The Dfm1 Derlin Is Required for ERAD Retrotranslocation of Integral Membrane Proteins

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Substrate specificity of α‐humulene synthase from Zingiber zerumbet Smith and determination of kinetic constants by a spectrophotometric assay

Terpene synthases are the key enzymes in terpene biosynthesis that provide a structurally complex and highly diverse product spectrum. A suitable and reliable analytical assay is indispensable to measure terpene synthase activity accurately and precisely. In this study, a malachite green assay (MG) was adapted to rapidly assay terpene synthase activity and was validated in comparison to an already established gas chromatography assay. A linear correlation between both assays was observed. Kinetic properties for the previously described sesquiterpene synthase α‐humulene synthase (HUM) from Zingiber zerumbet Smith were investigated for the bioconversion of the monoterpene precursors geranyl pyrophosphate (2E‐GPP) and neryl pyrophosphate (2Z‐NPP) as well as for the sesquiterpene precursor farnesyl pyrophosphate (2E,6E‐FPP). Also, gas chromatography mass spectrometry (GS‐MS) was carried out to identify the products of the bioconversion of (2E)‐GPP and (2Z)‐NPP.     

P-31 nuclear magnetic resonance analysis of brain: Normoxic and anoxic brain slices

Perchloric acid extracts were prepared from liquid-N₂-frozen gerbil and guinea pig brain slices studied under one of three conditions: O₂-incubated, N₂-incubated or O₂-incubated recovery following N₂ incubation. Mole percentages of the various phosphatic components contained in the extracts were determined by phosphorus-31 nuclear magnetic resonance spectroscopy. The brain slice extract spectrum revealed a previously unreported group of brain phosphodiesters at –0.73 relative to 85% orthophosphoric acid Although the phosphatic profiles from O₂-incubated slices fromgerbils and guinea pigs revealed only minor species variations, which differed quantitatively rather than qualitatively, species-specific differences were made readily apparent and amplified by incubating brain slices under oxygen-deficient conditions. Despite these differences which were most prevalent during the recovery phase, the overall metabolic changes described herein in response to N₂-incubation were in accord with the results obtained by other analytical techniques. Inorganic orthophosphate (2.63 ) was increased, while nucleoside (principally, adenosine) triphosphate (-, –10.92 , -, –21.45 , and -, –5.80 ) and phosphocreatine (–3.12 ) levels were decreased in response to N₂ incubation. In addition, inosine monophosphate (3.78 ) was increased and the levels of a partially characterized acid-labile phosphate (0.85 , guinea pig) were decreased upon N₂ incubation. Phosphoglyceride metabolism also appeared to be altered by oxygen deprivation (gerbil). These latter findings provide additional information concerning the metabolic responses of cerebral tissue to oxygen deficient conditions.Dedicated to Henry McIlwain.     

In vitro propagation of Achillea asplenifolia VENT. through multiple shoot regeneration

A method for the micropropagation of Achillea asplenifolia VENT. (Asteraceae) is described. Axillary shoots regenerated from nodal explants of adult plants could be stimulated to multiple shoot formation on the basal medium of Murashige and Skoog (1962), supplemented with different cytokinins. The best proliferation (5.33 shoots per culture), together with a healthy appearance of the cultures, was achieved with N-benzyl-9-(2-tetrahydropyranyl)adenine at a concentration of 1 mg/l. Shoots could be easily rooted on hormone-free MS basal medium and subsequently acclimatized to greenhouse and field conditions with 100% survival rate. DNA-fingerprinting, chromosome counts, the proazulene pattern as well as the essential oil analysis proved the true to type character of the micropropagated plants.Abbreviations 2iP 6-(,-Dimethylallylamino)purine - MS Murashige and Skoog (1962) medium - BAP 6-Benzylaminopurine - GA₃ Gibberellic acid - GC gas chromatography - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - Kin Kinetin - PBA N-Benzyl-9-(2-tetrahydropyranyl)adenine - TLC thin layerchromatography - WPM Lloyd and McCown (1981) woody plant medium - Zea Zeatin     

A cell-permeable fusion toxin as a tool to study the consequences of actin-ADP-ribosylation caused by the salmonella enterica virulence factor SpvB in intact cells

The virulence factor SpvB is a crucial component for the intracellular growth and infection process of Salmonella enterica. The SpvB protein mediates the ADP-ribosylation of actin in infected cells and is assumed to be delivered directly from the engulfed bacteria into the host cell cytosol. Here we used the binary Clostridium botulinum C2 toxin as a transport system for the catalytic domain of SpvB (C/SpvB) into the host cell cytosol. A recombinant fusion toxin composed of the enzymatically inactive N-terminal domain of C. botulinum C2 toxin (C2IN) and C/SpvB was cloned, expressed, and characterized in vitro and in intact cells. When added together with C2II, the C2IN-C/SpvB fusion toxin was efficiently delivered into the host cell cytosol and ADP-ribosylated actin in various cell lines. The cellular uptake of the fusion toxin requires translocation from acidic endosomes into the cytosol and is facilitated by Hsp90. The N- and C-terminal domains of SpvB are linked by 7 proline residues. To elucidate the function of this proline region, fusion toxins containing none, 5, 7, and 9 proline residues were constructed and analyzed. The existence of the proline residues was essential for the translocation of the fusion toxins into host cell cytosol and thereby determined their cytopathic efficiency. No differences concerning the mode of action of the C2IN-C/SpvB fusion toxin and the C2 toxin were obvious as both toxins induced depolymerization of actin filaments, resulting in cell rounding. The acute cellular responses following ADP-ribosylation of actin did not immediately induce cell death of J774.A1 macrophage-like cells.     

An activated glutamate residue identified in photosystem II at the interface between the manganese-stabilizing subunit and the D2 polypeptide

Photosystem II (PSII) catalyzes the oxidation of water during oxygenic photosynthesis. PSII is composed both of intrinsic subunits, such as D1, D2, and CP47, and extrinsic subunits, such as the manganese-stabilizing subunit (MSP). Previous work has shown that amines covalently bind to amino acid residues in the CP47, D1, and D2 subunits of plant and cyanobacterial PSII, and that these covalent reactions are prevented by the addition of chloride in plant preparations depleted of the 18- and 24-kDa extrinsic subunits. It has been proposed that these reactive groups are carbonyl-containing, post-translationally modified amino acid side chains (Ouellette, A. J. A., Anderson, L. B., and Barry, B. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2204-2209 and Anderson, L. B., Ouellette, A. J. A., and Barry, B. A. (2000) J. Biol. Chem. 275, 4920-4927). To identify the amino acid binding site in the spinach D2 subunit, we have employed a biotin-amine labeling reagent, which can be used in conjunction with avidin affinity chromatography to purify biotinylated peptides from the PSII complex. Multidimensional chromato-graphic separation and multistage mass spectrometry localizes a novel post-translational modification in the D2 subunit to glutamate 303. We propose that this glutamate is activated for amine reaction by post-translational modification. Because the modified glutamate is located at a contact site between the D2 and manganese-stabilizing subunits, we suggest that the modification is important in vivo in stabilizing the interaction between these two PSII subunits. Consistent with this conclusion, mutations at the modified glutamate alter the steady-state rate of photosynthetic oxygen evolution.     

Chemosystematic value of flavonoids from Crataegus x macrocarpa (Rosaceae) with special emphasis on (R)- and (S)-eriodictyol-7-O-glucuronide and luteolin-7-O-glucuronide

The chemotaxonomic investigation of Crataegus x macrocarpa, a hybrid of C. laevigata and C. rhipidophylla, presents the qualitative and quantitative composition of its flavonoid pattern in relationship to its parent species for the first time. Six flavonoids were identified as vitexin-2'-O-rhamnoside (1), vitexin (2), isovitexin (3), rutin (4), hyperoside (5), and isoquercitrin (6). Furthermore, two flavonoids were isolated from C. x macrocarpa and identified as a diastereoisomeric mixture of (R)- and (S)-eriodictyol-7-O-beta-D-glucuronide (7) and luteolin-7-O-beta-D-glucuronide (8) by means of 1D- and 2D-NMR, MS, and UV experiments. Compounds 7 and 8 were isolated for the first time from Crataegus species. While missing in C. laevigata, their occurrence in C. rhipidophylla additionally emphasizes its chemotaxonomic relationship to C. x macrocarpa.     

First evidence of the presence of chitin in skeletons of marine sponges. Part II. Glass sponges (Hexactinellida: Porifera)

Sponges (Porifera) are presently gaining increased scientific attention because of their secondary metabolites and specific skeleton structures. In contrast to demosponges, whose skeletons are formed from biopolymer spongin, glass sponges (hexactinellids) possess silica-organic composites as the main natural material for their skeletal fibres. Chitin has a crystalline structure and it constitutes a network of organized fibres. This structure confers rigidity and resistance to organisms that contain it, including monocellular (yeast, amoeba, diatoms) and multicellular (higher fungi, arthropods, nematodes, molluscs) organisms. In contrast to different marine invertebrates whose exoskeletons are built of chitin, this polysaccharide has not been found previously as an endogenous biopolymer within glass sponges (Hexactinellida). We hypothesized that glass sponges, which are considered to be the most basal lineage of multicellular animals, must possess chitin. Here, we present a detailed study of the structural and physico-chemical properties of skeletal fragments of the glass sponge Farrea occa. We show that these fibres have a layered design with specific compositional variations in the chitin/silica composite. We applied an effective approach for the demineralization of glass sponge skeletal formations based on an etching procedure using alkali solutions. The results show unambiguously that alpha-chitin is an essential component of the skeletal structures of Hexactinellida. This is the first report of a silica-chitin's composite biomaterial found in nature. From this perspective, the view that silica-chitin scaffolds may be key templates for skeleton formation also in ancestral unicellular organisms, rather than silica-protein composites, emerges as a viable alternative hypothesis.