On the Mechanism of Cationic Amphiphile-mediated Transfection. To Fuse or not to Fuse: Is that the Question?

Due to charge interaction, cationic lipids spontaneously associate with nucleic acids, resulting in the formation of so-called lipoplexes. Lipoplexes are membranous structures that are capable of transducing genes into cells, eventually leading to expression of the genes (transfection). The mechanism involved in the cellular uptake of lipoplexes is most likely endocytosis, which occurs after nonspecific charge-mediated binding to cellular receptors. An important step in the transfection process following the actual internalization of lipoplexes is the release of the lipoplex and/or its DNA into the cytoplasm in order to evade lysosomal degradation. Here, the membranous nature of the lipoplex seems to be crucial in that it allows the exchange of lipids between the endosomal membrane and the lipoplex, which results in membrane perturbations that are a prerequisite in the endosomal escape of DNA. Interestingly, a hexagonal phase of the lipoplexes has been correlated with efficient transfection and it can be envisaged that such a phase could be instrumental in the creation of membrane perturbations. Subsequent to its release into the cytoplasm, the DNA has to be transferred into the nucleus. The nuclear import of DNA is most likely a protein-mediated process. In addition, the nuclear uptake of DNA may be facilitated at the time of nuclear envelope disassembly during mitosis. Currently, cationic liposomes are widely used as gene carrier system to deliver nucleic acids into cells in culture to study the cell-biological functioning of genes plus accompanying proteins. Ultimately, cationic lipids may be used in gene therapeutic protocols.     

Sphingolipid trafficking and protein sorting in epithelial cells

Sphingolipids represent a minor, but highly dynamic subclass of lipids in all eukaryotic cells. They are involved in functions that range from structural protection to signal transduction and protein sorting, and participate in lipid raft assembly. In polarized epithelial cells, which display an asymmetric apical and basolateral membrane surface, rafts have been proposed as a sorting principle for apical resident proteins, following their biosynthesis. However, raft-mediated trafficking is ubiquitous in cells. Also, sphingolipids per se, which are strongly enriched in the apical domain, are subject to sorting in polarity development. Next to the trans Golgi network, a subapical compartment called SAC or common endosome appears instrumental in regulating these sorting events.     

Biosynthesis of anthraquinones in cell cultures of Cinchona 'Robusta' proceeds via the methylerythritol 4-phosphate pathway.

Robustaquinone B was found as a major anthraquinone in cell cultures of Cinchona 'Robusta' after treatment with a fungal elicitor. Anthraquinones in Cinchona are considered to be of the Rubia type, i.e. rings A and B are derived from chorismate and alpha-ketoglutarate, whereas ring C is formed from isopentenyl diphosphate (IPP). To determine the origin of IPP, either formed via the mevalonic acid pathway or the 2-C-methyl-D-erythritol 4-phosphate pathway, the incorporation of [1-13C]glucose into robustaquinone B was studied. The 13C labeling of robustaquinone B was analyzed by one- and two-dimensional NMR spectroscopy and the labeling pattern was compared with the hypothetical labeling patterns obtained via the different biosynthetic pathways. The results clearly show that the IPP, constituting the ring C of robustaquinone B, is biosynthesized via the 2-C-methyl-D-erythritol 4-phosphate pathway. Moreover, the data also confirm that rings A and B of robustaquinone B are formed from chorismate and alpha-ketoglutarate via o-succinylbenzoate.     

Starch biosynthesis and intermediary metabolism in maize kernels. Quantitative analysis of metabolite flux by nuclear magnetic resonance

The seeds of cereals represent an important sink for metabolites during the accumulation of storage products, and seeds are an essential component of human and animal nutrition. Understanding the metabolic interconversions (networks) underpinning storage product formation could provide the foundation for effective metabolic engineering of these primary nutritional sources. In this paper, we describe the use of retrobiosynthetic nuclear magnetic resonance analysis to establish the metabolic history of the glucose (Glc) units of starch in maize (Zea mays) kernels. Maize kernel cultures were grown with [U-(13)C(6)]Glc, [U-(13)C(12)]sucrose, or [1,2-(13)C(2)]acetate as supplements. After 19 d, starch was hydrolyzed, and the isotopomer composition of the resulting Glc was determined by quantitative nuclear magnetic resonance analysis. [1,2-(13)C(2)]Acetate was not incorporated into starch. [U-(13)C(6)]Glc or [U-(13)C(12)]sucrose gave similar labeling patterns of polysaccharide Glc units, which were dominated by [1,2,3-(13)C(3)]- and [4,5,6-(13)C(3)]-isotopomers, whereas the [U-(13)C(6)]-, [3,4,5,6-(13)C(4)]-, [1,2-(13)C(2)]-, [5,6-(13)C(2)], [3-(13)C(1)], and [4-(13)C(1)]-isotopomers were present at lower levels. These isotopomer compositions indicate that there is extensive recycling of Glc before its incorporation into starch, via the enzymes of glycolytic, glucogenic, and pentose phosphate pathways. The relatively high abundance of the [5,6-(13)C(2)]-isotopomer can be explained by the joint operation of glycolysis/glucogenesis and the pentose 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.     

Membrane fluidity adjustments in ethanol-stressed Oenococcus oeni cells

The effect of ethanol on the cytoplasmic membrane of Oenococcus oeni cells and the role of membrane changes in the acquired tolerance to ethanol were investigated. Membrane tolerance to ethanol was defined as the resistance to ethanol-induced leakage of preloaded carboxyfluorescein (cF) from cells. To probe the fluidity of the cytoplasmic membrane, intact cells were labeled with doxyl-stearic acids and analyzed by electron spin resonance spectroscopy. Although the effect of ethanol was noticeable across the width of the membrane, we focused on fluidity changes at the lipid-water interface. Fluidity increased with increasing concentrations of ethanol. Cells responded to growth in the presence of 8% (vol/vol) ethanol by decreasing fluidity. Upon exposure to a range of ethanol concentrations, these adapted cells had reduced fluidity and cF leakage compared with cells grown in the absence of ethanol. Analysis of the membrane composition revealed an increase in the degree of fatty acid unsaturation and a decrease in the total amount of lipids in the cells grown in the presence of 8% (vol/vol) ethanol. Preexposure for 2 h to 12% (vol/vol) ethanol also reduced membrane fluidity and cF leakage. This short-term adaptation was not prevented in the presence of chloramphenicol, suggesting that de novo protein synthesis was not involved. We found a strong correlation between fluidity and cF leakage for all treatments and alcohol concentrations tested. We propose that the protective effect of growth in the presence of ethanol is, to a large extent, based on modification of the physicochemical state of the membrane, i.e., cells adjust their membrane permeability by decreasing fluidity at the lipid-water interface.     

Interleukin-1beta activates specific populations of enteric neurons and enteric glia in the guinea pig ileum and colon

Fos expression was used to assess whether the proinflammatory cytokine interleukin-1beta (IL-1beta) activated specific, chemically coded neuronal populations in isolated preparations of guinea pig ileum and colon. Whether the effects of IL-1beta were mediated through a prostaglandin pathway and whether IL-1beta induced the expression of cyclooxygenase (COX)-2 was also examined. Single- and double-labeling immunohistochemistry was used after treatment of isolated tissues with IL-1beta (0.1-10 ng/ml). IL-1beta induced Fos expression in enteric neurons and also in enteric glia in the ileum and colon. For enteric neurons, activation was concentration-dependent and sensitive to indomethacin, in both the myenteric and submucosal plexuses in both regions of the gut. The maximum proportion of activated neurons differed between the ileal (approximately 15%) and colonic (approximately 42%) myenteric and ileal (approximately 60%) and colonic (approximately 75%) submucosal plexuses. The majority of neurons activated in the myenteric plexus of the ileum expressed nitric oxide synthase (NOS) or enkephalin immunoreactivity. In the colon, activated myenteric neurons expressed NOS. In the submucosal plexus of both regions of the gut, the majority of activated neurons were vasoactive intestinal polypeptide (VIP) immunoreactive. After treatment with IL-1beta, COX-2 immunoreactivity was detected in the wall of the gut in both neurons and nonneuronal cells. In conclusion, we have found that the proinflammatory cytokine IL-1beta specifically activates certain neurochemically defined neural pathways and that these changes may lead to disturbances in motility observed in the inflamed bowel.     

Protein-induced fusion can be modulated by target membrane lipids through a structural switch at the level of the fusion peptide

Regulatory features of protein-induced membrane fusion are largely unclear, particularly at the level of the fusion peptide. Fusion peptides being part of larger protein complexes, such investigations are met with technical limitations. Here, we show that the fusion activity of influenza virus or Golgi membranes is strongly inhibited by minor amounts of (lyso)lipids when present in the target membrane but not when inserted into the viral or Golgi membrane itself. To investigate the underlying mechanism, we employ a membrane-anchored peptide system and show that fusion is similarly regulated by these lipids when inserted into the target but not when present in the peptide-containing membrane. Peptide-induced fusion is regulated by a reversible switch of secondary structure from a fusion-permissive alpha-helix to a nonfusogenic beta-sheet. The "on/off" activation of this switch is governed by minor amounts of (lyso)-phospholipids in targets, causing a drop in alpha-helix and a dramatic increase in beta-sheet contents. Concomitantly, fusion is inhibited, due to impaired peptide insertion into the target membrane. Our observations in biological fusion systems together with the model studies suggest that distinct lipids in target membranes provide a means for regulating membrane fusion by causing a reversible secondary structure switch of the fusion peptides.     

Crystal structure of a conformation-selective casein kinase-1 inhibitor

Members of the casein kinase-1 family of protein kinases play an essential role in cell regulation and disease pathogenesis. Unlike most protein kinases, they appear to function as constitutively active enzymes. As a result, selective pharmacological inhibitors can play an important role in dissection of casein kinase-1-dependent processes. To address this need, new small molecule inhibitors of casein kinase-1 acting through ATP-competitive and ATP-noncompetitive mechanisms were isolated on the basis of in vitro screening. Here we report the crystal structure of 3-[(2,4,6-trimethoxyphenyl) methylidenyl]-indolin-2-one (IC261), an ATP-competitive inhibitor with differential activity among casein kinase-1 isoforms, in complex with the catalytic domain of fission yeast casein kinase-1 refined to a crystallographic R-factor of 22.4% at 2.8 A resolution. The structure reveals that IC261 stabilizes casein kinase-1 in a conformation midway between nucleotide substrate liganded and nonliganded conformations. We propose that adoption of this conformation by casein kinase-1 family members stabilizes a delocalized network of side chain interactions and results in a decreased dissociation rate of inhibitor.     

A study of water relations in neem (Azadirachta indica) seed that is characterized by complex storage behaviour

Neem (Azadirachta indica) seed is reputed to have limited tolerance to desiccation, to be sensitive to chilling and imbibitional stress, and to display intermediate storage behaviour. To understand this behaviour the properties of water in seed tissues were studied. Water sorption isotherms showed that at similar relative humidity (RH), the water content was consistently higher in axes than in cotyledons, mainly due to the elevated lipid content (51%) in the cotyledons. Using differential scanning calorimetry, melting transitions of water were observed at water contents higher than 0.14 g H2O g-1 DW in the cotyledons and 0.23 g H2O g-1 DW in the axes. Beside melting transitions of lipid, as verified by infrared spectroscopy, changes in heat capacity were observed which shifted with water content, indicative of glass-to-liquid transitions. State diagrams are given on the basis of the water content of seed tissues, and also on the basis of the RH at 20 degrees C. Longevity was considerably improved, and the sensitivity to chilling/subzero temperatures was reduced when axis and cotyledons were dehydrated to moisture contents < or = of approximately 0.05 g H2O g-1 DW. However, longevity during storage at very low water contents was limited. A possible mechanism for the loss of sensitivity to chilling/subzero temperatures at low water contents is discussed. The results suggest that dry neem seeds in the glassy state have great potential for extended storability, also at subzero temperatures.