Purification and functional characterization of human 11beta hydroxylase expressed in Escherichia coli

The human 11beta-hydroxylase (hCYP11B1) is responsible for the conversion of 11-deoxycortisol into the major mammalian glucocorticoid, cortisol. The reduction equivalents needed for this reaction are provided via a short electron transfer chain consisting of a [2Fe-2S] ferredoxin and a FAD-containing reductase. On the biochemical and biophysical level, little is known about hCYP11B1 because it is very unstable for analyses performed in vitro. This instability is also the reason why it has not been possible to stably express it so far in Escherichia coli and subsequently purify it. In the present study, we report on the successful and reproducible purification of recombinant hCYP11B1 coexpressed with molecular chaperones GroES/GroEL in E. coli. The protein was highly purified to apparent homogeneity, as observed by SDS/PAGE. Upon mass spectrometry, the mass-to-charge ratio (m/z) of the protein was estimated to be 55 761, which is consistent with the value 55 760.76 calculated for the form lacking the translational initiator Met. The functionality of hCYP11B1 was analyzed using different methods (substrate conversion assays, stopped-flow, Biacore). The results clearly demonstrate that the enzyme is capable of hydroxylating its substrates at position 11-beta. Moreover, the determined NADPH coupling percentage for the hCYP11B1 catalyzed reactions using either 11-deoxycortisol or 11-deoxycorticosterone as substrates was approximately 75% in both cases. Biacore and stopped-flow measurements indicate that hCYP11B1 possesses more than one binding site for its redox partner adrenodoxin, possibly resulting in the formation of more than one productive complexes. In addition, we performed CD measurements to obtain information about the structure of hCYP11B1.     

Costs of induced volatile production in maize

Herbivore‐induced plant volatiles have been shown to serve as indirect defence signals that attract natural enemies of herbivores. Parasitoids and predators exploit these plant‐provided cues to locate their victims and several herbivores are repelled by the volatiles. Recently, benefits, in terms of plant fitness, from the action of the parasitoids were shown for a few systems. However, the cost of production of herbivore‐induced volatiles for the plant remains unknown. Here, we estimate the fitness cost of the production of induced volatiles in maize, Zea mays. Plants were treated with regurgitant of Spodoptera littoralis or with the elicitor volicitin and we measured dry weight of plant parts at specific times after treatments. After a two‐week treatment period, the dry‐weight of leaves of induced plants was lower than that of un‐induced plants, suggesting a metabolic cost for induced defence. However, maize plants seem to compensate for this loss during subsequent growth, since seed production at maturity was not different for unharmed plants and plants treated with caterpillar regurgitant. For volicitin treated plants a small but significant reduction in seed production was found. It is likely that the treatments also induced the production of other defence compounds, which will contribute to the cost. Yet, a comparison of six maize inbred lines with distinct differences in volatile emissions showed a strong correlation between the intensity of induced emissions and reduction in plant performance. An analysis of the terpenoids that accumulated in the leaves of the inbred lines revealed non‐volatilised compounds are constitutively present in maize and only the volatilised compounds are induced. Interestingly, the lines that released the largest amounts of induced volatiles also contained more of the non‐volatile terpenoids. Based on these results and results from a previous study on the benefits of attracting parasitoids, we conclude that costs of induced volatile production in plants are counterbalanced by the benefits as long as natural enemies of the herbivores are present in the environment.     

Tollip, a negative regulator of TLR-signalling, is encoded by twin genes in salmonid fish

The factor Tollip is known to dampen TLR2- and TLR4-mediated signalling in mammals. No negative regulator of the piscine TLR-signalling cascade has been described so far, albeit a sizable collection of factors contributing to this ancient pathogen-sensing system are known from fish to date. We identified two closely related Tollip-encoding genes in Atlantic salmon (Salmo salar) and the respective ortholog mRNA molecules in rainbow trout (Oncorhynchus mykiss). The salmonid Tollip genes are segmented into 6 exons, similar to the human orthologous gene. The protein-encoding sequences are homologous to >97% among the twin factors and also between the species. Both encoded proteins contain a C2 domain and an ubiquitin system component, which are also characteristic features of the mammalian Tollip factor. We analysed the expression of these genes in trout. Both Tollip-encoding genes are ubiquitously and also equally expressed, as indicated by similar mRNA concentrations of both factors in any one tissue. Tollip expression was found to be up-regulated by viral infection. Our data suggest that the Tollip genes were duplicated before salmon and trout were evolutionary separated. Moreover, pathways dampening the activity of the TLR-cascade may have been conserved from lower vertebrates to mammals since Tollip, as a respective key factor has been highly conserved from fish to human.     

Psychiatric genetics: progress amid controversy

Several psychiatric disorders--such as bipolar disorder, schizophrenia and autism--are highly heritable, yet identifying their genetic basis has been challenging, with most discoveries failing to be replicated. However, inroads have been made by the incorporation of intermediate traits (endophenotypes) and of environmental factors into genetic analyses, and through the identification of rare inherited variants and novel structural mutations. Current efforts aim to increase sample sizes by gathering larger samples for case-control studies or through meta-analyses of such studies. More attention on unique families, rare variants, and on incorporating environment and the emerging knowledge of biological function and pathways into genetic analysis is warranted.     

Aldosterone: from biosynthesis to non-genomic action onto the proteome

An increased aldosterone concentration can lead to a progression of heart diseases and to myocardial fibrosis. These fatal processes can be prevented by e.g. inhibiting the mineralocorticoid receptor (MR), which is nowadays part of a commonly applied standard therapy. Moreover, selective inhibition of aldosterone synthase (CYP11B2) is a straightforward goal whereby CYP11B1, a key enzyme in glucocorticoid biosynthesis exhibiting a high structure identity with CYP11B2 should not be inhibited. Therefore, effective test systems have been developed and rather potent and selective CYP11B2 compounds like SIAS-1 have been identified by our group. In addition to finding new inhibitors, we investigated which proteins are directly influenced by aldosterone focussing on non-genomic effects. Schizosaccharomyces pombe was chosen as a model organism, since this yeast does not contain nuclear steroid receptors, but many genes and regulatory mechanisms that are close to those of mammals. Besides creating a reference map for this organism, protein spots affected by aldosterone as well as deoxycorticosterone (DOC) and corticosterone have been identified. In case of aldosterone, a regulatory effect of proteins that are connected with structural proteins, signal cascades, osmoregulation and calcium pathway as well as to general metabolism have been discovered. DOC causes overlapping but also different effects compared with aldosterone. As shown exemplarily for GAPDH, the aldosterone-mediated effects in S. pombe can also be verified in mammalian cells. These and further investigations contribute to a deeper understanding of so-called non-genomic aldosterone effects.     

Das Kopulationsverhalten von Kiebitzen (Vanellus vanellus) im Verlauf der Brutsaison

Zusammenfassung Von 1996 bis 2000 wurden insgesamt 43 verschiedene und 58 des Kiebitz jeweils vom 1. März bis 15. Juni beobachtet. Während 79 Ganztagsbeobachtungen mit insgesamt 1109 Stunden wurden 767 Kopulationen registriert. Eine Kopulation dauerte in allen Phasen des Brutgeschäftes durchschnittlich 1,7 s. Die ersten Begattungen wurden acht bis zehn Tage nach Erstbeobachtung der im Revier der und je vier bis sechs Tage vor Legebeginn ab Anfang/Mitte März festgestellt. Die letzten Begattungen waren noch Anfang Juni zu beobachten. Die Kopulationen verteilten sich über den ganzen Tag mit Maxima in den frühen Morgen- und späten Nachmittag- bzw. frühen Abendstunden. Bis zum vollständigen Gelege wurde jedes 40- bis 70-mal begattet. Diese häufigen Kopulationen könnten zur Festigung und zum Erhalt von Paarbindungen beitragen. Auch während der gesamten Gelegebebrütung kopulierten die Brutpartner durchschnittlich zwei- bis dreimal täglich. Hier erfolgten 51,5 % aller Kopulationen bei der Ablösung des brütenden durch das . Mit zunehmender Zahl an Brutablösungen nahm die Anzahl der Ablösungen mit Kopulation zu. Insgesamt wurde im Durchschnitt bei jeder vierten Brutablösung kopuliert. Nur an 18 (13,5 %) von insgesamt 133 beobachteten Bebrütungstagen eines konnte den ganzen Tag über keine Begattung festgestellt werden. Polygyne hielten zu all ihren sexuellen Kontakt. Mit dem Schlupf der Küken wurden die Kopulationen eingestellt. Erneut einsetzende Begattungen während des Jungeführens oder nach Kükenverlust erfolgten im Rahmen der Ablage eines Zweitgeleges. Zwischen Gelegealter und Kopulationshäufigkeit wurde kein Zusammenhang gefunden. Fremdkopulationen (EPCs) wurden nicht beobachtet. Kopulationen während der Gelegebebrütung dienen daher wohl nicht zur Verhinderung von Fremdbefruchtung eines Ersatz- oder Zweitgeleges. Sie sind wohl während der Bebrütung ebenso nicht nötig, um das Follikelwachstum und die Keimdrüsen für ein weiteres Gelege zu stimulieren, da auch nach mehreren Wochen ohne Kopulation innerhalb weniger Tage der Follikelsprung ausgelöst wurde. Offenbar sind Kopulationen während der Bebrütungsphase unabhängig von ihrer eigentlichen Befruchtungsfunktion. Die sehr häufigen Kopulationen scheinen Kommunikationsfunktion im Sinne der Aufforderung zur Brutablösung zu haben und können die Paarbindung stärken und so eine optimale Bebrütung des Geleges ermöglichen.

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The copulation behaviour of Lapwings (Vanellus vanellus) throughout the breeding season

Summary From 1996 to 2000 43 male and 58 female Lapwings were observed from March 1st until June 15th. During 79 full day observations over a total of 1109 hours, 767 copulations were recorded. Viewed over the whole breeding period a copulation took on average 1.7 seconds. The first matings (beginning of March) were registered eight to ten days after the arrival of the female in the male territory; four to six days later egg laying started. The last copulations were still being observed in early June. Matings were observed at all times of the day with peaks in the early morning and late afternoon. By the time the clutch was completed each female lapwing had taken part in 40 to 70 copulations. Frequent copulation may promote pair bonding. Over the complete incubation period the partners copulated on average two to three times a day. 51.5 % of copulations occured during brood relief of the male by the female. With an increasing number of brood reliefs the number of male to female reliefs with copulations became more frequent. All in all matings took place on average every fourth brood relief. Over 133 days of observation of an incubating fermale on only 18 days (13.5 %) were no copulations at all recorded. Polygamous males kept up contact to all their mates. The copulations finished on the hatching of the chicks. During chick leading or after the loss of chicks, matings started again for a second clutch. No connection was found between the age of a clutch and the frequency of copulations. Extra-pair copulations (EPCs) were not observed. Therefore matings during the incubation period would not appear to serve to prevent the extra-pair fertilization of a replacement or second clutch. Copulations during incubation time seem to be unnecessary to stimulate follicle growth and the germ glandular for a second clutch, because even after several weeks of abstention, an ovulation could be provoked within a few days. Copulations during the breeding period are obviously not connected to their regular function of fertilization. The very large number of copulations may have a communicative function stimulating turn-taking at brooding and helping to stabilize the pair bond, with a positive effect on the breeding success.

     

Coupling of coat assembly and vesicle budding to packaging of putative cargo receptors

COPI-coated vesicle budding from lipid bilayers whose composition resembles mammalian Golgi membranes requires coatomer, ARF, GTP, and cytoplasmic tails of putative cargo receptors (p24 family proteins) or membrane cargo proteins (containing the KKXX retrieval signal) emanating from the bilayer surface. Liposome-derived COPI-coated vesicles are similar to their native counterparts with respect to diameter, buoyant density, morphology, and the requirement for an elevated temperature for budding. These results suggest that a bivalent interaction of coatomer with membrane-bound ARF[GTP] and with the cytoplasmic tails of cargo or putative cargo receptors is the molecular basis of COPI coat assembly and provide a simple mechanism to couple uptake of cargo to transport vesicle formation.