43 research outputs found
Biosynthesis of fucoxanthin and diadinoxanthin and function of initial pathway genes in Phaeodactylum tricornutum
The biosynthesis pathway to diadinoxanthin and fucoxanthin was elucidated in Phaeodactylum tricornutum by a combined approach involving metabolite analysis identification of gene function. For the initial steps leading to ÎČ-carotene, putative genes were selected from the genomic database and the function of several of them identified by genetic pathway complementation in Escherichia coli. They included genes encoding a phytoene synthase, a phytoene desaturase, a ζ-carotene desaturase, and a lycopene ÎČ-cyclase. Intermediates of the pathway beyond ÎČ-carotene, present in trace amounts, were separated by TLC and identified as violaxanthin and neoxanthin in the enriched fraction. Neoxanthin is a branching point for the synthesis of both diadinoxanthin and fucoxanthin and the mechanisms for their formation were proposed. A single isomerization of one of the allenic double bounds in neoxanthin yields diadinoxanhin. Two reactions, hydroxylation at C8 in combination with a keto-enol tautomerization and acetylation of the 3âČ-HO group results in the formation of fucoxanthin
Funktionelle Expression, Reinigung und biochemische Charakterisierung der z-Carotin-Desaturase aus Capsicum annuum (Paprika)
Erstmals konnte eine zÂCarotinÂDesaturase einer höheren Pflanze nach heterologer Expression in E. coli in nativer Form gereinigt und enzymatisch charakterisiert werden. Dazu wurde die cDNA der CapsicumÂZDS in einen Expressionsvektor kloniert, der die ZDS als rekombinantes Polypeptid mit 6 NÂterminalen Histidinen exprimierte. Dadurch konnte das Enzym in nur zwei Schritten ĂŒber eine Kombination von AmmoniumsulfatfĂ€llung und MetallionenÂAffinitĂ€tschromatographie selektiv aus E. coli separiert werden. Die ZDS wurde ohne eine mutmaĂliche Transitsequenz als ein Polypeptid von 59 kDa exprimiert. Das pH Optimum der ZDSÂAktivtitĂ€t liegt bei 7,2 in der NĂ€he der rechnerisch ermittelten pIÂWertes von 7,4. Die ZDS fĂŒhrt zwei Desaturierungsschritte ausgehend von zÂCarotin zu Lycopin als ein monomeres Protein durch. Unter Verwendung des TwoÂHybridÂSystems, einer Gelelektrophorese unter nativen Bedingungen und einer Gelfiltration der nativen ZDS, konnte gezeigt werden, daĂ die ZDS als Monomer und als Dimer vorliegen kann. Die Dimerisierung der ZDS ist jedoch fĂŒr deren enzymatischer AktivitĂ€t und fĂŒr die DurchfĂŒhrung beider Desaturierungsschritte nicht notwendig. FĂŒr die Substratcarotinoide zÂCarotin und Neurosporin, wurden die Km ÂWerte von 8,4 ”M und 9,0 ”M bestimmt. Die CapsicumÂZDS zeigt von ihrer AminosĂ€uresequenz her eine groĂe Ăhnlichkeit zu den cyanobakteriellen zÂCarotinÂDesaturasen und eine geringere Ăhnlichkeit zu den pflanzlichen Phytoendesaturasen. Eine diskutierte phylogenetische Verwandtschaft der zÂCarotin und Phytoendesaturase aus höheren Pflanzen und Cyanobakterien wird durch die Verwendung des gleichen Kofaktors Plastochinon und durch die gemeinsame Hemmbarkeit mit den zÂCarotinÂDesaturaseÂHemmstoffen J852 und LS80707 unterstĂŒtzt. Eine Kofaktoruntersuchung ergab, daĂ Plastochinon sowohl der Kofaktor der ZDS aus Capsicum, als auch der Phytoendesaturasen aus Gentiana lutea (gelber Entian), aus dem Cyanobakterium Synechococcus sp. PCC 7942, sowie der z CarotinÂDesaturase aus Synechocystis sp. PCC 6803 ist. Der Km ÂWert von Decyl Plastochinon wurde fĂŒr die CapsicumÂZDS zu 0,4 ”M bestimmt. Der Kofaktor der z CarotinÂDesaturase Plastochinon, sowie die Entdeckung einer plastidĂ€ren terminalen Oxidase (Carol et al., 1999) ermöglicht die Entwicklung eines Modells der Ăbertragung der bei der Desaturierung von zÂCarotin gewonnenen Elektronen ĂŒber Plastochinon auf Sauerstoff, wie es bereits fĂŒr die pflanzliche Phytoendesaturase postuliert wurde (Caro
Anleitung fĂŒr Benutzer des Rechenprogramms STASIP (statics of single-point moorings) = Instruction manual for the computer program STASIP (statics of single-point moorings)
ALs ErgĂ€nzung zum IfM-Bericht Nr. 108 wird in dem vorliegenden Bericht fĂŒr den Benutzer eine Anleitung zu dem Dialogprogramm STASIP (Statistics of Single-Point Moorings) gegeben. Ein vereinfachter Programmlauf stellt die Funktionsweise des Programms dar und vermittelt die wichtigsten Grundlagen. Durch einen weiteren umfassenderen Rechenlauf wird dann die Anwendungsvielfalt des Programms erlĂ€utert. Im Anhang befindet sich neben wichtigen Tabellen ein vollstĂ€ndiger Programmausdruck. (AUT
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Seeâsaw relationship of the Holocene East AsianâAustralian summer monsoon
The East AsianâIndonesianâAustralian summer monsoon (EAIASM) links the Earthâs hemispheres and provides a heat source that drives global circulation. At seasonal and inter-seasonal timescales, the summer monsoon of one hemisphere is linked via outflows from the winter monsoon of the opposing hemisphere. Long-term phase relationships between the East Asian summer monsoon (EASM) and the IndonesianâAustralian summer monsoon (IASM) are poorly understood, raising questions of long-term adjustments to future greenhouse-triggered climate change and whether these changes could âlock inâ possible IASM and EASM phase relationships in a region dependent on monsoonal rainfall. Here we show that a newly developed nonlinear time series analysis technique allows confident identification of strong versus weak monsoon phases at millennial to sub-centennial timescales. We find a seeâsaw relationship over the last 9,000 yearsâwith strong and weak monsoons opposingly phased and triggered by solar variations. Our results provide insights into centennial- to millennial-scale relationships within the wider EAIASM regime
See-saw relationship of the Holocene East Asian-Australian summer monsoon
D.E. and N.M. acknowledge support by the Leibniz Association (WGL) under Grant No. SAW-2013-IZW-2. F.H.M.âs research is funded through an Australian Postgraduate Award. I.O. is financially supported from TUBITAK under 2214/A program and by Ege University under the Research Project number 2015FEN028. This study received funding from the European Unionâs Horizon 2020 Research and Innovation programme under the Marie SkĆodowska-Curie grant agreement No 691037. The publication of this article was funded by the Open Access Fund of the Leibniz Association. K.H.W. thank Rhawn F. Denniston for his wider involvement in the northwest Australian monsoon project and the Kimberley Foundation Australia for financial support for this project and Paul Wyrwoll for helpful comments. We are also grateful to Yanjun Cai for providing the Lake Qinghai record.Peer reviewedPublisher PD
Metabolic engineering of astaxanthin biosynthesis in maize endosperm and characterization of a prototype high oil hybrid
Maize was genetically engineered for the biosynthesis of the high value carotenoid astaxanthin in the kernel endosperm. Introduction of a ÎČ-carotene hydroxylase and a ÎČ-carotene ketolase into a white maize genetic background extended the carotenoid pathway to astaxanthin. Simultaneously, phytoene synthase, the controlling enzyme of carotenogenesis, was over-expressed for enhanced carotenoid production and lycopene Δ-cyclase was knocked-down to direct more precursors into the ÎČ-branch of the extended ketocarotenoid pathway which ends with astaxanthin. This astaxanthin-accumulating transgenic line was crossed into a high oil- maize genotype in order to increase the storage capacity for lipophilic astaxanthin. The high oil astaxanthin hybrid was compared to its astaxanthin producing parent. We report an in depth metabolomic and proteomic analysis which revealed major up- or down- regulation of genes involved in primary metabolism. Specifically, amino acid biosynthesis and the citric acid cycle which compete with the synthesis or utilization of pyruvate and glyceraldehyde 3-phosphate, the precursors for carotenogenesis, were down-regulated. Nevertheless, principal component analysis demonstrated that this compositional change is within the range of the two wild type parents used to generate the high oil producing astaxanthin hybrid
Somatostatin Inhibits Cell Migration and Reduces Cell Counts of Human Keratinocytes and Delays Epidermal Wound Healing in an Ex Vivo Wound Model
The peptide hormone somatostatin (SST) and its five G protein-coupled receptors
(SSTR1-5) were described to be present in the skin, but their cutaneous
function(s) and skin-specific signalling mechanisms are widely unknown. By using
receptor specific agonists we show here that the SSTRs expressed in
keratinocytes are functionally coupled to the inhibition of adenylate cyclase.
In addition, treatment with SSTR4 and SSTR5/1 specific agonists significantly
influences the MAP kinase signalling pathway. As epidermal hormone receptors in
general are known to regulate re-epithelialization following skin injury, we
investigated the effect of SST on cell counts and migration of human
keratinocytes. Our results demonstrate a significant inhibition of cell
migration and reduction of cell counts by SST. We do not observe an effect on
apoptosis and necrosis. Analysis of signalling pathways showed that somatostatin
inhibits cell migration independent of its effect on cAMP. Migrating
keratinocytes treated with SST show altered cytoskeleton dynamics with delayed
lamellipodia formation. Furthermore, the activity of the small GTPase Rac1 is
diminished, providing evidence for the control of the actin cytoskeleton by
somatostatin receptors in keratinocytes. While activation of all receptors leads
to redundant effects on cell migration, only treatment with a SSTR5/1 specific
agonist resulted in decreased cell counts. In accordance with reduced cell
counts and impaired migration we observe delayed re-epithelialization in an
ex vivo wound healing model. Consequently, our experiments
suggest SST as a negative regulator of epidermal wound healing