Two novel types of O-glycans on the mugwort pollen allergen Art v 1 and their role in antibody binding

Art v 1, the major allergen of mugwort (Artemisia vulgaris) pollen contains galactose and arabinose. As the sera of some allergic patients react with natural but not with recombinant Art v 1 produced in bacteria, the glycosylation of Art v 1 may play a role in IgE binding and human allergic reactions. Chemical and enzymatic degradation, mass spectrometry, and 800 MHz (1)H and (13)C nuclear magnetic resonance spectroscopy indicated the proline-rich domain to be glycosylated in two ways. We found a large hydroxyproline-linked arabinogalactan composed of a short beta1,6-galactan core, which is substituted by a variable number (5-28) of alpha-arabinofuranose residues, which form branched side chains with 5-, 2,5-, 3,5-, and 2,3,5-substituted arabinoses. Thus, the design of the Art v 1 polysaccharide differs from that of the well known type II arabinogalactans, and we suggest it be named type III arabinogalactan. The other type of glycosylation was formed by single (but adjacent) beta-arabinofuranoses linked to hydroxyproline. In contrast to the arabinosylation of Ser-Hyp(4) motifs in other hydroxyproline-rich glycoproteins, such as extensins or solanaceous lectins, no oligo-arabinosides were found in Art v 1. Art v 1 and parts thereof produced by alkaline degradation, chemical deglycosylation, proteolytic degradation, and/or digestion with alpha-arabinofuranosidase were used in enzyme-linked immunosorbent assay and immunoblot experiments with rabbit serum and with the sera of patients. Although we could not observe antibody binding by the polysaccharide, the single hydroxyproline-linked beta-arabinose residues appeared to react with the antibodies. Mono-beta-arabinosylated hydroxyproline residues thus constitute a new, potentially cross-reactive, carbohydrate determinant in plant proteins.     

Mdm31 and Mdm32 are inner membrane proteins required for maintenance of mitochondrial shape and stability of mitochondrial DNA nucleoids in yeast

The MDM31 and MDM32 genes are required for normal distribution and morphology of mitochondria in the yeast Saccharomyces cerevisiae. They encode two related proteins located in distinct protein complexes in the mitochondrial inner membrane. Cells lacking Mdm31 and Mdm32 harbor giant spherical mitochondria with highly aberrant internal structure. Mitochondrial DNA (mtDNA) is instable in the mutants, mtDNA nucleoids are disorganized, and their association with Mmm1-containing complexes in the outer membrane is abolished. Mutant mitochondria are largely immotile, resulting in a mitochondrial inheritance defect. Deletion of either one of the MDM31 and MDM32 genes is synthetically lethal with deletion of either one of the MMM1, MMM2, MDM10, and MDM12 genes, which encode outer membrane proteins involved in mitochondrial morphogenesis and mtDNA inheritance. We propose that Mdm31 and Mdm32 cooperate with Mmm1, Mmm2, Mdm10, and Mdm12 in maintenance of mitochondrial morphology and mtDNA.     

Specific formation of arachidonic acid and eicosapentaenoic acid by a front-end Delta5-desaturase from Phytophthora megasperma

The biosynthesis of arachidonic acid (20:4(Delta5Z,8Z,11Z,14Z)) from linoleic acid in plants by transgenic means requires the sequential and specific action of two desaturation reactions and one elongation reaction. Here, we describe the isolation of a specific acyl-lipid-desaturase catalyzing the formation of the double bond at position 5 from a cDNA library from Phytophthora megasperma. The isolated full-length cDNA harbors a sequence of 1740 bp encoding a protein of 477 amino acids with a calculated molecular weight of 53.5 kDa. The desaturase sequence contained a predicted N-terminal cytochrome b(5)-like domain, as well as three histidine-rich domains. For functional identification, the cDNA was expressed in Saccharomyces cerevisiae, and the formation of newly formed fatty acids was analyzed. The expression of the heterologous enzyme resulted in the formation of arachidonic acid after di-homo-gamma-linolenic acid supplementation and in the formation of eicosapentaenoic acid synthesis from omega3-arachidonic acid. Results presented here on the substrate specificity identify this expressed protein as a classical Delta5-acyl-lipid-desaturase, capable of specifically introducing a double bond at the Delta5 position solely in 20-carbon-atom chain length fatty acids containing a double bond at position Delta8. Detailed analysis of the different lipid species showed a preferential occurrence of the desaturation reaction for fatty acids esterified to phosphatidylcholine.     

Characterization of the putative native and recombinant rat sterol transporter Niemann-Pick C1 Like 1 (NPC1L1) protein

The exact mechanistic pathway of cholesterol absorption in the jejunum of the small intestines is a poorly understood process. Recently, a relatively novel gene, Niemann-Pick C1 Like 1 (NPC1L1), was identified as being critical for intestinal sterol absorption in a pathway which is sensitive to sterol absorption inhibitors such as ezetimibe. NPC1L1 is a multi-transmembrane protein, with a putative sterol sensing domain. Very little else is known about the NPC1L1 protein. In this report, we characterize the native and recombinant rat NPC1L1 protein. We show that NPC1L1 is a 145 kDa membrane protein, enriched in the brush border membrane of the intestinal enterocyte and is highly glycosylated. In addition, sequential detergent extraction of enterocytes result in highly enriched preparations of NPC1L1. An engineered Flag epitope tagged rat NPC1L1 cDNA was expressed as recombinant protein in CHO cells and demonstrated cell surface expression, similar to the native rat protein. These biochemical data indicate that NPC1L1 exists as a predominantly cell surface membrane expressed protein, consistent with its proposed role as the putative intestinal sterol transporter.     

Characterization of advanced glycation end products for biochemical studies: side chain modifications and fluorescence characteristics

Advanced glycation end products (AGEs) are known to be involved in the pathogenesis of several diseases and therefore effects of AGEs on cells are the objective of numerous investigations. Since AGEs used in biochemical studies are usually not chemically characterized, comparison of data is difficult if not impossible. To find a suitable characterization protocol, human serum albumin was reacted with different concentrations of glucose, methyl glyoxal, and glyoxylic acid. The obtained AGEs were characterized with respect to the extent of side chain modifications (lysine and arginine), the carboxymethyl lysine and carbonyl content, and the fibrillar state. Additionally, their fluorescence and absorbance characteristics were extensively studied. Although we found significant differences in the degree of modification and in AGE-specific fluorescence when using different modifiers, the results provide important information and allow comparing AGEs derived from different modifier concentrations. The results also suggest strong conformational changes within the modified proteins. In the present paper we propose a set of parameters that is sufficient to partially characterize AGEs used for biochemical studies.     

Prototype foamy virus envelope glycoprotein leader peptide processing is mediated by a furin-like cellular protease, but cleavage is not essential for viral infectivity

Analogous to cellular glycoproteins, viral envelope proteins contain N-terminal signal sequences responsible for targeting them to the secretory pathway. The prototype foamy virus (PFV) envelope (Env) shows a highly unusual biosynthesis. Its precursor protein has a type III membrane topology with both the N and C terminus located in the cytoplasm. Coexpression of FV glycoprotein and interaction of its leader peptide (LP) with the viral capsid is essential for viral particle budding and egress. Processing of PFV Env into the particle-associated LP, surface (SU), and transmembrane (TM) subunits occur posttranslationally during transport to the cell surface by yet-unidentified cellular proteases. Here we provide strong evidence that furin itself or a furin-like protease and not the signal peptidase complex is responsible for both processing events. N-terminal protein sequencing of the SU and TM subunits of purified PFV Env-immunoglobulin G immunoadhesin identified furin consensus sequences upstream of both cleavage sites. Mutagenesis analysis of two overlapping furin consensus sequences at the PFV LP/SU cleavage site in the wild-type protein confirmed the sequencing data and demonstrated utilization of only the first site. Fully processed SU was almost completely absent in viral particles of mutants having conserved arginine residues replaced by alanines in the first furin consensus sequence, but normal processing was observed upon mutation of the second motif. Although these mutants displayed a significant loss in infectivity as a result of reduced particle release, no correlation to processing inhibition was observed, since another mutant having normal LP/SU processing had a similar defect.     

Flow-cytometric assessment of cellular poly(ADP-ribosyl)ation capacity in peripheral blood lymphocytes

Background  

Poly(ADP-ribosyl)ation is a posttranslational modification of nuclear proteins catalysed by poly(ADP-ribose) polymerases (PARPs), using NAD⁺ as a substrate. Activation of PARP-1 is in immediate response to DNA damage generated by endogenous and exogenous damaging agents. It has been implicated in several crucial cellular processes including DNA repair and maintenance of genomic stability, which are both intimately linked with the ageing process. The measurement of cellular poly(ADP-ribosyl)ation capacity, defined as the amount of poly(ADP-ribose) produced under maximal stimulation, is therefore relevant for research on ageing, as well as for a variety of other scientific questions.     

Identification of serotonin 5-HT1A receptor partial agonists in ginger

Animal studies suggest that ginger (Zingiber officinale Roscoe) reduces anxiety. In this study, bioactivity-guided fractionation of a ginger extract identified nine compounds that interact with the human serotonin 5-HT_1A receptor with significant to moderate binding affinities (K_i = 3–20 μΜ). [³⁵S]-GTPγS assays indicated that 10-shogaol, 1-dehydro-6-gingerdione, and particularly the whole lipophilic ginger extract (K_i = 11.6 μg/ml) partially activate the 5-HT_1A receptor (20–60% of maximal activation). In addition, the intestinal absorption of gingerols and shogaols was simulated and their interactions with P-glycoprotein were measured, suggesting a favourable pharmacokinetic profile for the 5-HT_1A active compounds.     

Reducing the impact of pesticides on biological control in Australian vineyards: pesticide mortality and fecundity effects on an indicator species, the predatory mite Euseius victoriensis (Acari: Phytoseiidae)

Laboratory bioassays on detached soybean, Glycine max (L.) Merr., leaves were used to test 23 fungicides, five insecticides, two acaricides, one herbicide, and two adjuvants on a key Australian predatory mite species Euseius victoriensis (Womersley) in "worst-case scenario" direct overspray assays. Zero- to 48-h-old juveniles, their initial food, and water supply were sprayed to runoff with a Potter tower; spinosad and wettable sulfur residues also were tested. Tests were standardized to deliver a pesticide dose comparable with commercial application of highest label rates at 1,000 liter/ha. Cumulative mortality was assessed 48 h, 4 d, and 7 d after spraying. Fecundity was assessed for 7 d from start of oviposition. No significant mortality or fecundity effects were detected for the following compounds at single-use application at 1,000 liter/ha: azoxystrobin, Bacillus thuringiensis (Bt) subsp. kurstaki, captan, chlorothalonil, copper hydroxide, fenarimol, glyphosate, hexaconazole, indoxacarb, metalaxyl/copper hydroxide, myclobutanil, nonyl phenol ethylene oxide, phosphorous acid, potassium bicarbonate, pyraclostrobin, quinoxyfen, spiroxamine, synthetic latex, tebufenozide, triadimenol, and trifloxystrobin. Iprodione and penconazole had some detrimental effect on fecundity. Canola oil as acaricide (2 liter/100 liter) and wettable sulfur (200 g/100 liter) had some detrimental effect on survival and fecundity and cyprodinil/fludioxonil on survivor. The following compounds were highly toxic (high 48-h mortality): benomyl, carbendazim, emamectin benzoate, mancozeb, spinosad (direct overspray and residue), wettable sulfur (> or = 400 g/100 liter), and pyrimethanil; pyrimethanil had no significant effect on fecundity of surviving females. Indoxacarb safety to E. victoriensis contrasts with its toxicity to key parasitoids and chrysopid predators. Potential impact of findings is discussed.