Dieter Seidel‐65 Jahre

Fruit extract of Solanum xanthocarpum was evaluated for its toxicity against Alternaria brassicae, the causal agent of Alternaria blight of Indian mustard [Brassica juncea (L.) Czern. &; Coss]. The mass obtained after vacuum drying of the crude methanolic extract was utilised for further sequential fractionation using n-hexane, ethyl acetate, n-butanol and methanol. Among the crude and different fractions tested, methanolic fraction was most effective with a minimum inhibitory concentration (MIC) of 62.5 μg/ml. The methanolic fraction was further fractionated using open column liquid chromatography into five subfractions (I–V) to identify the antifungal bioactive compounds. Among the five subfractions (SFs) tested SF IV was most effective at inhibiting A. brassicae conidial germination and thereby inhibited lesion development of Alternaria blight at a concentration of 15.625 μg/ml or higher. Furthermore, bioautography of SF IV with Alternaria alternata and diagnosis with Dragendorff reagent indicated that SF IV contains a mixture of bioactive alkaloids, namely a₁ (R_f = 0.12) and a₂ (R_f = 0.22). The potential of using S. xanthocarpum as a resource for the development of biofungicides is discussed.     

Buchbesprechungen

Karl Esser: Kryptogamen 1. Cyanobacterien, Algen, Pilze, Flechten. Praktikum und Lehrbuch. Dritte, wesentlich überarbeitete Auflage. 585 S., 300 Abb., Springer‐Verlag Berlin, Heidelberg 2000. Preis: 129.00 DM, ISBN 3–540–66451–3

Jain, S. M., Gupta, R. J., Newton, R. J. (Eds.): Somatic Embryogenesis in Woody Plants. Kluwer Academic Publishers Dordrecht 1999, Vol. 2, 547 S

Vanneste, J. L. (Ed.): FIRE Blight: The Disease and its Causative Agent, Erwinia amylovora. CABI Publishing, CAB International, Oxon, UK, 2000, 370 p., 16 color plates, 38 figures, 25 tables, 6 boxes, Price US$120.00, ISBN 0 85199 294 3

Heitefuss, R. Pflanzenschutz. Grundlagen der praktischen Phytomedi‐zin. 3., neubearbeitete und erweiterte Auflage. Georg Thieme Verlag Stuttgart. 2000, 399 S., 94 Abb., 22 Tab., Preis 49.90 DM, ISBN 313 5133036/650

L. Benzing, Der sachkundige Vorratsschützer. Sachkunde für Anwender und Abgebende von Vorratsschutzmitteln. Agrimedia Spithal, 2000, 158 S., 32 farbige Abb., 14 schwarz ‐ weiße Abb., 23 Tab.. Preis: 78 DM, ISBN 3–86037–115–0     

Fibulin-3, -4, and -5 Are Highly Susceptible to Proteolysis,Interact with Cells and Heparin,and Form Multimers

Extracellular short fibulins, fibulin-3, -4, and -5, are components of the elastic fiber/microfibril system and are implicated in the formation and homeostasis of elastic tissues. In this study, we report new structural and functional properties of the short fibulins. Full-length human short fibulins were recombinantly expressed in human embryonic kidney cells and purified by immobilized metal ion affinity chromatography. All three fibulins showed various levels of degradation after the purification procedure. N-terminal sequencing revealed that all three fibulins are highly susceptible to proteolysis within the N-terminal linker region of the first calcium-binding epidermal growth factor domain. Proteolytic susceptibility of the linker correlated with its length. Exposure of these fibulins to matrix metalloproteinase (MMP)-1, -2, -3, -7, -9, and -12 resulted in similar proteolytic fragments with MMP-7 and -12 being the most potent proteases. Fibulin-3 proteolysis was almost completely inhibited in cell culture by the addition of 25 μm doxycycline (a broad spectrum MMP inhibitor). Reducible fibulin-4 dimerization and multimerization were consistently observed by SDS-PAGE, Western blotting, and mass spectrometry. Atomic force microscopy identified monomers, dimers, and multimers in purified fibulin-4 preparations with sizes of ∼10–15, ∼20–25, and ∼30–50 nm, respectively. All short fibulins strongly adhered to human fibroblasts and smooth muscle cells. Although only fibulin-5 has an RGD integrin binding site, all short fibulins adhere at a similar level to the respective cells. Solid phase binding assays detected strong calcium-dependent binding of the short fibulins to immobilized heparin, suggesting that these fibulins may bind cell surface-located heparan sulfate.     

High-level production of the non-cariogenic sucrose isomer palatinose in transgenic tobacco plants strongly impairs development

Palatinose (isomaltulose, 6-O-alpha-D-glucopyranosyl-D-fructose) is a structural isomer of sucrose which is produced from sucrose by some bacterial strains as a reserve material during periods of low carbon availability. The ability to synthesise palatinose is not only advantageous for the bacteria but is also of industrial interest since palatinose is used as a sucrose substitute in food production. To explore the possibility of palatinose production in plants a recently isolated sucrose isomerase gene (palI; EC 5.4.99.11) from Erwinia rhapontici [F. B?rnke et al. (2001) J Bacteriol 183: 2425-2430] was cloned into a plant expression vector between the constitutive 35S CaMV promoter and the octopine synthase polyadenylation signal. To allow secretion of the protein into the apoplast the signal peptide of the potato proteinase inhibitor II was N-terminally fused to the pall coding region. Expression of the protein was verified by northern and western blot analyses. Efficient secretion of the protein was demonstrated by palI detection in intercellular fluids. Transgenic plants expressing palI accumulated high levels of palatinose. As a consequence, transgenic plants showed severe phenotypic alterations. Young leaves were curled and developed bleached areas during maturation. Flowers were misshapen and sterile. Based on nonaqueous fractionation experiments palatinose was found in several subcellular compartments, indicating limited membrane transport of the sugar. In contrast to results obtained with short-term feeding experiments, no evidence for palatinose-mediated regulation of photosynthetic or defence genes could be obtained in the transgenic palI-expressing tobacco plants. Based on our results we conclude that plants can efficiently be used as bioreactors for the production of palatinose. Furthermore, tissue-specific expression of palI should allow carbon allocation to specific tissues and/or cell-types to be modulated.     

A short story about a big magic bug

Bacillus megaterium, the "big beast," is a Gram-positive bacterium with a size of 4 × 1.5 μm. During the last years, it became more and more popular in the field of biotechnology for its recombinant protein production capacity. For the purpose of intra- as well as extracellular protein synthesis several vectors were constructed and commercialized (MoBiTec GmbH, Germany). On the basis of two compatible vectors, a T7 RNA polymerase driven protein production system was established. Vectors for chromosomal integration enable the direct manipulation of the genome. The vitamin B(12) biosynthesis of B. megaterium served as a model for the systematic development of a production strain using these tools. For this purpose, the overexpression of chromosomal and plasmid encoded genes and operons, the synthesis of anti-sense RNA for gene silencing, the removal of inhibitory regulatory elements in combination with the utilization of strong promoters, directed protein design, and the recombinant production of B(12) binding proteins to overcome feedback inhibition were successfully employed. For further system biotechnology based optimization strategies the genome sequence will provide a closer look into genomic capacities of B. megaterium. DNA arrays are available. Proteome, fluxome and metabolome analyses are possible. All data can be integrated by using a novel bioinformatics platform. Finally, the size of the "big beast" B. megaterium invites for cell biology research projects. All these features provide a solid basis for challenging biotechnological approaches.     

The Cdc48-Ufd1-Npl4 complex is central in ubiquitin-proteasome triggered catabolite degradation of fructose-1,6-bisphosphatase

The switch from gluconeogenesis to glycolysis in yeast has been shown to require ubiquitin-proteasome dependent elimination of the key enzyme fructose-1,6-bisphosphatase (FBPase). Prior to proteasomal degradation, polyubiquitination of the enzyme occurs via the ubiquitin-conjugating enzymes Ubc1, Ubc4, Ubc5 and Ubc8 in conjunction with a novel multi-subunit ubiquitin ligase, the Gid complex. As an additional machinery required for the catabolite degradation process, we identified the trimeric Cdc48^Ufd1-Npl4 complex and the ubiquitin receptors Dsk2 and Rad23. We show that this machinery acts between polyubiquitination of FBPase and its degradation by the proteasome.     

Dodecins: a family of lumichrome binding proteins

Dodecin is a small dodecameric flavoprotein from Halobacterium salinarum that contains two flavins stacked between two tryptophan residues to form an aromatic tetrade. The functional properties of heterologously expressed dodecin were investigated by fluorescence spectroscopy, which allowed the determination of dissociation constants for a number of protein-ligand complexes. The values obtained were in the nanomolar to micromolar range and correlate positively with the ligand size. These data were supplemented by X-ray crystal structures of the apododecin and holocomplexes with lumichrome, lumiflavin, riboflavin and FMN at resolutions between 1.55 to 1.95 A to unravel a gating mechanism as the structural basis for the preferential binding of the small ligands lumichrome and lumiflavin. The detailed analysis of the dodecin manifold for preferential binding of lumichrome and lumiflavin provides insight on a subatom level into a protein's strategy to gain selectivity for low molecular mass compounds by steric restrictions rather than specific interactions. Investigations on the ligand composition of a wild-type dodecin crystal (1.32 A resolution) support conclusions of functional and structural investigations on heterologously expressed dodecin, and strongly suggest that lumichrome, a molecule associated with the flavin metabolism, is a ligand of dodecin in vivo. Studies on mutant protein and a Halorhodospira halophila homologue spread the idea of a lumichrome binding system as a possible "waste"-trapping device, widely distributed in prokaryotes.     

ER arrival sites for COPI vesicles localize to hotspots of membrane trafficking

COPI‐coated vesicles mediate retrograde membrane traffic from the cis‐Golgi to the endoplasmic reticulum (ER) in all eukaryotic cells. However, it is still unknown whether COPI vesicles fuse everywhere or at specific sites with the ER membrane. Taking advantage of the circumstance that the vesicles still carry their coat when they arrive at the ER, we have visualized active ER arrival sites (ERAS) by monitoring contact between COPI coat components and the ER‐resident Dsl tethering complex using bimolecular fluorescence complementation (BiFC). ERAS form punctate structures near Golgi compartments, clearly distinct from ER exit sites. Furthermore, ERAS are highly polarized in an actin and myosin V‐dependent manner and are localized near hotspots of plasma membrane expansion. Genetic experiments suggest that the COPI?Dsl BiFC complexes recapitulate the physiological interaction between COPI and the Dsl complex and that COPI vesicles are mistargeted in dsl1 mutants. We conclude that the Dsl complex functions in confining COPI vesicle fusion sites.     

Reduced hnRNPA3 increases C9orf72 repeat RNA levels and dipeptide‐repeat protein deposition

Intronic hexanucleotide (G₄C₂) repeat expansions in C9orf72 are genetically associated with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The repeat RNA accumulates within RNA foci but is also translated into disease characterizing dipeptide repeat proteins (DPR). Repeat‐dependent toxicity may affect nuclear import. hnRNPA3 is a heterogeneous nuclear ribonucleoprotein, which specifically binds to the G₄C₂ repeat RNA. We now report that a reduction of nuclear hnRNPA3 leads to an increase of the repeat RNA as well as DPR production and deposition in primary neurons and a novel tissue culture model that reproduces features of the C9orf72 pathology. In fibroblasts derived from patients carrying extended C9orf72 repeats, nuclear RNA foci accumulated upon reduction of hnRNPA3. Neurons in the hippocampus of C9orf72 patients are frequently devoid of hnRNPA3. Reduced nuclear hnRNPA3 in the hippocampus of patients with extended C9orf72 repeats correlates with increased DPR deposition. Thus, reduced hnRNPA3 expression in C9orf72 cases leads to increased levels of the repeat RNA as well as enhanced production and deposition of DPR proteins and RNA foci.     

Normal giants? Temporal and latitudinal shifts of Palaeozoic marine invertebrate gigantism and global change

During and after the Cambrian explosion, very large marine invertebrate species have evolved in several groups. Gigantism in Carboniferous land invertebrates has been explained by a peak in atmospheric oxygen concentrations, but Palaeozoic marine invertebrate gigantism has not been studied empirically and explained comprehensively. By quantifying the spatiotemporal distribution of the largest representatives of some of the major marine invertebrate clades (orthoconic cephalopods, ammonoids, trilobites, marine eurypterids), we assessed possible links between environmental parameters (atmospheric or oceanic oxygen concentrations, ocean water temperature or sea level) and maximum body size, but we could not find a straightforward relationship between both. Nevertheless, marine invertebrate gigantism within these groups was temporally concentrated within intervals of high taxonomic diversity (Ordovician, Devonian) and spatially correlated with latitudes of high occurrence frequency. Regardless of whether temporal and spatial variation in sampled diversity and occurrence frequency reflect true biological patterns or sampling controls, we find no evidence that the occurrences of giants in these groups were controlled by optimal conditions other than those that controlled the group as a whole; if these conditions shift latitudinally, occurrences of giants will shift as well. It is tempting to attribute these shifts to contemporary changes in temperature, oxygen concentrations in the atmosphere and the oceans as well as global palaeogeography over time, but further collection‐based studies are necessary on finer stratigraphic and phylogenetic resolution to corroborate such hypotheses and rule out sampling or collection biases.