long C-terminal sequence.

Abstract The action of cyanogen bromide on the quite insoluble bovine proteolipid apoprotein allowed the determination of four peptide fragments: two of them constituted a 19 amino acid long C-terminal sequence of the apoprotein. Our results were in favour of the existence of only one subunit presenting a molecular weight closely related to 25,000 for which a schematic representation is given.

Organization and dynamics of lipids in bovine brain coated and uncoated vesicles

Three characteristics have been demonstrated by the chemical analysis of bovine brain coated vesicles following removal of the coat proteins: a high protein content, a high cholesterol/lipid ratio and a high percentage of phosphatidylethanolamine amongst the phospholipids.The study of lipid bilayer organization and dynamics has been performed using the fluorescent probes pyrene and parinaric acid (cis and trans). This has allowed the study of both lateral mobility and rotational motion in the lipid bilayer of the coated and uncoated vesicles.Lateral mobility in the fluid phase of the lipid is slightly reduced by the presence of the clathrin coat, as indicated by the lower diffusion coefficient of pyrene in coated compared with uncoated vesicles.At all temperatures from 6° to 30°C, solid-phase domains, probed by trans parinaric acid, coexist with fluid-phase domains in the lipid bilayer. The temperature dependence of the parinaric acid lifetimes and of their amplitudes strongly suggests that the solid phase domains decrease in size with temperature, both in coated and uncoated vesicles.However, the difference in the value of the anisotropy at long times (r∞), between coated and uncoated vesicles (a difference which is more pronounced for cis than for trans parinaric acid), indicates that the presence of the clathrin coat introduces disorder in the surrounding lipids, thus suggesting a possible role of the clathrin in the formation of the pits on the plasma membrane.

Solubilization and separation of Δ5,3β-hydroxysteroid dehydrogenase and 3-oxosteroid-Δ4–Δ5-isomerase from bovine adrenal cortex microsomes

Abstract A physical separation of Δ 5 ,3β-hydroxysteroid dehydrogenase and 3-oxosteroid Δ 4 –Δ 5 -isomerase solubilized from bovine adrenocortical microsomes is described for the first time. The solubilization as well as the separation was carried out with a mixture of a detergent: a substituted betaine (Empigen BB/P) and sodium cholate. This latter detergent protects isomerase from complete inactivation by Empigen and is necessary for the recovery of a significant amount of soluble isomerase. Separation of dehydrogenase and isomerase was successfully accomplished by the use of a DEAE-Biogel A anion-exchanger. Dehydrogenase activity was eluted, while the isomerase was retained. Measurements of dehydrogenase activity with androst-5-en-3β-ol-17-one, pregnen-3β-ol-20-one and pregn-5-en-(3β,17α)-diol-20-one and of isomerase activity with androst-5-en-(3,17)-dione and pregn-5-en-(3,20)-dione suggested that more than one isomerase and more than one dehydrogenase form were present.

Clustering in coated vesicles of polyunsaturated phospholipids segregated from plasma and Golgi membranes of adrenocortical cells

In bovine adrenocortical cells, the fatty acyl chains of the phospholipids have been identified in the membranes of the different cell compartments: plasma membranes, Golgi complex and coated vesicle membranes. An increase in the total number of unsaturation in the fatty acid is demonstrated in the coated vesicle membranes as compared with the plasma and Golgi membranes. Furthermore, it appears that phosphatidylcholine and phosphatidylethanolamine are both enriched in polyunsaturated fatty acyl chains, namely arachidonic and adrenic acids in both types of coated vesicles. Only two of the fatty acids are characteristic of Golgi complex and small coated vesicles, 22:5 (n−6) in PC and 22:6 (n−3) in PE, suggesting that the SCV could originate from the Golgi stacks. A high value of the ratio 22:5 (n−3)/22:6 (n−3) is observed which is, as far as we know, characteristic of adrenal cells.

Studies on Δ5→4-3-oxosteroid isomerases III. Effect of solvent on the enzymatic proton transfer reaction

Abstract The kinetics of the isomerization of different substrates and the binding of inhibitors to Pseudomonas testosteroniΔ5→4-3-oxosteroid isomerase have been determined by the calculation of the parameters, Km, Ki and kcat (the catalytic constant) in mixtures of water and several organic solvents (i.e. methanol, ethanol, propanol and butanol isomers, and dioxane) at low concentrations. The partition coefficient of steroids between isooctane and an aqueous phase containing increasing concentrations of organic solvent has been determined. In order to correlate the free energy change of partition to the free energy change of binding of the same steroid to the enzymatic protein, thermodynamic studies on the different steps of the reaction have been performed with increasing concentrations of alcohol. With the straight chain aliphatic alcohols, the main effect is a decrease in affinity of substrates and inhibitors for the enzyme, and a decrease of kcat with increasing concentrations of the alcohol. The inhibition is competitive only with dioxane, where kcat is unchanged. A linear relationship between the logarithm of the partition coefficient of a given steroid and the logarithm of 1/Km and 1/Ki has been found. With branched alcohols, no change of Km is observed and kcat increases with chain branching. The relation between the kinetic parameters and the dielectric constant (e) of the reaction medium has been studied. The changes in entropy and enthalpy for the activation step with increasing concentrations of organic solvent (methanol and ethanol) suggest the participation of liquid water structure in the enzymatic reaction.