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 β-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 β-cyclase. Intermediates of the pathway beyond β-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 3′-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 z­Carotin­Desaturase einer höheren Pflanze nach heterologer Expression in E. coli in nativer Form gereinigt und enzymatisch charakterisiert werden. Dazu wurde die cDNA der Capsicum­ZDS in einen Expressionsvektor kloniert, der die ZDS als rekombinantes Polypeptid mit 6 N­terminalen Histidinen exprimierte. Dadurch konnte das Enzym in nur zwei Schritten über eine Kombination von Ammoniumsulfatfällung und Metallionen­Affinitätschromatographie selektiv aus E. coli separiert werden. Die ZDS wurde ohne eine mutmaßliche Transitsequenz als ein Polypeptid von 59 kDa exprimiert. Das pH­ Optimum der ZDS­Aktivtität liegt bei 7,2 in der Nähe der rechnerisch ermittelten pI­Wertes von 7,4. Die ZDS führt zwei Desaturierungsschritte ausgehend von z­Carotin zu Lycopin als ein monomeres Protein durch. Unter Verwendung des Two­Hybrid­Systems, einer Gelelektrophorese unter nativen Bedingungen und einer Gelfiltration der nativen ZDS, konnte gezeigt werden, daß die ZDS als Monomer und als Dimer vorliegen kann. Die Dimerisierung der ZDS ist jedoch für deren enzymatischer Aktivität und für die Durchführung beider Desaturierungsschritte nicht notwendig. Für die Substratcarotinoide z­Carotin und Neurosporin, wurden die Km ­Werte von 8,4 µM und 9,0 µM bestimmt. Die Capsicum­ZDS zeigt von ihrer Aminosäuresequenz her eine große Ähnlichkeit zu den cyanobakteriellen z­Carotin­Desaturasen und eine geringere Ähnlichkeit zu den pflanzlichen Phytoendesaturasen. Eine diskutierte phylogenetische Verwandtschaft der z­Carotin­ und Phytoendesaturase aus höheren Pflanzen und Cyanobakterien wird durch die Verwendung des gleichen Kofaktors Plastochinon und durch die gemeinsame Hemmbarkeit mit den z­Carotin­Desaturase­Hemmstoffen J852 und LS80707 unterstützt. Eine Kofaktoruntersuchung ergab, daß 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 Carotin­Desaturase aus Synechocystis sp. PCC 6803 ist. Der Km ­Wert von Decyl­ Plastochinon wurde für die Capsicum­ZDS zu 0,4 µM bestimmt. Der Kofaktor der z­ Carotin­Desaturase Plastochinon, sowie die Entdeckung einer plastidären terminalen Oxidase (Carol et al., 1999) ermöglicht die Entwicklung eines Modells der Übertragung der bei der Desaturierung von z­Carotin gewonnenen Elektronen über Plastochinon auf Sauerstoff, wie es bereits für die pflanzliche Phytoendesaturase postuliert wurde (Caro

Anleitung für Benutzer des Rechenprogramms STASIP (statics of single-point moorings) = Instruction manual for the computer program STASIP (statics of single-point moorings)

ALs Ergänzung zum IfM-Bericht Nr. 108 wird in dem vorliegenden Bericht für 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 erläutert. Im Anhang befindet sich neben wichtigen Tabellen ein vollständiger Programmausdruck. (AUT

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.’s 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 Union’s Horizon 2020 Research and Innovation programme under the Marie Skłodowska-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

See–saw relationship of the Holocene East Asian–Australian summer monsoon

The East Asian–Indonesian–Australian summer monsoon (EAIASM) links the Earth’s 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 Indonesian–Australian summer monsoon (IASM) are poorly understood, raising questions of long-term adjustments to future greenhouse-triggered climate change and whether these changes could ‘lock in’ 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 see–saw relationship over the last 9,000 years—with 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

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 β-carotene hydroxylase and a β-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 ε-cyclase was knocked-down to direct more precursors into the β-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