Ganglioside GM(1) biphasically regulates the activity of protein kinase C by the effects on the structure of the lipid bilayer

Addition of a small amount of ganglioside GM(1) to phosphatidylserine (PS) liposomes, a gradual increase of protein kinase C (PKC) activity was recorded up to about 2 mol% GM(1) where the maximal enzyme activity was obtained. Then the activity of PKC began to decline and even turned to be inhibited with the further increase of GM(1) content. It was also indicated that GM(1)/PS binary liposomes had the highest membrane fluidity and very low spatial density of lipid headgroups which was demonstrated in the MC-540 studies due to the interposition of GM(1) when the liposomes contained about 2 mol% GM(1). Besides, the liposomes containing about 2 mol% GM(1) provided a more hydrophobic environment for PKC than the liposomes containing less or more GM(1) which was indicated in the Acrylodan experiments. These factors commonly induced PKC to be stimulated maximally. Whether at the lower or higher GM(1) content, the membrane structure was not the most suitable to support the activity of PKC, which declined as a consequence.     

Diapause development and acclimation regulating enzymes associated with glycerol synthesis in the Shonai ecotype of the rice stem borer larva,Chilo suppressalis walker

Overwintering larvae of the Shonai ecotype of the rice stem borer, Chilo suppressalis, enter diapause in early September and terminate diapause at the end of October. Cold acclimation at 0°C did not influence glycerol, trehalose or glycogen content in larvae collected on 22 September. Acclimation at 0°C increased the glycerol content and reduced the glycogen content significantly in larvae collected on 2 October and 22 November compared with acclimation at 15°C. These results indicate that overwintering larvae at different phases of diapause development respond differently to the low temperature stimulus for glycerol synthesis. Thus, we evaluated the metabolic rearrangements associated with glycerol synthesis during diapause development and after temperature acclimation. Larvae collected on 2 October were acclimated at 15°C for 15 and 60 days. Some of those acclimated at 15°C were then moved to 0°C for 15 days. The larvae acclimated at 15°C for 15 days were in deep diapause and accumulated little glycerol, while larvae acclimated at 15°C for 60 days were nearly ready to emerge from diapause and accumulated glycerol at 155.5 μmol/g. When larvae acclimated to 15°C for 15 days were transferred to 0°C, glycerol accumulation was stimulated to the same extent (ca 140 μmol/g) as it was in larvae that were acclimated to 15°C for 60 days and then transferred to 0°C. These results indicate that low temperature has a cumulative effect on glycerol production in larvae at different phases of diapause development. Glycerol accumulation was accomplished by activation of glycogen phosphorylase and inhibition of fructose-1,6-bisphosphatase, and activation of enzymes associated with glycerol synthesis, mainly glyceraldehyde-3-phosphatase and polyol dehydrogenase with glyceraldehyde activity.     

Presenilin 1 is required for maturation and cell surface accumulation of nicastrin

Proteolytic processing of amyloid precursor protein generates beta-amyloid (Abeta) peptides that are deposited in senile plaques in brains of aged individuals and patients with Alzheimer's disease. Presenilins (PS1 and PS2) facilitate the final step in Abeta production, the intramembranous gamma-secretase cleavage of amyloid precursor protein. Biochemical and pharmacological evidence support a catalytic or accessory role for PS1 in gamma-secretase cleavage, as well as a regulatory role in select membrane protein trafficking. In this report, we demonstrate that PS1 is required for maturation and cell surface accumulation of nicastrin, an integral component of the multimeric gamma-secretase complex. Using kinetic labeling studies we show that in PS1(-/-)/PS2(-/-) cells nicastrin fails to reach the medial Golgi compartment, and as a consequence, is incompletely glycosylated. Stable expression of human PS1 restores these deficiencies in PS1(-/-) fibroblasts. Moreover, membrane fractionation studies show co-localization of PS1 fragments with mature nicastrin. These results indicate a novel chaperone-type role for PS1 and PS2 in facilitating nicastrin maturation and transport in the early biosynthetic compartments. Our findings are consistent with PS1 influencing gamma-secretase processing at multiple steps, including maturation and intracellular trafficking of substrates and component(s) of the gamma-secretase complex.     

Evaluation of the role of phosphatidylserine translocase activity in ABCA1-mediated lipid efflux

The following two theories for the mechanism of ABCA1 in lipid efflux to apolipoprotein acceptors have been proposed: 1) that ABCA1 directly binds the apolipoprotein ligand and then facilitates lipid efflux and 2) that ABCA1 acts as a phosphatidylserine (PS) translocase, increasing PS levels in the plasma membrane exofacial leaflet, and that this is sufficient to facilitate apolipoprotein binding and lipid assembly. Upon induction of ABCA1 in RAW264.7 cells by cAMP analogues there was a moderate increase in cell surface PS as detected by annexin V binding, whereas apoAI binding was increased more robustly. Apoptosis induced large increases in annexin V and apoAI binding; however, apoptotic cells did not efflux lipids to apoAI. Annexin V did not act as a cholesterol acceptor, and it did not compete for the cholesterol acceptor or cell binding activity of apoAI. ApoAI binds to ABCA1-expressing cells, and with incubation at 37 degrees C apoAI is co-localized within the cells in ABCA1-containing endosomes. Fluorescent recovery after photobleaching demonstrated that apoAI bound to ABCA1-expressing cells was relatively immobile, suggesting that it was bound either directly or indirectly to an integral membrane protein. Although ABCA1 induction was associated with a small increase in cell surface PS, these results argue against the notion that this cell surface PS is sufficient to mediate cellular apoAI binding and lipid efflux.     

Expression of the mel gene from Pseudomonas maltophilia in Bacillus thuringiensis

AIMS: The objective of this work was to express a novel mel gene, responsible for melanin formation, in Bacillus thuringiensis. METHODS AND RESULTS: A novel mel gene from Pseudomonas maltophilia was sub-cloned into B. thuringiensis using a shuttle vector plasmid and electroporation. Results revealed that the mel gene was expressed under the control of the CryIIIA promoter in B. thuringiensis and conferred u.v. protection on the recipient strain. CONCLUSIONS: The novel mel gene from Ps. maltophilia expressed in B. thuringiensis conferred u.v. protection on the recipient strain. SIGNIFICANCE AND IMPACT OF THE STUDY: Products containing B. thuringiensis for pest control are sensitive to u.v. degradation. As melanin has the ability to act as a u.v. absorber, a recombinant B. thuringiensis strain producing melanin provides a new stability for B. thuringiensis preparations.     

Origin of two isoprenoid units in a lavandulyl moiety of sophoraflavanone G from Sophora flavescens cultured cells

Cell suspension cultures of Sophora flavescens produced large amounts of sophoraflavanone G, an 8-lavandulylated flavanone and lupalbigenin, a 6,3'-di-dimethylallylated isoflavone, by the simultaneous addition of cork tissues and methyl jasmonate. The labeling pattern of the isoprene units resulting after administration of [1-13C] glucose into the cell cultures in the presence of the above additives revealed that two isoprene units in the lavandulyl group of sophoraflavanone G and two dimethylallyl groups of lupalbigenin were biosynthesized via the 1-deoxy-D-xylulose-5-phosphate pathway.     

Influence of different light intensities on the content of diosgenin, lipids, carotenoids and fatty acids in leaves of Dioscorea zingiberensis

Cultivation of the climbing plant Dioscorea zingiberensis at a light intensity of 100 microE. m(-2) sec(-1) yields three different phenotypes. Most of the plants grow as green phenotype (DzW). Two further forms differ in their leaf shape and leaf color. Whereas one type exhibits a more pointed leaf shape in the upper part of the plant with leaves appearing yellow-green with white stripes or hatchings (DzY), the other type shows a more round leaf shape with an intensive yellow-green color (DzT). These three plant types differ in their diosgenin content not only in their rhizomes but also in the chloroplasts. In the rhizomes the diosgenin content in the green form is 0.4%, in the DzY-form 0.6% and in the DzT-form even 1.3% of the dry weight. Furthermore, even in chloroplasts of the green DzW-form and of the DzY-form the presence of diosgenin was demonstrated. It occurs there as the epimeric form yamogenin. The DzT-form contains no yamogenin in its chloroplasts. Besides this, these plant forms differ in their chlorophyll and carotenoid content and in their fatty acid composition. Carotenoids increase from 1.3% of total lipids in the green phenotype to 3.3% in the DzY- and to 4.2% in the DzT-form. This increase refers to beta-carotene as well as to lutein and neoxanthin. The chlorophyll content in the green type is 8.1% and lower in the DzY-form with 7%. The highest chlorophyll content is found in the DzT-form with 12%. Fatty acids in the DzY-form and in the DzT-form have a more unsaturated character than in the green phenotype. The content of the monoenoic acid trans-hexadecenoic acid is considerably lower in both phenotypes when compared to the green phenotype. In both phenotypes the quantity of fatty acids with 16 carbon atoms is reduced, whereas fatty acids with 18 carbon atoms occur in higher concentration. Cultivation of the green phenotype (DzW) at the three light intensities of 10, 100 and 270 microE x m(-2) x sec(-1) leads to changes of the diosgenin content in rhizomes, to an increase of leaf dry weight, to a reduction of the grana structure in chloroplasts and therewith to a decrease of the chlorophyll content. The total lipid content is highest under the cultivation at 100 microE x m(-2) x sec(-1) and reduced by 30% at 10 and 270 microE x m(-2) x sec(-1). Carotenoids, however, are highest in shaded plants (10 microE x m(-2) x sec(-1)) and plants grown under high light conditions of 270 microE x m(-2) x sec(-1). At 100 microE x m(-2) x sec(-1) a decrease of saturated fatty acids is observed in comparison to plants grown under shaded conditions.     

The extraction of imperialine and imperialine-3 beta-glucoside from Fritillaria pallidiflora Schrenk and quantitative determination by HPLC-evaporative light scattering detection

The extraction procedure and quantitative determination by HPLC-evaporative light scattering detection (ELSD) of the main bioactive components, namely, imperialine (1) and imperialine-3 beta-glucoside (2), of bulbs of Fritillaria pallidiflora Schrenk have been investigated. The most efficient method for the simultaneous extraction of 1 and 2 involved pre-treatment of the bulb powder with ammonia, followed by reflux with dichloromethane:methanol at 90 degrees C for 4 h. Simultaneous determination of non-chromophoric 1 and 2 by HPLC-ELSD employed a Kromasil C18 column eluted with acetonitrile:water:diethylamine. The assay was accurate and reproducible with an overall variation lower than 4% and a sample recovery higher than 98%. The methods described have been successfully used to evaluate the quality of three batches of the crude traditional Chinese medicinal herb derived from the bulbs of F. pallidiflora.