Structural investigations on betacyanin pigments by LC NMR and 2D NMR spectroscopy

Four betacyanin pigments were analysed by LC NMR and subjected to extensive NMR characterisation after isolation. Previously, low pH values were applied for NMR investigations of betalains resulting in rapid degradation of the purified substances thus preventing extensive NMR studies. Consequently, up to now only one single (13)C NMR spectrum of a betalain pigment, namely that of neobetanin (=14,15-dehydrobetanin), was available. Because of its sufficient stability under highly acidic conditions otherwise detrimental for betacyanins, this pigment remained an exemption. Since betalains are most stable in the pH range of 5-7, a new solvent system has been developed allowing improved data acquisition through improved pigment stability at near neutral pH. Thus, not only (1)H, but for the first time also partial (13)C data of betanin, isobetanin, phyllocactin and hylocerenin isolated from red-purple pitaya [Hylocereus polyrhizus (Weber) Britton & Rose, Cactaceae] could be indirectly obtained by gHSQC- and gHMQC-NMR experiments.     

Production of recombinant protein therapeutics in cultivated mammalian cells

Cultivated mammalian cells have become the dominant system for the production of recombinant proteins for clinical applications because of their capacity for proper protein folding, assembly and post-translational modification. Thus, the quality and efficacy of a protein can be superior when expressed in mammalian cells versus other hosts such as bacteria, plants and yeast. Recently, the productivity of mammalian cells cultivated in bioreactors has reached the gram per liter range in a number of cases, a more than 100-fold yield improvement over titers seen for similar processes in the mid-1980s. This increase in volumetric productivity has resulted mainly from improvements in media composition and process control. Opportunities still exist for improving mammalian cell systems through further advancements in production systems as well as through vector and host cell engineering.     

AliasServer: a web server to handle multiple aliases used to refer to proteins

SUMMARY: AliasServer provides services that facilitate the assembly of data or datasets that make use of different identifiers for refering to the same protein. This resource relies on a database which contains, for a given organism, a non-redundant list of protein sequences associated with a set of aliases. AVAILABILITY: AliasServer is available as an interactive Web server at http://cbi.labri.fr/outils/alias/ and as a web service using a SOAP interface. The complete tool, including sources and data, is available for local installations upon request. SUPPLEMENTARY INFORMATION: Technical documentation is available at http://cbi.labri.fr/outils/alias/asdoc.pdf     

Functional characterization of the postulated intramolecular sphingolipid activator protein domain of human acid sphingomyelinase

Degradation of membrane-bound sphingomyelin to phosphorylcholine and ceramide is catalyzed by the water-soluble lysosomal acid sphingomyelinase (A-SMase). The presence of sphingolipid activator proteins (Saps: saposins A-D; GM2 activator) is not essential to mediate this reaction at the water-lipid interface in vivo . A hypothesis based on amino acid sequence alignments suggests that the enzyme possesses an N-terminal saposin-homologous domain, which may facilitate the enzymatic reaction at the interface. We mutated one homologous and three conserved amino acid residues of this domain and studied the activity of the variant enzymes using different sphingomyelin degradation assays. A variant with an exchange of a conserved amino acid residue, Pro153Ala, still exhibited enzyme activity of approximately 52% of normal in a detergent-containing micellar assay, but only 13% of normal in a detergent-free liposomal assay system, which suggests that the Sap-homologous domain fulfills membrane-disturbing functions. Addition of saposin C to the liposomal assay mixtures increased the Pro153Ala variant sphingomyelinase activity to 46% of normal, indicating that the variant saposin-like domain can be substituted by the presence of the sphingolipid activator protein. On the other hand, the addition of saposin C did not result in complete restoration of the variant activity. Thus, the Sap-like domain may also have another role, e.g., to stabilize the fold of acid sphingomyelinase, which cannot be compensated by the presence of saposin C or a detergent. Such an essential second function of the saposin-like domain as an integral part of acid sphingomyelinase is confirmed by our observation that the Lys118Glu, Cys120Ser and Cys131Ser variants were almost completely devoid of activity in the detergent-containing micellar assay system as well as in the liposomal assay system in the presence of saposin C.     

Effect of Vibrio parahaemolyticus haemolysin on human erythrocytes

Haemolysin Kanagawa, a toxin from Vibrio parahaemolyticus, is known to trigger haemolysis. Flux studies indicated that haemolysin forms a cation channel. In the present study, channel properties were elucidated by patch clamp and functional significance of ion fluxes by fluorescence-activated cell sorting (FACS) analysis. Treatment of human erythrocytes with 1 U ml-1 haemolysin within minutes induces a non-selective cation permeability. Moreover, haemolysin activates clotrimazole-sensitive K+ channels, pointing to stimulation of Ca2+-sensitive Gardos channels. Haemolysin (1 U ml-1) leads within 5 min to slight cell shrinkage, which is reversed in Ca2+-free saline. Erythrocytes treated with haemolysin (0.1 U ml-1) do not undergo significant haemolysis within the first 60 min. Replacement of extracellular Na+ with NMDG+ leads to slight cell shrinkage, which is potentiated by 0.1 U ml-1 haemolysin. According to annexin binding, treatment of erythrocytes with 0.1 U ml-1 haemolysin leads within 30 min to breakdown of phosphatidylserine asymmetry of the cell membrane, a typical feature of erythrocyte apoptosis. The annexin binding is significantly blunted at increased extracellular K+ concentrations and by K+ channel blocker clotrimazole. In conclusion, haemolysin Kanagawa induces cation permeability and activates endogenous Gardos K+ channels. Consequences include breakdown of phosphatidylserine asymmetry, which depends at least partially on cellular loss of K+.     

Evidence for a size-sensing mechanism in animal cells

Continuously proliferating cells exactly double their mass during each cell cycle. Here we have addressed the controversial question of if and how cell size is sensed and regulated. We used erythroblasts that proliferate under the control of a constitutively active oncogene (v-ErbB) or under the control of physiological cytokines (stem cell factor, erythropoietin and v-ErbB inhibitor). The oncogene-driven cells proliferated 1.7 times faster and showed a 1.5-fold increase in cell volume. The two phenotypes could be converted into each other 24 h after altering growth factor signalling. The large cells had a higher rate of protein synthesis, together with a shortened G1 phase. Additional experiments with chicken erythroblasts and mouse fibroblasts, synchronized by centrifugal elutriation, provided further evidence that vertebrate cells can respond to cell size alterations (induced either through different growth factor signalling or DNA synthesis inhibitors) by compensatory shortening of the subsequent G1 phase. Taken together, these data suggest that an active size threshold mechanism exists in G1, which induces adjustment of cell-cycle length in the next cycle, thus ensuring maintenance of a proper balance between growth and proliferation rates in vertebrates.     

ARF6-dependent interaction of the TWIK1 K+ channel with EFA6, a GDP/GTP exchange factor for ARF6

TWIK1 belongs to a family of K(+) channels involved in neuronal excitability and cell volume regulation. Its tissue distribution suggests a role in epithelial potassium transport. Here we show that TWIK1 is expressed in a subapical compartment in renal proximal tubules and in polarized MDCK cells. In nonpolarized cells, this compartment corresponds to pericentriolar recycling endosomes. We identified EFA6, an exchange factor for the small G protein ADP-ribosylation factor 6 (ARF6), as a protein binding to TWIK1. EFA6 interacts with TWIK1 only when it is bound to ARF6. Because ARF6 modulates endocytosis at the apical surface of epithelial cells, the ARF6/EFA6/TWIK1 association is probably important for channel internalization and recycling.     

Characterization of the microheterogeneity of transthyretin in plasma and urine using SELDI-TOF-MS immunoassay

Background  

It has been shown that transthyretin (TTR) exists in different molecular variants. Besides point mutations associated with different diseases such as amyloidosis, other posttranslational modifications occur that might be of diagnostic interest.     

Involvement of putative SNF2 chromatin remodeling protein DRD1 in RNA-directed DNA methylation

In plants, the mechanism by which RNA can induce de novo cytosine methylation of homologous DNA is poorly understood. Cytosines in all sequence contexts become modified in response to RNA signals. Recent work has implicated the de novo DNA methyltransferases (DMTases), DRM1 and DRM2, in establishing RNA-directed methylation of the constitutive nopaline synthase promoter, as well as the DMTase MET1 and the putative histone deacetylase HDA6 in maintaining or enhancing CpG methylation induced by RNA. Despite the identification of enzymes that catalyze epigenetic modifications in response to RNA signals, it is unclear how RNA targets DNA for methylation. A screen for mutants defective in RNA-directed DNA methylation identified a novel putative chromatin-remodeling protein, DRD1. This protein belongs to a previously undefined, plant-specific subfamily of SWI2/SNF2-like proteins most similar to the RAD54/ATRX subfamily. In drd1 mutants, RNA-induced non-CpG methylation is almost eliminated at a target promoter, resulting in reactivation, whereas methylation of centromeric and rDNA repeats is unaffected. Thus, unlike the SNF2-like proteins DDM1/Lsh1 and ATRX, which regulate methylation of repetitive sequences, DRD1 is not a global regulator of cytosine methylation. DRD1 is the first SNF2-like protein implicated in an RNA-guided, epigenetic modification of the genome.