Analytische Charakterisierung von Struktur und Biosynthese der Nudicauline aus gelben Papaver nudicaule L. Blüten

Bei den Nudicaulinen handelt es sich um Farbstoffe aus gelben Papaver nudicaule L. Blüten, deren Strukturaufklärung 1939 begann. Im Jahr 2006 zeigte ein erster Vor-schlag, dass die Nudicauline zur Substanzklasse der Indolalkaloide gehören, jedoch eine flavonoidähnliche Teilstruktur aufweisen. Daraus folgte das wissenschaftliche Interesse an ihrer Biosynthese und ökologischer Funktion. Um den Strukturvorschlag von 2006 zu verifizieren, wurden zunächst intensive NMR-, CD- und MS-Analysen an den Nudicaulinen und ihren synthetisierten Derivaten durchgeführt. Die Ergebnisse führten zur Revision der Aglykonstruktur und der Be-stimmung der absoluten Konfiguration. An diese Untersuchungen schlossen sich Markierungsexperimente mit 13C-haltigen Vorstufen der bekannten Biosyntheserouten an. Hierbei gelang es zu zeigen, dass nicht wie erwartet Tryptophan oder Tryptamin sondern Indol eine der beiden finalen Vorstufen der Nudicaulinbildung ist. Weiterhin ergab die Auswertung von Duftsammlungen an den weiß-, gelb-, orange- und rotblühenden P. nudicaule-Pflanzen einige Unterschiede in der Bouquetzusam-mensetzung. Insbesondere das Auftreten von Indol im Duft der gelben bzw. sein Fehlen bei den übrigen Kultivaren weist auf einen Zusammenhang zwischen Nudicaulin-biosynthese und Duftkomposition hin. Es wurde außerdem gezeigt, dass Bouquet und Blütenfarbe das Pollinationsverhalten von Honigbienen beeinflussen. Bei der finalen Nudicaulinvorstufe aufseiten des Flavonoidbiosynthesewegs handelt es sich um Pelargonidinderivate, die nur während des Wachstums der Petalen auftre-ten. Durch HPLC-DAD- sowie Cluster- und Diskriminanzanalysen gelang die Eintei-lung der Blütenblattentwicklung in fünf Stadien. Die gewonnenen Kenntnisse fanden Anwendung in allen folgenden Probennahmen, insbesondere für die der Proteom- und Transkriptomanalysen, deren Ergebnisse nun als Basis für die weiterführende Untersuchung der Nudicaulinbiosynthese und ihrer Regulation zur Verfügung stehen.Nudicaulins are the pigments of yellow Papaver nudicaule L. petals. Their structure determination began in 1939. A first structure proposal published in 2006 showed that the nudicaulins belong to the indole alkaloids and contain a flavonoid-related subunit in the aglycone. This unique composition raised the interest in their biosynthesis and ecological function. To verify the structure proposal intensive NMR-, CD- and MS- analyses were carried out using the nudicaulins and synthetic derivatives. As a result the nudicaulin structure was revised and the absolute configuration determined. Experiments using 13C labeled precursors were carried out to probe for biosynthetic incorporation into the nudicaulins. Indole instead of tryptophan or tryptamine was found to be the immediate precursor of the indole substructure of the nudicaulin aglycone. Additionally the analysis of odour collections from white, yellow, orange and red flowering cultivars revealed differences in the bouquet composition. Especially the high abundance of indole in the headspace of yellow flowers and its absence in the other cultivars indicated a correlation of the nudicaulin biosynthesis and odour composition. Behavioural experiments with honey bees showed their ability to distinguish both the colours and the scent of different P. nudicaule cultivars. The final flavonoid precursors of the nudicaulin aglycone are pelargonidin derivatives, which appear only during the maturing of the petals. Five petal developmental stages were determined by means of HPLC-DAD-, Cluster- and Discriminant function analyses. The results were applied to all following samplings, especially for the proteome and transcriptome analyses. The obtained findings form the basis for further studies of the nudicaulin biosynthesis and regulation

An integrated—omics/chemistry approach unravels enzymatic and spontaneous steps to form flavoalkaloidal nudicaulin pigments in flowers of Papaver nudicaule L.

Flower colour is an important trait for plants to attract pollinators and ensure their reproductive success. Among yellow flower pigments, the nudicaulins in Papaver nudicaule L. (Iceland poppy) are unique due to their rarity and unparalleled flavoalkaloid structure. Nudicaulins are derived from pelargonidin glycoside and indole, products of the flavonoid and indole/tryptophan biosynthetic pathway, respectively. To gain insight into the molecular and chemical basis of nudicaulin biosynthesis, we combined transcriptome, differential gel electrophoresis (DIGE)-based proteome, and ultra-performance liquid chromatography–high resolution mass spectrometry (UPLC-HRMS)-based metabolome data of P. nudicaule petals with chemical investigations. We identified candidate genes and proteins for all biosynthetic steps as well as some key metabolites across five stages of petal development. Candidate genes of amino acid biosynthesis showed a relatively stable expression throughout petal development, whereas most candidate genes of flavonoid biosynthesis showed increasing expression during development followed by downregulation in the final stage. Notably, gene candidates of indole-3-glycerol-phosphate lyase (IGL), sharing characteristic sequence motifs with known plant IGL genes, were co-expressed with flavonoid biosynthesis genes, and are probably providing free indole. The fusion of indole with pelargonidin glycosides was retraced synthetically and promoted by high precursor concentrations, an excess of indole, and a specific glycosylation pattern of pelargonidin. Thus, nudicaulin biosynthesis combines the enzymatic steps of two different pathways with a spontaneous fusion of indole and pelargonidin glycoside under precisely tuned reaction conditions

ORAI1 calcium channel orchestrates skin homeostasis

To achieve and maintain skin architecture and homeostasis, keratinocytes must intricately balance growth, differentiation, and polarized motility known to be governed by calcium. Orai1 is a pore subunit of a store-operated Ca(2+) channel that is a major molecular counterpart for Ca(2+) influx in nonexcitable cells. To elucidate the physiological significance of Orai1 in skin, we studied its functions in epidermis of mice, with targeted disruption of the orai1 gene, human skin sections, and primary keratinocytes. We demonstrate that Orai1 protein is mainly confined to the basal layer of epidermis where it plays a critical role to control keratinocyte proliferation and polarized motility. Orai1 loss of function alters keratinocyte differentiation both in vitro and in vivo. Exploring underlying mechanisms, we show that the activation of Orai1-mediated calcium entry leads to enhancing focal adhesion turnover via a PKCβ-Calpain-focal adhesion kinase pathway. Our findings provide insight into the functions of the Orai1 channel in the maintenance of skin homeostasis

γ-Aminobutyric Acid Transporter 2 Mediates the Hepatic Uptake of Guanidinoacetate, the Creatine Biosynthetic Precursor, in Rats

Guanidinoacetic acid (GAA) is the biosynthetic precursor of creatine which is involved in storage and transmission of phosphate-bound energy. Hepatocytes readily convert GAA to creatine, raising the possibility that the active uptake of GAA by hepatocytes is a regulatory factor. The purpose of this study is to investigate and identify the transporter responsible for GAA uptake by hepatocytes. The characteristics of [14C]GAA uptake by hepatocytes were elucidated using the in vivo liver uptake method, freshly isolated rat hepatocytes, an expression system of Xenopus laevis oocytes, gene knockdown, and an immunohistochemical technique. In vivo injection of [14C]GAA into the rat femoral vein and portal vein results in the rapid uptake of [14C]GAA by the liver. The uptake was markedly inhibited by γ-aminobutyric acid (GABA) and nipecotinic acid, an inhibitor of GABA transporters (GATs). The characteristics of Na+- and Cl−-dependent [14C]GAA uptake by freshly isolated rat hepatocytes were consistent with those of GAT2. The Km value of the GAA uptake (134 µM) was close to that of GAT2-mediated GAA transport (78.9 µM). GABA caused a marked inhibition with an IC50 value of 8.81 µM. The [14C]GAA uptake exhibited a significant reduction corresponding to the reduction in GAT2 protein expression. GAT2 was localized on the sinusoidal membrane of the hepatocytes predominantly in the periportal region. This distribution pattern was consistent with that of the creatine biosynthetic enzyme, S-adenosylmethionine∶guanidinoacetate N-methyltransferase. GAT2 makes a major contribution to the sinusoidal GAA uptake by periportal hepatocytes, thus regulating creatine biosynthesis in the liver

A Genome Wide Association Scan of Bovine Tuberculosis Susceptibility in Holstein-Friesian Dairy Cattle

peer-reviewedBackground: Bovine tuberculosis is a significant veterinary and financial problem in many parts of the world. Although many factors influence infection and progression of the disease, there is a host genetic component and dissection of this may enlighten on the wider biology of host response to tuberculosis. However, a binary phenotype of presence/absence of infection presents a noisy signal for genomewide association study. Methodology/Principal Findings: We calculated a composite phenotype of genetic merit for TB susceptibility based on disease incidence in daughters of elite sires used for artificial insemination in the Irish dairy herd. This robust measure was compared with 44,426 SNP genotypes in the most informative 307 subjects in a genome wide association analysis. Three SNPs in a 65 kb genomic region on BTA 22 were associated (i.e. p,1025, peaking at position 59588069, p = 4.0261026) with tuberculosis susceptibility. Conclusions/Significance: A genomic region on BTA 22 was suggestively associated with tuberculosis susceptibility; it contains the taurine transporter gene SLC6A6, or TauT, which is known to function in the immune system but has not previously been investigated for its role in tuberculosis infection

Dazap2 modulates transcription driven by the Wnt effector TCF-4

A major outcome of the canonical Wnt/β-catenin-signalling pathway is the transcriptional activation of a specific set of target genes. A typical feature of the transcriptional response induced by Wnt signalling is the involvement of Tcf/Lef factors that function in the nucleus as the principal mediators of signalling. Vertebrate Tcf/Lef proteins perform two well-characterized functions: in association with β-catenin they activate gene expression, and in the absence of Wnt ligands they bind TLE/Groucho proteins to act as transcriptional repressors. Although the general characteristics of Tcf/Lef factors are well understood, the mechanisms that control their specific roles in various cellular backgrounds are much less defined. In this report we reveal that the evolutionary conserved Dazap2 protein functions as a TCF-4 interacting partner. We demonstrate that a short region proximal to the TCF-4 HMG box mediates the interaction and that all Tcf/Lef family members associate with Dazap2. Interestingly, knockdown of Dazap2 not only reduced the activity of Wnt signalling as measured by Tcf/β-catenin reporters but additionally altered the expression of Wnt-signalling target genes. Finally, chromatin immunoprecipitation studies indicate that Dazap2 modulates the affinity of TCF-4 for its DNA-recognition motif

Transcriptomic Analysis of Human Retinal Detachment Reveals Both Inflammatory Response and Photoreceptor Death

Background Retinal detachment often leads to a severe and permanent loss of vision and its therapeutic management remains to this day exclusively surgical. We have used surgical specimens to perform a differential analysis of the transcriptome of human retinal tissues following detachment in order to identify new potential pharmacological targets that could be used in combination with surgery to further improve final outcome. Methodology/Principal Findings Statistical analysis reveals major involvement of the immune response in the disease. Interestingly, using a novel approach relying on coordinated expression, the interindividual variation was monitored to unravel a second crucial aspect of the pathological process: the death of photoreceptor cells. Within the genes identified, the expression of the major histocompatibility complex I gene HLA-C enables diagnosis of the disease, while PKD2L1 and SLCO4A1 -which are both down-regulated- act synergistically to provide an estimate of the duration of the retinal detachment process. Our analysis thus reveals the two complementary cellular and molecular aspects linked to retinal detachment: an immune response and the degeneration of photoreceptor cells. We also reveal that the human specimens have a higher clinical value as compared to artificial models that point to IL6 and oxidative stress, not implicated in the surgical specimens studied here. Conclusions/Significance This systematic analysis confirmed the occurrence of both neurodegeneration and inflammation during retinal detachment, and further identifies precisely the modification of expression of the different genes implicated in these two phenomena. Our data henceforth give a new insight into the disease process and provide a rationale for therapeutic strategies aimed at limiting inflammation and photoreceptor damage associated with retinal detachment and, in turn, improving visual prognosis after retinal surgery

Alterations of Blood Brain Barrier Function in Hyperammonemia: An Overview

Ammonia is a neurotoxin involved in the pathogenesis of neurological conditions associated with hyperammonemia, including hepatic encephalopathy, a condition associated with acute—(ALF) or chronic liver failure. This article reviews evidence that apart from directly affecting the metabolism and function of the central nervous system cells, ammonia influences the passage of different molecules across the blood brain barrier (BBB). A brief description is provided of the tight junctions, which couple adjacent cerebral capillary endothelial cells to each other to form the barrier. Ammonia modulates the transcellular passage of low-to medium-size molecules, by affecting their carriers located at the BBB. Ammonia induces interrelated aberrations of the transport of the large neutral amino acids and aromatic amino acids (AAA), whose influx is augmented by exchange with glutamine produced in the course of ammonia detoxification, and maybe also modulated by the extracellularly acting gamma-glutamyl moiety transferring enzyme, gamma-glutamyl-transpeptidase. Impaired AAA transport affects neurotransmission by altering intracerebral synthesis of catecholamines (serotonin and dopamine), and producing “false neurotransmitters” (octopamine and phenylethylamine). Ammonia also modulates BBB transport of the cationic amino acids: the nitric oxide precursor, arginine, and ornithine, which is an ammonia trap, and affects the transport of energy metabolites glucose and creatine. Moreover, ammonia acting either directly or in synergy with liver injury-derived inflammatory cytokines also evokes subtle increases of the transcellular passage of molecules of different size (BBB “leakage”), which appears to be responsible for the vasogenic component of cerebral edema associated with ALF