Curvature of double-membrane organelles generated by changes in membrane size and composition

Transient double-membrane organelles are key players in cellular processes such as autophagy, reproduction, and viral infection. These organelles are formed by the bending and closure of flat, double-membrane sheets. Proteins are believed to be important in these morphological transitions but the underlying mechanism of curvature generation is poorly understood. Here, we describe a novel mechanism for this curvature generation which depends primarily on three membrane properties: the lateral size of the double-membrane sheets, the molecular composition of their highly curved rims, and a possible asymmetry between the two flat faces of the sheets. This mechanism is evolutionary advantageous since it does not require active processes and is readily available even when resources within the cell are restricted as during starvation, which can induce autophagy and sporulation. We identify pathways for protein-assisted regulation of curvature generation, organelle size, direction of bending, and morphology. Our theory also provides a mechanism for the stabilization of large double-membrane sheet-like structures found in the endoplasmic reticulum and in the Golgi cisternae.     

Polysaccharide elicitors enhance anthocyanin and phenolic acid accumulation in cell suspension cultures of <Emphasis Type="Italic">Vitis vinifera</Emphasis>

The effects of yeast extract and selected polysaccharide elicitors on secondary metabolite production, particularly of anthocyanin and phenolic acid, in cell suspension cultures of Vitis vinifera were investigated. All elicitors either maintained or promoted cell growth in culture. Overall, secondary metabolite production in V. vinifera cell suspension cultures responded differently to different elicitors. Chitosan, pectin, and alginate enhanced production of anthocyanin within 13 days of culture with levels of 2.5-, 2.5-, and 2.6-fold increase, respectively, over that of control. Chitosan, alginate, and gum arabic significantly promoted accumulation of phenolic acids, particularly 3-O-glucosyl-resveratrol, in V. vinifera cultures, as well as in the culture medium. Intracellular phenolic acid production was significantly enhanced by alginate and chitosan, with 1.7- and 1.5-fold levels, respectively, of that of control. Extracellular phenolic acid production was also significantly increased in the presence of chitosan and gum arabic, with levels of 3.3- and 1.7-fold higher, respectively, than those of control. In addition, DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity was enhanced in the presence of elicitors, and this was positively correlated with increased accumulation of anthocyanin in V. vinifera cell suspension cultures.     

Predictive Model for Inactivation of Feline Calicivirus, a Norovirus Surrogate, by Heat and High Hydrostatic Pressure

Noroviruses, which are members of the Caliciviridae family, represent the leading cause of nonbacterial gastroenteritis in developed countries; such norovirus infections result in high economic costs for health protection. Person-to-person contact, contaminated water, and foods, especially raw shellfish, vegetables, and fruits, can transmit noroviruses. We inactivated feline calicivirus, a surrogate for the nonculturable norovirus, in cell culture medium and mineral water by heat and high hydrostatic pressure. Incubation at ambient pressure and 75°C for 2 min as well as treatment at 450 MPa and 15°C for 1 min inactivated more than 7 log₁₀ PFU of calicivirus per ml in cell culture medium or mineral water. The heat and pressure time-inactivation curves obtained with the calicivirus showed tailing in the logarithmic scale. Modeling by nth-order kinetics of the virus inactivation was successful in predicting the inactivation of the infective virus particles. The developed model enables the prediction of the calicivirus reduction in response to pressures up to 500 MPa, temperatures ranging from 5 to 75°C, and various treatment times. We suggest high pressure for processing of foods to reduce the health threat posed by noroviruses.     

Rapid activation of human platelets by low concentrations of low-density lipoprotein via phosphatidylinositol cycle

The interaction of low-density lipoprotein (LDL) with the human platelet was investigated with regard to saturable high-affinity binding, shape change, cytosolic free Ca2+ concentration, phosphatidylinositol (PtdIns) turnover, and thromboxane B2 biosynthesis. The experiments show that LDL, at a concentration approximately 100 times lower than in plasma, causes platelet activation concomitantly with stimulation of the PtdIns cycle and thromboxane B2 formation, similarly to other activators of platelets. The effects of LDL were inhibited by high-density lipoprotein. The results suggest that activation of platelets by low concentrations of LDL may play a role in pathophysiological conditions and that platelet can serve as a model for studying the influence of LDL on various target cells.     

Effect of High-Pressure-Induced Ice I-to-Ice III Phase Transitions on Inactivation of Listeria innocua in Frozen Suspension

The inactivation of Listeria innocua BGA 3532 at subzero temperatures and pressures up to 400 MPa in buffer solution was studied to examine the impact of high-pressure treatments on bacteria in frozen matrices. The state of aggregation of water was taken into account. The inactivation was progressing rapidly during pressure holding under liquid conditions, whereas in the ice phases, extended pressure holding times had comparatively little effect. The transient phase change of ice I to other ice polymorphs (ice II or ice III) during pressure cycles above 200 MPa resulted in an inactivation of about 3 log cycles, probably due to the mechanical stress associated with the phase transition. This effect was independent of the applied pressure holding time. Flow cytometric analyses supported the assumption of different mechanisms of inactivation of L. innocua in the liquid phase and ice I (large fraction of sublethally damaged cells due to pressure inactivation) in contrast to cells subjected to ice I-to-ice III phase transitions (complete inactivation due to cell rupture). Possible applications of high-pressure-induced phase transitions include cell disintegration for the recovery of intracellular components and inactivation of microorganisms in frozen food.     

Stoichiometric noncovalent interaction in molecular imprinting

In this review article the function of the binding site monomers in the molecular imprinting procedure is discussed. Especially, new developments towards stoichiometric noncovalent interactions are highlighted. In stoichiometric noncovalent interactions template and binding site monomer in an 1:1 molar ratio are nearly completely bound to each other. This is only possible if the association constants are considerably high (K _ass < 900 M^–1). Using this type of interaction in molecular imprinting no excess of binding sites is necessary and binding sites are only located inside the imprinted cavity. Since all cavities can be reloaded these polymers show high capacity (e.g., for preparative application) and are especially suited for the synthesis of catalytically active imprinted polymers. Discussed are binding site interactions based on amidines (and guanidines), multiple hydrogen bonding, charge-transfer interactions, and host–guest inclusion. The systematic investigation of the underlying binding reaction is described in detail. With low-molecular weight model substances the thermodynamics of the association can be conveniently investigated, e.g., by NMR spectroscopy.     

Application of AFLP markers to genome mapping in poultry

The amplified fragment length polymorphism (AFLP) technique has been used to enhance marker density in the East Lansing reference chicken genome map, using a backcross family derived from a Red Jungle Fowl by White Leghorn mating with White Leghorn as the recurrent parent. To date, 204 AFLP markers have been added, expanding overall map coverage by about 25%. To the limits of our resolution, AFLP markers are distributed relatively evenly across the EL reference map. AFLP are about 60% as frequent in a cross within White Leghorns (line 7(2) x 6(3)) in comparison to the more divergent reference map population. Based on apparent identity of size, about 40% of the 7(2) x 6(3) cross AFLP fragments were also polymorphic in the reference map cross. Primer pairs in which one primer contains 3' extensions of three selective nucleotides and the other has two selective nucleotides successfully generated AFLP from chicken DNA, but such pairs appeared to amplify only a subset of those fragments to which they have an exact sequence match. Three different restriction enzymes with 4 bp recognition sites (TaqI, HinP1I and MspI) were found to work well with EcoRI as the rarer of the two AFLP restriction enzymes used, with HinP1I being the most effective of the three. AFLP markers are likely to provide an economical method with which to enhance framework linkage maps of chicken and probably other avian genomes.     

Sequence analysis of the equine SLC26A2 gene locus on chromosome 14q15-->q21

The solute carrier family 26, member 2 (SLC26A2) gene belongs to a family of multifunctional anion exchangers. Mutations in the human SLC26A2 gene are associated with autosomal recessively inherited chondrodysplasias. Hence, we postulate that the equine SLC26A2 could be a candidate gene for conformational traits in horses. An equine BAC clone harboring the SLC26A2 gene was isolated. The complete 142,625 bp insert sequence of this clone was determined by transposon sequencing. Together with the SLC26A2 gene the BAC clone contains four genes, i.e. the macrophage colony stimulating factor 1 receptor precursor (CSF1R), KIAA0194 protein gene similar to the SMF protein (KIAA0194), a tigger transposable element derived 14 (TIGD14), the 3'-5'-cyclic GMP phosphodiesterase alpha-chain (EC 3.1.4.35) and one unidentified open reading frame. The equine SLC26A2 gene encompassing 6,152 bp consists of two exons. The complete open reading frame of 2,211 bp encodes a protein of 736 amino acids. A comparison of the amino acid sequence with other mammalian orthologs revealed homologies with identity in a range between 80% and 88%. By contrast, the equine SLC26A2 protein lacks five C-terminal amino acids. Four single nucleotide polymorphisms (SNP) were identified (three synonymous and one non-synonymous variant Ser210Leu) in the coding region by comparative sequencing of 50 DNA samples representing the German Riding horse. Allele frequencies and distribution were further evaluated in a variety of different breeds: Arabians (for all four SNPs), Old Kladrub Horses, Draught Horses (including Westphalian Draught Horses, Rheinish Westphalian Draught Horses, Saxon-Thuringia Coldbloods, Altmarker Coldbloods), American Saddlebreds, Miniature Horses, Australian Riding Ponies, Appaloosa, Morgan Horses, and Lipizzaner for C629T (Ser210Leu) alone. No animal carrying the homozygous genotype TT has been detected. The overall frequency of the newly described variant T is low (between 2% and 6%). Simulation studies on the protein conformation predict structural protein changes mediated by the SNP.