34 results on '"Schinkel H"'
Search Results
2. Biotic and Abiotic Controls Over Ecosystem Cycling of Stable Natural Nitrogen, Carbon and Sulphur Isotopes
- Author
-
Bauer, G. A., Gebauer, G., Harrison, A. F., Högberg, P., Högbom, L., Schinkel, H., Taylor, A. F. S., Novak, M., Buzek, F., Harkness, D., Persson, T., Schulze, E.-D., Caldwell, M. M., editor, Heldmaier, G., editor, Lange, O. L., editor, Mooney, H. A., editor, Schulze, E.-D., editor, Sommer, U., editor, and Schulze, Ernst-Detlef, editor
- Published
- 2000
- Full Text
- View/download PDF
3. Polyamines delay leaf maturation in low‐alkaloid tobacco varieties
- Author
-
Nölke, G., Volke, D., Chudobová, I., Houdelet, M., Lusso, M., Frederick, J., Adams, A., Kudithipudi, C., Warek, U., Strickland, J.A., Xu, D., Schinkel, H., Schillberg, S., and Publica
- Abstract
The development of low‐alkaloid (LA) tobacco varieties is an important target in the tobacco breeding industry. However, LA Burley 21 plants, in which the Nic1 and Nic2 loci controlling nicotine biosynthesis are deleted, are characterized by impaired leaf maturation that leads to poor leaf quality before and after curing. Polyamines are involved in key developmental, physiological, and metabolic processes in plants, and act as anti‐senescence and anti‐ripening regulators. We investigated the role of polyamines in tobacco leaf maturation by analyzing the free and conjugated polyamine fractions in the leaves and roots of four Burley 21 varieties: NA (normal alkaloid levels, wild‐type control), HI (high intermediates, nic2−), LI (low intermediates, nic1−), and LA (nic1−nic2−). The pool of conjugated polyamines increased with plant age in the roots and leaves of all four varieties, but the levels of free and conjugated putrescine and spermidine were higher in the LI and LA plants than NA controls. The increase in the polyamine content correlated with delayed maturation and senescence, i.e., LA plants with the highest polyamine levels showed the most severe impaired leaf maturation phenotype, characterized by higher chlorophyll content and more mesophyll cells per unit leaf area. Treatment of LA plants with inhibitors of polyamine biosynthesis and/or the growth regulator Ethephon® reduced accumulation of polyamines, achieving a partial amelioration of the LA phenotype. Our data show that the regulation of polyamine homeostasis is strongly disrupted in LA plants, and that free and conjugated polyamines contribute to the observed impairment of leaf maturation.
- Published
- 2018
4. Optimization of a multi-stage, multi-subunit malaria vaccine candidate for the production in Pichia pastoris by the identification and removal of protease cleavage sites
- Author
-
Spiegel, H., Schinkel, H., Kastilan, R., Dahm, P., Boes, A., Scheuermayer, M., Chudobova, I., Maskus, D., Fendel, R., Schillberg, S., Reimann, A., Fischer, R., and Publica
- Abstract
We demonstrated the successful optimization of a recombinant multi-subunit malaria vaccine candidate protein for production in the methylotrophic yeast Pichia pastoris by the identification and subsequent removal of two protease cleavage sites. After observing protein degradation in the culture supernatant of a fed-batch fermentation, the predominant proteolytic fragment of the secreted recombinant protein was analyzed by mass spectrometry. The MS data indicated the cleavage of an amino acid sequence matching the yeast KEX2-protease consensus motif EKRE. The cleavage in this region was completely abolished by the deletion of the EKRE motif in a modified variant. This modified variant was produced, purified, and used for immunization of rabbits, inducing high antigen specific antibody titers (2x10(6)). Total IgG from rabbit immune sera recognized different stages of Plasmodium falciparum parasites in immunofluorescence assays, indicating native folding of the vaccine candidate. However, the modified variant was still degraded, albeit into different fragments. Further analysis by mass spectrometry and N-terminal sequencing revealed a second cleavage site downstream of the motif PEVK. We therefore removed a 17-amino-acid stretch including the PEVK motif, resulting in the subsequent production of the full-length recombinant vaccine candidate protein without significant degradation, with a yield of 53mg per liter culture volume. We clearly demonstrate that the proteolytic degradation of recombinant proteins by endogenous P. pastoris proteases can be prevented by the identification and removal of such cleavage sites. This strategy is particularly relevant for the production of recombinant subunit vaccines, where product yield and stability play a more important role than for the production of a stringently-defined native sequence which is necessary for most therapeutic molecules.
- Published
- 2015
5. Beitrag zur Beseitigung der Anregungsbeeinflussung bei flammenspektralanalytischen Untersuchungen: Eine Universalvorschrift zur Bestimmung von Kalium, Natrium und Lithium in Proben jeder Zusammensetzung
- Author
-
Schuhknecht, W. and Schinkel, H.
- Published
- 1963
- Full Text
- View/download PDF
6. Beitrag zur Deutung von Verdampfungs-, Zersetzungs- und Anregungsvorgängen in Flammen: Der Einfluß von Aluminium, Kieselsäure und Phosphorsäure auf die flammenphotometrische Erdalkalibestimmung
- Author
-
Schuhknecht, W. and Schinkel, H.
- Published
- 1958
- Full Text
- View/download PDF
7. Die flammenphotometrische Bestimmung von Calcium, Strontium und Barium nebeneinander
- Author
-
Schuhknecht, W. and Schinkel, H.
- Published
- 1958
- Full Text
- View/download PDF
8. Die flammenphotometrische Bestimmung geringer Mengen von Kalium, Natrium und Lithium neben großen Mengen von Erdalkalien
- Author
-
Schuhknecht, W. and Schinkel, H.
- Published
- 1954
- Full Text
- View/download PDF
9. Holistic approach to malaria elimination
- Author
-
Boes, A, Spiegel, H, Beiss, V, Edgü, G, Schinkel, H, Houdelet, M, Schillberg, S, Sack, M, Rademacher, T, Dahm, P, Kastilan, R, Buyel, J, Holland, T, Blessing, D, Vöpel, N, Hellwig, S, Drossard, J, Fendel, R, Kapelski, S, Maskus, D, Addai-Mensah, O, Seidel, M, Barth, S, Geese, M, Reiss, K, Wenzel, C, Hügel, C, Scholz, O, Kostka, G, Benz, M, Haßlmeyer, E, Münzenmayer, C, Weigand, C, Scheuermayer, M, Pradel, G, Remarque, E, Faber, B, Horstmann, R, Tannich, E, Fischer, R, Reimann, A, Boes, A, Spiegel, H, Beiss, V, Edgü, G, Schinkel, H, Houdelet, M, Schillberg, S, Sack, M, Rademacher, T, Dahm, P, Kastilan, R, Buyel, J, Holland, T, Blessing, D, Vöpel, N, Hellwig, S, Drossard, J, Fendel, R, Kapelski, S, Maskus, D, Addai-Mensah, O, Seidel, M, Barth, S, Geese, M, Reiss, K, Wenzel, C, Hügel, C, Scholz, O, Kostka, G, Benz, M, Haßlmeyer, E, Münzenmayer, C, Weigand, C, Scheuermayer, M, Pradel, G, Remarque, E, Faber, B, Horstmann, R, Tannich, E, Fischer, R, and Reimann, A
- Published
- 2014
10. Downregulation of high-isoelectric-point extracellular superoxide dismutase mediates alterations in the metabolism of reactive oxygen species and developmental disturbances in hybrid aspen
- Author
-
Srivastava, V., Schinkel, H., Witzell, J., Hertzberg, M., Torp, M., Srivastava, M.K., Karpinska, B., Melzer, M., Wingsle, G., and Publica
- Published
- 2007
11. Production of Desmodus rotundus plasminogen activator alpha1 (DSPA alpha1) in tobacco is hampered by proteolysis
- Author
-
Schiermeyer, A., Schinkel, H., Apel, S., Fischer, R., Schillberg, S., and Publica
- Published
- 2005
12. Nitrogen-related root variables of trees along an N-deposition gradient in Europe
- Author
-
Hogberg, P., primary, Hogbom, L., additional, and Schinkel, H., additional
- Published
- 1998
- Full Text
- View/download PDF
13. Multiple forms of extracellular superoxide dismutase in needles, stem tissues and seedlings of Scots pine
- Author
-
Schinkel, H., primary, Streller, S., additional, and Wingsle, G., additional
- Published
- 1998
- Full Text
- View/download PDF
14. A novel superoxide dismutase with a high isoelectric point in higher plants. expression, regulation, and protein localization.
- Author
-
Karpinska, B, Karlsson, M, Schinkel, H, Streller, S, Süss, K H, Melzer, M, and Wingsle, G
- Abstract
Several isoforms of superoxide dismutase (SOD) with a high isoelectric point (pI) have been identified by isoelectric focusing chromatography in protein extracts from Scots pine (Pinus sylvestris) needles. One of these isoforms, a CuZn-SOD with a pI of about 10 and thus denoted hipI-SOD, has been isolated and purified to apparent homogeneity. A cDNA encoding the hipI-SOD protein was cloned and sequenced. Northern hybridization of mRNA isolated from different organs and tissues showed that hipI-SOD has a markedly different pattern of expression compared with chloroplastic and cytosolic SOD. Furthermore, the transcript levels of hipI-SOD and cytosolic SOD were found to respond differently to mechanical wounding, treatment with oxidized glutathione, paraquat, and ozone. Immunogold electron microscopy localized the hipI-SOD in the plasma membrane of sieve cells and the Golgi apparatus of albuminous cells. Moreover, high protein density was also detected in extracellular spaces such as secondary cell wall thickenings of the xylem and sclerenchyma and in intercellular spaces of parenchyma cells.
- Published
- 2001
- Full Text
- View/download PDF
15. Beitrag zur Deutung von Verdampfungs-, Zersetzungs- und Anregungsvorgängen in Flammen
- Author
-
Schuhknecht, W. and Schinkel, H.
- Abstract
1.Unsere Auffassung, daß die Störung der Calciumemission durch Aluminiumverbindungen nicht als echte Anregungsbeeinflussung sondern mit der Annahme einer Verdampfungsblockierung durch Bildung schwerflüchtiger bzw. schlecht wärmeleitender Verbindungen zu deuten ist, wird erneut bestätigt.2.Es wird sichergestellt, daß die Bildung dieser Verbindungen nicht in der Gasphase erfolgt, sondern daß sie vielmehr in der Flamme direkt aus den festen oder flüssigen Teilchen des Aerosols entstehen.3.Hinsichtlich des Reaktionsmechanismus besteht kein grundsätz-licher, sondern nur ein quantitativer Unterschied zwischen verschiedenen Aluminiumsalzen.4.Die Beeinflussung der Calciurnemission durch Kieselsäure und Phos-phorsäure ist nach unseren Versuchsergebnissen ebenfalls als Folge einer Verdampfungsblockierung zu deuten.5.Es wird durch Versuche in verschiedenen Flammen gezeigt, daß die Störung der Calciumemission mit Zunahme der Temperatur, welche die Teilchen beim Durchlaufen der Flamme im Mittel erreichen, abnimmt.6.Die von den Teilchen erreichte Temperatur ist nicht nur von der Flammentemperatur, sondern wesentlich auch von der Verweilzeit der Teilchen in der Flamme und dem Tröpfchenspektrum abhängig. Aus diesem Grund ist die Störung unter sonst gleichen Bedingungen — z.B. bei Verwendung von Wasserstoff und Sauerstoff — in der Flamme eines Vorkammerzerstüberbrenners bedeutend geringer als in der Flamme eines Direktzerstüberbrenners.7.Es wird eine Arbeitsvorschrift zur flammenphotometrischen Bestimmung des Calciumgehaltes mitgeteilt, welche auf tonerde-, kieselsäure- und phosphorsürereiche Proben ohne Einschrünkung anwendbar ist.
- Published
- 1958
- Full Text
- View/download PDF
16. Beitrag zur Beseitigung der Anregungsbeeinflussung bei flammenspektralanalytischen Untersuchungen
- Author
-
Schuhknecht, W. and Schinkel, H.
- Abstract
1.Die im Schrifttum verschiedentlich erörterte spektroskopische Pufferung wurde unter Verwendung des leichtionisierbaren Caesiums als Puffer am Beispiel der Bestimmung von Kalium, Natrium und Li-thium erstmalig quantitativ untersucht.2.Die Messungen ergaben, daß die gegenseitige Anregungsbeeinflussung von Kalium, Natrium und Lithium selbst für das besonders anfällige Kalium durch einen Zusatz von Caesiumchlorid völlig beseitigt werden kann.3.Die Pufferung mit Caesiumsalz bietet ferner den Vorteil, daß auch alle anderen, auf Ionisationsvorgängen beruhenden Störungen flammenspektralanalytischer Messungen behoben werden. Unter anderem ergibt sich für verschiedene Zonen der Flamme ein identischer Verlauf der Intensitäts-Konzentrationskurven.4.Bei gemeinsamer Anwendung von Caesiumchlorid und Aluminiumnitrat, welches die Emission der Erdalkalien und verschiedener anderer Elemente unterdrückt und zusammen mit dem Caesiumchlorid die physikalischen Eigenschaften der Meßlösung festlegt, lassen sich Proben jeder Zusammensetzung nach einer einzigen Vorschrift analysieren. Diese Universalvorschrift wird mitgeteilt und durch Analysenreihen belegt.5.Es wird gezeigt, daß die Universalvorschrift nicht nur auf Proben jeder Zusammensetzung, sondern auch auf Zerstäuberbrenner jeder Konstruktion und mit allen üblichen Brenngasgemischen angewendet werden kann.
- Published
- 1963
- Full Text
- View/download PDF
17. Photocycloadditions of aromatic thiones to allenes
- Author
-
Bos, H.J.T., primary, Schinkel, H., additional, and Wijsman, Th.C.M., additional
- Published
- 1971
- Full Text
- View/download PDF
18. ChemInform Abstract: PHOTOCYCLOADDITIONSRK. VON AROMATISCHEN THIONEN AN ALLENE
- Author
-
BOS, H. J. T., primary, SCHINKEL, H., additional, and WIJSMAN, T. C. M., additional
- Published
- 1972
- Full Text
- View/download PDF
19. Impact of nicotine pathway downregulation on polyamine biosynthesis and leaf ripening in tobacco.
- Author
-
Nölke G, Chudobova I, Houdelet M, Volke D, Lusso M, Frederick J, Kudithipudi C, Shen Y, Warek U, Strickland JA, Xu D, Schinkel H, and Schillberg S
- Abstract
Traditional breeding and molecular approaches have been used to develop tobacco varieties with reduced nicotine and secondary alkaloid levels. However, available low-alkaloid tobacco varieties have impaired leaf quality likely due to the metabolic consequences of nicotine biosynthesis downregulation. Recently, we found evidence that the unbalanced crosstalk between nicotine and polyamine pathways is involved in impaired leaf ripening of a low-alkaloid (LA) Burley 21 line having a mutation at the Nic1 and Nic2 loci, key biosynthetic regulators of nicotine biosynthesis. Since the Nic1 and Nic2 loci are comprised of several genes, all phenotypic changes seen in LA Burley 21 could be due to a mixture of genetics-based responses. Here, we investigated the commercial burley variety TN90 LC and its transgenic versions with only one downregulated gene, either putrescine methyl transferase (PMT-RNAi) or PR50-protein (PR50-RNAi). Nicotine levels of cured lamina of TN90 LC, TN90 PMT-RNAi and TN90 PR50-RNAi, were 70.5 ± 3.8, 2.4 ± 0.5, and 6.0 ± 1.1 mg/g dry weight, respectively. Low-alkaloid transgenic lines showed delayed leaf maturation and impaired leaf quality. We analyzed polyamine contents and ripening markers in wild-type TN90 control plants (WT) and the two transgenic lines. The ripening markers revealed that the PMT-RNAi line showed the most pronounced impaired leaf maturation phenotype at harvest, characterized by higher chlorophyll (19%) and glucose (173%) contents and more leaf mesophyll cells per area (25%), while the ripening markers revealed that maturation of PR50-RNAi plants was intermediate between PMT-RNAi and WT lines. Comparative polyamine analyses showed an increase in free and conjugated polyamines in roots of both transgenic lines, this being most pronounced in the PMT-RNAi plants. For PMT-RNAi plants, there were further perturbations of polyamine content in the leaves, which mirrored the general phenotype, as PR50-RNAi transgenic plants looked more similar to the WT than PMT-RNAi transgenic plants. Activity of ornithine decarboxylase, the enzyme that catalyzes the committing step of polyamine biosynthesis, was significantly higher in roots and mature leaves of PMT-RNAi plants in comparison to WT, while there was no increase observed for arginine decarboxylase. Treatment of both transgenic lines with polyamine biosynthesis inhibitors decreased the polyamine content and ameliorated the phenotype, confirming the intricate interplay of polyamine and nicotine biosynthesis in tobacco and the influence of this interplay on leaf ripening., Competing Interests: M.L., J.F., C.K., Y.S., U.W., J.A.S., and D.X. were employed by Altria Client Services, and Altria Client Services provided funding for the research. All authors declare no conflict of interest., (© 2021 The Authors. Plant Direct published by American Society of Plant Biologists, Society for Experimental Biology and John Wiley & Sons Ltd.)
- Published
- 2021
- Full Text
- View/download PDF
20. Targeted insertion of large DNA sequences by homology-directed repair or non-homologous end joining in engineered tobacco BY-2 cells using designed zinc finger nucleases.
- Author
-
Schiermeyer A, Schneider K, Kirchhoff J, Schmelter T, Koch N, Jiang K, Herwartz D, Blue R, Marri P, Samuel P, Corbin DR, Webb SR, Gonzalez DO, Folkerts O, Fischer R, Schinkel H, Ainley WM, and Schillberg S
- Abstract
Targeted integration of recombinant DNA fragments into plant genomes by DNA double-strand break (DSB) repair mechanisms has become a powerful tool for precision engineering of crops. However, many targeting platforms require the screening of many transgenic events to identify a low number of targeted events among many more random insertion events. We developed an engineered transgene integration platform (ETIP) that uses incomplete marker genes at the insertion site to enable rapid phenotypic screening and recovery of targeted events upon functional reconstitution of the marker genes. The two marker genes, encoding neomycin phosphotransferase II ( nptII ) and Discosoma sp . red fluorescent protein ( DsRed ) enable event selection on kanamycin-containing selective medium and subsequent screening for red fluorescent clones. The ETIP design allows targeted integration of donor DNA molecules either by homology-directed repair (HDR) or non-homologous end joining (NHEJ)-mediated mechanisms. Targeted donor DNA integration is facilitated by zinc finger nucleases (ZFN). The ETIP cassette was introduced into Nicotiana tabacum BY-2 suspension cells to generate target cell lines containing a single copy locus of the transgene construct. The utility of the ETIP platform has been demonstrated by targeting DNA constructs containing up to 25-kb payload. The success rate for clean targeted DNA integration was up to 21% for HDR and up to 41% for NHEJ based on the total number of calli analyzed by next-generation sequencing (NGS). The rapid generation of targeted events with large DNA constructs expands the utility of the nuclease-mediated gene addition platform both for academia and the commercial sector., Competing Interests: K.J., R.B., P.M., P.S., D.R.C, S.R.W., D.O.G., W.M.A., and O.F. were employed by Dow AgroSciences LLC, a wholly owned subsidiary of The Dow Chemical Company, at the time the work was performed, and Dow AgroSciences provided funding for the research. Dow AgroSciences is now part of Corteva Agrisciences, which has filed a patent application on the ETIP concept and the cell lines containing the targeting cassette. All authors declare no conflict of interest.
- Published
- 2019
- Full Text
- View/download PDF
21. Polyamines delay leaf maturation in low-alkaloid tobacco varieties.
- Author
-
Nölke G, Volke D, Chudobová I, Houdelet M, Lusso M, Frederick J, Adams A, Kudithipudi C, Warek U, Strickland JA, Xu D, Schinkel H, and Schillberg S
- Abstract
The development of low-alkaloid (LA) tobacco varieties is an important target in the tobacco breeding industry. However, LA Burley 21 plants, in which the Nic1 and Nic2 loci controlling nicotine biosynthesis are deleted, are characterized by impaired leaf maturation that leads to poor leaf quality before and after curing. Polyamines are involved in key developmental, physiological, and metabolic processes in plants, and act as anti-senescence and anti-ripening regulators. We investigated the role of polyamines in tobacco leaf maturation by analyzing the free and conjugated polyamine fractions in the leaves and roots of four Burley 21 varieties: NA (normal alkaloid levels, wild-type control), HI (high intermediates, nic2
- ), LI (low intermediates, nic1- ), and LA ( nic1- nic2- ). The pool of conjugated polyamines increased with plant age in the roots and leaves of all four varieties, but the levels of free and conjugated putrescine and spermidine were higher in the LI and LA plants than NA controls. The increase in the polyamine content correlated with delayed maturation and senescence, i.e., LA plants with the highest polyamine levels showed the most severe impaired leaf maturation phenotype, characterized by higher chlorophyll content and more mesophyll cells per unit leaf area. Treatment of LA plants with inhibitors of polyamine biosynthesis and/or the growth regulator Ethephon® reduced accumulation of polyamines, achieving a partial amelioration of the LA phenotype. Our data show that the regulation of polyamine homeostasis is strongly disrupted in LA plants, and that free and conjugated polyamines contribute to the observed impairment of leaf maturation.- Published
- 2018
- Full Text
- View/download PDF
22. Genome editing: intellectual property and product development in plant biotechnology.
- Author
-
Schinkel H and Schillberg S
- Subjects
- CRISPR-Cas Systems, Genetic Engineering methods, Mutagenesis, Site-Directed, Plants, Genetically Modified, Biotechnology methods, Gene Editing methods, Genome, Plant genetics, Intellectual Property, Plants genetics
- Abstract
Genome editing is a revolutionary technology in molecular biology. While scientists are fascinated with the unlimited possibilities provided by directed and controlled changes in DNA in eukaryotes and have eagerly adopted such tools for their own experiments, an understanding of the intellectual property (IP) implications involved in bringing genome editing-derived products to market is often lacking. Due to the ingenuity of genome editing, the time between new product conception and its actual existence can be relatively short; therefore knowledge about IP of the various genome editing methods is relevant. This point must be regarded in a national framework as patents are instituted nationally. Therefore, when designing scientific work that could lead to a product, it is worthwhile to consider the different methods used for genome editing not only for their scientific merits but also for their compatibility with a speedy and reliable launch into the desired market.
- Published
- 2016
- Full Text
- View/download PDF
23. Targeted gene exchange in plant cells mediated by a zinc finger nuclease double cut.
- Author
-
Schneider K, Schiermeyer A, Dolls A, Koch N, Herwartz D, Kirchhoff J, Fischer R, Russell SM, Cao Z, Corbin DR, Sastry-Dent L, Ainley WM, Webb SR, Schinkel H, and Schillberg S
- Subjects
- Blotting, Southern, Deoxyribonucleases genetics, Flow Cytometry methods, Kanamycin Resistance genetics, Luminescent Proteins genetics, Luminescent Proteins metabolism, Plant Cells, Plants, Genetically Modified, Recombinational DNA Repair genetics, Nicotiana cytology, Zinc Fingers, Red Fluorescent Protein, Deoxyribonucleases metabolism, Gene Targeting methods, Nicotiana genetics
- Abstract
Genome modification by homology-directed repair (HDR) is an attractive tool for the controlled genetic manipulation of plants. Here, we report the HDR-mediated gene exchange of expression cassettes in tobacco BY-2 cells using a designed zinc finger nuclease (ZFN). The target contained a 7-kb fragment flanked by two ZFN cutting sites. That fragment was replaced with a 4-kb donor cassette, which integrates gene markers for selection (kanamycin resistance) and for scoring targeting (red fluorescent protein, RFP). Candidates resulting from cassette exchange were identified by molecular analysis of calli generated by transformation via direct DNA delivery. The precision of HDR-mediated donor integration was evaluated by Southern blot analysis, sequencing of the integration locus and analysis of RFP fluorescence by flow cytometry. Screening of 1326 kanamycin-resistant calli yielded 18 HDR events, 16 of which had a perfect cassette exchange at the insert junction and 13 of which produced functional RFP. Our results demonstrate that ZFN-based HDR can be used for high frequency, precise, targeted exchange of fragments of sizes that are commercially relevant in plants., (© 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)
- Published
- 2016
- Full Text
- View/download PDF
24. Acquired immune responses to three malaria vaccine candidates and their relationship to invasion inhibition in two populations naturally exposed to malaria.
- Author
-
Addai-Mensah O, Seidel M, Amidu N, Maskus DJ, Kapelski S, Breuer G, Franken C, Owusu-Dabo E, Frempong M, Rakotozandrindrainy R, Schinkel H, Reimann A, Klockenbring T, Barth S, Fischer R, and Fendel R
- Subjects
- Adult, Antigens, Protozoan immunology, Chromatography, Affinity, Enzyme-Linked Immunosorbent Assay, Female, Humans, Male, Middle Aged, Plasmodium falciparum immunology, Protozoan Proteins immunology, Malaria Vaccines immunology, Malaria, Falciparum immunology, Malaria, Falciparum prevention & control
- Abstract
Background: Malaria still represents a major cause of morbidity and mortality predominantly in several developing countries, and remains a priority in many public health programmes. Despite the enormous gains made in control and prevention the development of an effective vaccine represents a persisting challenge. Although several parasite antigens including pre-erythrocytic antigens and blood stage antigens have been thoroughly investigated, the identification of solid immune correlates of protection against infection by Plasmodium falciparum or clinical malaria remains a major hurdle. In this study, an immuno-epidemiological survey was carried out between two populations naturally exposed to P. falciparum malaria to determine the immune correlates of protection., Methods: Plasma samples of immune adults from two countries (Ghana and Madagascar) were tested for their reactivity against the merozoite surface proteins MSP1-19, MSP3 and AMA1 by ELISA. The antigens had been selected on the basis of cumulative evidence of their role in anti-malarial immunity. Additionally, reactivity against crude P. falciparum lysate was investigated. Purified IgG from these samples were furthermore tested in an invasion inhibition assay for their antiparasitic activity., Results: Significant intra- and inter- population variation of the reactivity of the samples to the tested antigens were found, as well as a significant positive correlation between MSP1-19 reactivity and invasion inhibition (p < 0.05). Interestingly, male donors showed a significantly higher antibody response to all tested antigens than their female counterparts. In vitro invasion inhibition assays comparing the purified antibodies from the donors from Ghana and Madagascar did not show any statistically significant difference. Although in vitro invasion inhibition increased with breadth of antibody response, the increase was not statistically significant., Conclusions: The findings support the fact that the development of semi-immunity to malaria is probably contingent on the development of antibodies to not only one, but a range of antigens and that invasion inhibition in immune adults may be a function of antibodies to various antigens. This supports strategies of vaccination including multicomponent vaccines as well as passive vaccination strategies with antibody cocktails.
- Published
- 2016
- Full Text
- View/download PDF
25. Optimization of a multi-stage, multi-subunit malaria vaccine candidate for the production in Pichia pastoris by the identification and removal of protease cleavage sites.
- Author
-
Spiegel H, Schinkel H, Kastilan R, Dahm P, Boes A, Scheuermayer M, Chudobová I, Maskus D, Fendel R, Schillberg S, Reimann A, and Fischer R
- Subjects
- Animals, Antibodies, Protozoan blood, Binding Sites, Biotechnology methods, Fluorescent Antibody Technique, Direct, Immunization methods, Immunoglobulin G blood, Malaria Vaccines chemistry, Malaria Vaccines genetics, Mass Spectrometry, Mice, Mutant Proteins biosynthesis, Mutant Proteins chemistry, Mutant Proteins genetics, Mutant Proteins isolation & purification, Pichia genetics, Pichia metabolism, Plasmodium falciparum immunology, Proteolysis, Rabbits, Sequence Deletion, Technology, Pharmaceutical methods, Vaccines, Synthetic biosynthesis, Vaccines, Synthetic chemistry, Vaccines, Synthetic genetics, Vaccines, Synthetic isolation & purification, Malaria Vaccines biosynthesis, Malaria Vaccines isolation & purification, Peptide Hydrolases metabolism
- Abstract
We demonstrated the successful optimization of a recombinant multi-subunit malaria vaccine candidate protein for production in the methylotrophic yeast Pichia pastoris by the identification and subsequent removal of two protease cleavage sites. After observing protein degradation in the culture supernatant of a fed-batch fermentation, the predominant proteolytic fragment of the secreted recombinant protein was analyzed by mass spectrometry. The MS data indicated the cleavage of an amino acid sequence matching the yeast KEX2-protease consensus motif EKRE. The cleavage in this region was completely abolished by the deletion of the EKRE motif in a modified variant. This modified variant was produced, purified, and used for immunization of rabbits, inducing high antigen specific antibody titers (2 × 10(6) ). Total IgG from rabbit immune sera recognized different stages of Plasmodium falciparum parasites in immunofluorescence assays, indicating native folding of the vaccine candidate. However, the modified variant was still degraded, albeit into different fragments. Further analysis by mass spectrometry and N-terminal sequencing revealed a second cleavage site downstream of the motif PEVK. We therefore removed a 17-amino-acid stretch including the PEVK motif, resulting in the subsequent production of the full-length recombinant vaccine candidate protein without significant degradation, with a yield of 53 mg per liter culture volume. We clearly demonstrate that the proteolytic degradation of recombinant proteins by endogenous P. pastoris proteases can be prevented by the identification and removal of such cleavage sites. This strategy is particularly relevant for the production of recombinant subunit vaccines, where product yield and stability play a more important role than for the production of a stringently-defined native sequence which is necessary for most therapeutic molecules., (© 2014 Wiley Periodicals, Inc.)
- Published
- 2015
- Full Text
- View/download PDF
26. Plant-based production of recombinant Plasmodium surface protein pf38 and evaluation of its potential as a vaccine candidate.
- Author
-
Feller T, Thom P, Koch N, Spiegel H, Addai-Mensah O, Fischer R, Reimann A, Pradel G, Fendel R, Schillberg S, Scheuermayer M, and Schinkel H
- Subjects
- Animals, Antigens, Protozoan immunology, Enzyme-Linked Immunosorbent Assay, Fluorescent Antibody Technique, Plantibodies immunology, Plasmodium falciparum growth & development, Recombinant Proteins biosynthesis, Recombinant Proteins genetics, Recombinant Proteins immunology, Antigens, Protozoan genetics, Malaria Vaccines immunology, Plantibodies genetics, Plasmodium falciparum immunology, Nicotiana genetics
- Abstract
Pf38 is a surface protein of the malarial parasite Plasmodium falciparum. In this study, we produced and purified recombinant Pf38 and a fusion protein composed of red fluorescent protein and Pf38 (RFP-Pf38) using a transient expression system in the plant Nicotiana benthamiana. To our knowledge, this is the first description of the production of recombinant Pf38. To verify the quality of the recombinant Pf38, plasma from semi-immune African donors was used to confirm specific binding to Pf38. ELISA measurements revealed that immune responses to Pf38 in this African subset were comparable to reactivities to AMA-1 and MSP119. Pf38 and RFP-Pf38 were successfully used to immunise mice, although titres from these mice were low (on average 1∶11.000 and 1∶39.000, respectively). In immune fluorescence assays, the purified IgG fraction from the sera of immunised mice recognised Pf38 on the surface of schizonts, gametocytes, macrogametes and zygotes, but not sporozoites. Growth inhibition assays using αPf38 antibodies demonstrated strong inhibition (≥60%) of the growth of blood-stage P. falciparum. The development of zygotes was also effectively inhibited by αPf38 antibodies, as determined by the zygote development assay. Collectively, these results suggest that Pf38 is an interesting candidate for the development of a malaria vaccine.
- Published
- 2013
- Full Text
- View/download PDF
27. Monoclonal tobacco cell lines with enhanced recombinant protein yields can be generated from heterogeneous cell suspension cultures by flow sorting.
- Author
-
Kirchhoff J, Raven N, Boes A, Roberts JL, Russell S, Treffenfeldt W, Fischer R, Schinkel H, Schiermeyer A, and Schillberg S
- Subjects
- Biotechnology methods, Cell Line, Cells, Cultured, Drug Industry methods, Humans, Plants, Genetically Modified, Antibodies, Monoclonal biosynthesis, Flow Cytometry methods, Plant Cells metabolism, Recombinant Proteins biosynthesis, Nicotiana cytology, Nicotiana metabolism
- Abstract
Plant cell suspension cultures can be used for the production of recombinant pharmaceutical proteins, but their potential is limited by modest production levels that may be unstable over long culture periods, reflecting initial culture heterogeneity and subsequent genetic and epigenetic changes. We used flow sorting to generate highly productive monoclonal cell lines from a heterogeneous population of tobacco BY-2 cells expressing the human antibody M12 by selecting the co-expressed fluorescent marker protein DsRed located on the same T-DNA. Separation yielded ∼35% wells containing single protoplasts and ∼15% wells with monoclonal microcolonies that formed within 2 weeks. Thus, enriching the population of fluorescent cells from initially 24% to 90-96% in the six monoclonal lines resulted in an up to 13-fold increase in M12 production that remained stable for 10-12 months. This is the first straightforward procedure allowing the generation of monoclonal plant cell suspension cultures by flow sorting, greatly increasing the potential of plant cells as an economical platform for the manufacture of recombinant pharmaceutical proteins., (© 2012 The Authors. Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.)
- Published
- 2012
- Full Text
- View/download PDF
28. A monoclonal antibody that specifically binds chitosan in vitro and in situ on fungal cell walls.
- Author
-
Schubert M, Agdour S, Fischer R, Olbrich Y, Schinkel H, and Schillberg S
- Subjects
- Animals, Antibodies, Bacterial immunology, Antibodies, Monoclonal immunology, Cell Wall immunology, Chitosan immunology, Fungi immunology, Mice, Mice, Inbred BALB C, Antibodies, Bacterial analysis, Antibodies, Monoclonal analysis, Cell Wall chemistry, Chitosan analysis, Fungi chemistry
- Abstract
We report the generation of the first monoclonal antibody that specifically binds to the polysaccharide chitosan. Mice were immunized with a mixture of chitosans, and hybridoma clones were screened for specific binders resulting in the isolation of a single clone secreting a chitosan-specific IgM, mAbG7. In ELISAs, the antibody can bind to chitosans of varying composition, but demonstrates the highest affinity for chitosans with lower degrees of acetylation (DA) and very poor binding to chitin. We tested the ability of the antibody to bind to chitosan in situ, using preparations of fungal cell walls. Immunofluorescence microscopy confirmed that the antibody bound strongly to the cell walls of fungi with high levels of chitosan, whereas poor staining was observed in those species with cell walls of predominantly chitin or cellulose. The potential use of this antibody for the detection of fungal contamination and the protection of plants against fungal pathogens is discussed.
- Published
- 2010
- Full Text
- View/download PDF
29. Infrared picosecond laser for perforation of single plant cells.
- Author
-
Schinkel H, Jacobs P, Schillberg S, and Wehner M
- Subjects
- Cells, Cultured, Infrared Rays, Cell Membrane physiology, Cell Membrane radiation effects, Cell Membrane Permeability radiation effects, Electroporation methods, Lasers, Nicotiana physiology, Nicotiana radiation effects
- Abstract
The functional analysis of plant cells at the cellular and subcellular levels requires novel technologies for the directed manipulation of individual cells. In this report, we demonstrate the use of an infrared (1,064 nm) picosecond laser for the perforation of tobacco cell protoplasts. A single pulse was sufficient to perforate the plasma membrane enabling the uptake of dye from the surrounding medium into the cytosol. Moreover, the procedure was shown to be suitable for the efficient delivery of DNA expression constructs to the nucleus, as demonstrated by the subsequent expression and correct targeting of a recombinant fluorescent protein. Single cell perforation using this novel optoporation method shows that isolated plant cells can be permeabilized without direct manipulation. This is a valuable procedure for cell-specific applications, particularly where the import of specific molecules into plant cells is required for functional analysis., ((c) 2007 Wiley Periodicals, Inc.)
- Published
- 2008
- Full Text
- View/download PDF
30. Downregulation of high-isoelectric-point extracellular superoxide dismutase mediates alterations in the metabolism of reactive oxygen species and developmental disturbances in hybrid aspen.
- Author
-
Srivastava V, Schinkel H, Witzell J, Hertzberg M, Torp M, Srivastava MK, Karpinska B, Melzer M, and Wingsle G
- Subjects
- Amino Acid Sequence, Gene Expression Profiling, Gene Expression Regulation, Developmental, Hybridization, Genetic, Isoelectric Point, Lignin metabolism, Molecular Sequence Data, Phenotype, Phylogeny, Plant Proteins genetics, Plant Proteins metabolism, Plant Stems cytology, Plant Stems metabolism, Plants, Genetically Modified, Populus enzymology, Populus genetics, Down-Regulation, Gene Expression Regulation, Plant, Populus growth & development, Populus metabolism, Reactive Oxygen Species metabolism, Superoxide Dismutase chemistry, Superoxide Dismutase metabolism
- Abstract
Transgenic hybrid aspen (Populus tremula L. x P. tremuloides Michx.) plants expressing a high-isoelectric-point superoxide dismutase (hipI-SOD) gene in antisense orientation were generated to investigate its function. Immunolocalization studies showed the enzyme to be localized extracellularly, in the secondary cell wall of xylem vessels and phloem fibers. The antisense lines of hipI-SOD exhibited a distinct phenotype; growth rate was reduced, stems were thinner and leaves smaller than in wild-type (WT) plants. The abundance of hipI-SOD was reduced in the bark and xylem of plants from these antisense lines. The vascular tissue of transgenic lines became lignified earlier than in WT plants and also showed an increased accumulation of reactive oxygen species (ROS). Xylem fibers and vessels were shorter and thinner in the transgenic lines than in WT plants. The total phenolic content was enhanced in the antisense lines. Furthermore, microarray analysis indicated that several enzymes involved in cell signaling, lignin biosynthesis and stress responses were upregulated in apical vascular tissues of transgenic plants. The upregulation of selected genes involved in lignin biosynthesis was also verified by real-time PCR. The results suggest that, in the transgenic plants, a premature transition into maturation occurs and the process is discussed in terms of the effects of increased accumulation of ROS due to reduced expression of hipI-SOD during development and differentiation.
- Published
- 2007
- Full Text
- View/download PDF
31. Production of an active recombinant thrombomodulin derivative in transgenic tobacco plants and suspension cells.
- Author
-
Schinkel H, Schiermeyer A, Soeur R, Fischer R, and Schillberg S
- Subjects
- Animals, CHO Cells, Cricetinae, Cricetulus, Electrophoresis, Polyacrylamide Gel, Humans, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Thrombomodulin isolation & purification, Thrombomodulin metabolism, Nicotiana metabolism, Plants, Genetically Modified, Recombinant Proteins genetics, Thrombomodulin genetics, Nicotiana genetics
- Abstract
Thrombomodulin is a membrane-bound protein that plays an active role in the blood coagulation system by binding thrombin and initiating the protein C anticoagulant pathway. Solulin is a recombinant soluble derivative of human thrombomodulin. It is used for the treatment of thrombotic disorders. To evaluate the production of this pharmaceutical protein in plants, expression vectors were generated using four different N-terminal signal peptides. Immunoblot analysis of transiently transformed tobacco leaves showed that intact Solulin could be detected using three of these signal peptides. Furthermore transgenic tobacco plants and BY2 cells producing Solulin were generated. Immunoblot experiments showed that Solulin accumulated to maximum levels of 115 and 27 microg g(-1) plant material in tobacco plants and BY2 cells, respectively. Activity tests performed on the culture supernatant of transformed BY2 cells showed that the secreted Solulin was functional. In contrast, thrombomodulin activity was not detected in total soluble protein extracts from BY2 cells, probably due to inhibitory effects of substances in the cell extract. N-terminal sequencing was carried out on partially purified Solulin from the BY2 culture supernatant. The sequence was identical to that of Solulin produced in Chinese hamster ovary cells, confirming correct processing of the N-terminal signal peptide. We have demonstrated that plants and plant cell cultures can be used as alternative systems for the production of an active recombinant thrombomodulin derivative.
- Published
- 2005
- Full Text
- View/download PDF
32. Production of Desmodus rotundus salivary plasminogen activator alpha1 (DSPAalpha1) in tobacco is hampered by proteolysis.
- Author
-
Schiermeyer A, Schinkel H, Apel S, Fischer R, and Schillberg S
- Subjects
- Blotting, Western, Feasibility Studies, Histidine chemistry, Hydrolysis, Plant Leaves genetics, Plant Leaves metabolism, Plants, Genetically Modified, Plasminogen Activators chemistry, Plasminogen Activators genetics, Plasminogen Activators isolation & purification, Protein Processing, Post-Translational, Recombinant Proteins biosynthesis, Recombinant Proteins chemistry, Recombinant Proteins isolation & purification, Nicotiana genetics, Plants, Toxic, Plasminogen Activators biosynthesis, Nicotiana metabolism
- Abstract
The high fibrin specificity of Desmodus rotundus salivary plasminogen activator alpha1 (DSPAalpha1 or desmoteplase (INN)) makes it a promising candidate for the treatment of acute ischemic stroke. In the current study we explored the use of transgenic tobacco plants and BY-2 suspension cells as alternative production platforms for this drug. Four different N-terminal signal peptides, from plants and animals, were used to translocate the recombinant DSPAalpha1 protein to the endomembrane system. Intact recombinant DSPAalpha1 was produced in transgenic plants and BY-2 cells, although a certain degree of degradation was observed in immunoblotted extracts. The choice of signal peptide had no major influence on the degradation pattern or recombinant protein accumulation, which reached a maximum level of 38 microg/g leaf material. N-terminal sequencing of purified, His6-tagged DSPAalpha1 revealed only minor changes in the position of signal peptide cleavage compared to the same protein expressed in Chinese hamster ovary cells. However, correctly processed recombinant DSPAalpha1 was also detected. The enzymatic activity of the recombinant protein was confirmed using an in vitro assay with unpurified and purified samples, demonstrating that plants are suitable for the production of functional DSPAalpha1. In contrast to whole plant cell extracts, no recombinant DSPAalpha1 was detected in the culture supernatant of transgenic BY-2 cells. Further analysis showed that recombinant DSPAalpha1 is subject to proteolysis and that endogenous secreted BY-2 proteases are responsible for DSPAalpha1 degradation in the culture medium. The addition of a highly concentrated protease inhibitor mixture or 5 mM EDTA reduced DSPAalpha1 proteolysis, improving the accumulation of intact product in the culture medium. Strategies to improve the plant cell suspension system for the production of secreted recombinant proteins are discussed., (2005 Wiley Periodicals, Inc.)
- Published
- 2005
- Full Text
- View/download PDF
33. A small family of novel CuZn-superoxide dismutases with high isoelectric points in hybrid aspen.
- Author
-
Schinkel H, Hertzberg M, and Wingsle G
- Subjects
- Amino Acid Sequence, Chimera, DNA, Complementary, DNA, Plant, Gene Expression Regulation, Plant, Isoelectric Point, Molecular Sequence Data, Salicaceae enzymology, Superoxide Dismutase chemistry, Superoxide Dismutase metabolism, Salicaceae genetics, Superoxide Dismutase genetics
- Abstract
Several CuZn-superoxide dismutases (SODs; EC 1.15.1.1) were cloned from hybrid aspen (Populus tremula L. x tremuloides Michx.). Two of the cloned genes encode representatives of a novel type of CuZn-SOD and we named it HipI-SOD because of its high isoelectric point (> or =9). The SODs were cloned by screening a cDNA library with a probe based on a Scots pine (Pinus sylvestris L.) CuZn-SOD that is predominantly located extracellularly. The expression pattern of HipI-SOD was examined using a Northern blot technique and compared with the expression patterns of cytosolic and chloroplastic SODs. Distinct expression patterns were observed for the three types of CuZn-SOD, with HipI-SODs showing strong expression in apical tissues. Southern blots as well as protein analysis suggest that these novel HipI-SODs belong to a small gene family, one member of which might be monomeric.
- Published
- 2001
- Full Text
- View/download PDF
34. 15 N abundance of surface soils, roots and mycorrhizas in profiles of European forest soils.
- Author
-
Högberg P, Högbom L, Schinkel H, Högberg M, Johannisson C, and Wallmark H
- Abstract
15 N natural abundances of soil total N, roots and mycorrhizas were studied in surface soil profiles in coniferous and broadleaved forests along a transect from central to northern Europe. Under conditions of N limitation in Sweden, there was an increase in δ15 N of soil total N of up to 9% from the uppermost horizon of the organic mor layer down to the upper 0-5 cm of the mineral soil. The δ15 N of roots was only slightly lower than that of soil total N in the upper organic horizon, but further down roots were up to 5% depleted under such conditions. In experimentally N-enriched forest in Sweden, i.e. in plots which have received an average of c. 100 kg N ha-1 year-1 for 20 years and which retain less than 50% of this added N in the stand and the soil down to 20 cm depth, and in some forests in central Europe, the increase in δ15 N with depth in soil total N was smaller. An increase in δ15 N of the surface soil was even observed on experimentally N-enriched plots, although other data suggest that the N fertilizer added was depleted in15 N. In such cases roots could be enriched in15 N relative to soil total N, suggesting that labelling of the surface soil is via the pathway: - available pools of N-plant N-litter N. Under N-limiting conditions roots of different species sampled from the same soil horizon showed similar δ15 N. By contrast, in experimentally N-enriched forest δ15 N of roots increased in the sequence: ericaceous dwarf shrubs15 N enriched compounds in fungal material, which could contribute to explain the observed δ 15 N profiles if fungal material is enriched, because it is a precursor of stable organic matter and recalcitrant N. This could act in addition to the previous explanation of the isotopically lighter soil surface in forests: plant uptake of15 N-depleted N and its redeposition onto the soil surface by litter-fall.- Published
- 1996
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.