Hydrophilic carotenoids: surface properties and aggregation of an astaxanthin-lysine conjugate, a rigid, long-chain, highly unsaturated and highly water-soluble tetracationic bolaamphiphile

The surface and aggregation properties of a synthetic, highly water-soluble carotenoid, the tetracationic astaxanthin-lysine conjugate (Asly), have been examined through measurements of surface tension, optical absorption and dynamic light scattering. The following parameters were determined: critical aggregation concentration c(M), surface concentration Gamma, molecular area a(m), free energy of adsorption and aggregation (DeltaG(ad) degrees and DeltaG(M) degrees , respectively), and the aggregate size r(H). The compound forms true monomolecular solutions in water below c(M); aggregates emerge only at rather high concentrations (> or =2.18 mM).     

Synthesis of structured lipids and etherlipids with antioxidants: combination of a selena fatty acid and a selena fatty alcohol with a carotenoic acid in glyceride molecules

Selenium and carotenoids show similar and complementary properties and protect against a variety of pathological processes. Mixtures of both compounds are found in nutritional supplements and are used to prevent several diseases. The synthetic connection of carotenoids with selenium in glycerols may increase the chemopreventive activity of the individual compounds. Beta-apo-8'-carotenoic acid and 7-selenacapryloic acid were esterified with glycerol to highly unsaturated stable di- and triglycerides. Intramolecular selenium:carotenoid ratios of 1:1, 2:1 and 1:2 were obtained for 1-(7-selenaoctanoyl)-3-(3beta-apo-8 -carotenoyl)-glycerol, 1,3-di-(beta-apo-8'-carotenoyl)-2-(7-selenaoctanoyl)-glycero l and 1,2-di-(7-selenaoctanoyl)-3-(beta-apo-8'-carotenoyl)-glycero l, respectively. The carotenoic acid was likewise connected to the pharmacologically interesting 11-selenalaurylglycerolether forming an alkyl-acylglyceride: 1-(11-selenadodecyl)-3-(beta-apo-8'-carotenoyl)-glycerol.     

Hydrophilic carotenoids: surface properties and aggregation behavior of a highly unsaturated carotenoid lysophospholipid

The water dispersibility of a hydrophobic carotenoid has been greatly enhanced by using it as the acyl part in the synthesis of a highly unsaturated lysophospholipid. Dynamic light scattering has revealed the formation of stable aggregates with an average hydrodynamic radius of a few nanometers, and absorption spectra show that the aggregates can withstand the addition of ethanol or acetonitrile until the volume fraction of water falls below 70 and 62%, respectively. The properties of the carotenoid phospholipids have been characterized by determining surface tension, critical micelle concentration, surface concentration, molecular area, free energy of adsorption and micellation, adsorption-micellar energy relationship, and equilibrium constants.     

Molecular Characterization of Proteolytic Processing of the Gag Proteins of Human Spumavirus

Spumaviruses, or foamy viruses, express Gag proteins that are incompletely processed by the viral protease in cell cultures. To delineate the proteolytic cleavage sites between potential Gag subdomains, recombinant human spumaretrovirus (HSRV) Gag proteins of different lengths were expressed, purified by affinity chromatography, and subjected to HSRV protease assays. HSRV-specific proteolytic cleavage products were isolated and characterized by Western blotting. Peptides spanning potential cleavage sites, as deduced from the sizes of the proteolytic cleavage products, were chemically synthesized and assayed with HSRV protease. The cleaved peptides were then subjected to mass spectrometry. In control experiments, HSRV protease-deficient mutant proteins were used to rule out unspecific processing by nonviral proteases. The cleavage site junctions identified and the calculated sizes of the cleavage products were in agreement with those of the authentic cleavage products of the HSRV Gag proteins detectable in viral proteins from purified HSRV particles and in virus-infected cells. The biological significance of the data was confirmed by mutational analysis of the cleavage sites in a recombinant Gag protein and in the context of the infectious HSRV DNA provirus.     

Ether lipid biosynthesis: isolation and molecular characterization of human dihydroxyacetonephosphate acyltransferase

In this paper we describe isolation and molecular characterization of human dihydroxyacetonephosphate acyltransferase (DAP-AT). The enzyme was extracted from rabbit Harderian gland peroxisomes and isolated as a trimeric complex by sucrose density gradient centrifugation. From peptide sequences matching EST-clones were obtained which allowed cloning and sequencing of the cDNA from a human cDNA library. The nucleotide-derived amino acid sequence revealed a protein consisting of 680 amino acid residues of molecular mass 77 187 containing a C-terminal type 1 peroxisomal targeting signal. Monospecific antibodies raised against this polypeptide efficiently immunoprecipitated DAP-AT activity from solubilized peroxisomal preparations, thus demonstrating that the cloned cDNA codes for DAP-AT.     

Plakophilin 3 — a novel cell-type-specific desmosomal plaque protein

Desomosomes are cell-cell adhesion structures of epithelia and some non-epithelial tissues, such as heart muscle and the dendritic reticulum of lymph node follicles, which on their cytoplasmic side anchor intermediate filaments at the plasma membrane. Besides clusters of specific transmembrane glycoproteins of the cadherin family (desmogleins and desmocollins), they contain several desmosomal plaque proteins, such as desmoplakins, plakoglobin, and one or more plakophilins. Using recombinant DNA and immunological techniques, we have identified a novel desmosomal plaque protein that is closely related to plakophilins 1 and 2, both members of the "armadillo-repeat" multigene family, and have named it plakophilin 3 (PKP3). The product of the complete human cDNA defines a protein of 797 amino acids, with a calculated molecular weight of 87.081 kDa and an isoelectric point of pH 10.1. Northern blot analysis has shown that PKP3 mRNA has a size of approximately 2.9 kb and is detectable in the total RNA of cells of stratified and single-layered epithelia. With the help of specific poly- and monoclonal antibodies we have localized PKP3, by immunofluorescence or immunoelectron microscopy, to desmosomes of most simple and almost all stratified epithelia and cell lines derived therefrom, with the remarkable exception of hepatocytes and hepatocellular carcinoma cells. We have also determined the structure of the human PKP3 gene and compared it with that of plakophilin 1 (PKP1). Using fluorescence in situ hybridization, we have localized the human genes for the three known plakophilins to the chromosomes 1q32 (PKP1), 12p11 (PKP2) and 11p15 (PKP3). The similarities and differences of the diverse plakophilins are discussed.     

Hydrophilic carotenoids: surface properties and aqueous aggregation of a rigid, long-chain, highly unsaturated dianionic bolaamphiphile with a carotenoid spacer

The water dispersibility of astaxanthin was greatly enhanced by converting it to a disodium disuccinate salt. This carotenoid salt behaved as a bolaamphiphile in water; dynamic light scattering (DLS) revealed the formation of stable aggregates with an average hydrodynamic radius close to 1 microm. Larger aggregates were observed in solutions of increased osmolarity. Absorption spectra demonstrated that the aggregates could withstand the addition of 20% acetonitrile before disintegrating to monomers. The physicochemical properties of this astaxanthin derivative in solution were comprehensively studied by measuring surface tension, critical aggregate concentration, surface concentration, molecule area, free energy of adsorption and micellation, adsorption-aggregate energy relationship, and equilibrium constants, and then compared with similar compounds reported previously in the literature.     

Superlative carotenoids

A selection of carotenoids beyond normal appearance or properties has been presented at the 16(th) International Symposium on Carotenoids. Some of the exceptional compounds shown at the conference cannot be reproduced in this proceeding since they have not yet been published. In addition, editorial space limitation does not allow illustrating all of the previously mentioned carotenoids.     

Rapid restriction mapping of DNA cloned in lambda phage vectors

A protocol for the rapid restriction mapping of phage λ clones has been developed. Partial digestion products are selectively labelled at the right or left cohesive λ DNA termini by hybridisation with [³²P]oligonucleotides complementary to the single-stranded cos ends. After gel electrophoresis and autoradiography, the restriction map can be directly determined from the “ladder” of partial digestion products.