High-Level Transient Expression of ER-Targeted Human Interleukin 6 in Nicotiana benthamiana

Tobacco plants can be used to express recombinant proteins that cannot be produced in a soluble and active form using traditional platforms such as Escherichia coli. We therefore expressed the human glycoprotein interleukin 6 (IL6) in two commercial tobacco cultivars (Nicotiana tabacum cv. Virginia and cv. Geudertheimer) as well as the model host N. benthamiana to compare different transformation strategies (stable vs. transient expression) and subcellular targeting (apoplast, endoplasmic reticulum (ER) and vacuole). In T₀ transgenic plants, the highest expression levels were achieved by ER targeting but the overall yields of IL6 were still low in the leaves (0.005% TSP in the ER, 0.0008% in the vacuole and 0.0005% in the apoplast). The apoplast variant accumulated to similar levels in leaves and seeds, whereas the ER-targeted variant was 1.2-fold more abundant in seeds and the vacuolar variant was 6-fold more abundant in seeds. The yields improved in subsequent generations, with the best-performing T₂ plants producing the ER-targeted IL6 at 0.14% TSP in both leaves and seeds. Transient expression of ER-targeted IL6 in leaves using the MagnICON system resulted in yields of up to 7% TSP in N. benthamiana, but only 1% in N. tabacum cv. Virginia and 0.5% in cv. Geudertheimer. Although the commercial tobacco cultivars produced up to threefold more biomass than N. benthamiana, this was not enough to compensate for the lower overall yields. The recombinant IL6 produced by transient and stable expression in plants was biologically active and presented as two alternative bands matching the corresponding native protein.     

Oxidized phospatidylcholine but not native phosphatidylcholine inhibits inducible nitric oxide synthase in RAW 264.7 macrophages

This study was designed to compare the effects of oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (PAPC) and native PAPC on the inducible nitric oxide synthase (iNOS) in the macrophage cell line RAW 264.7. Macrophages stimulated by bacterial lipopolysaccharide (1 microg/ml) were incubated with increasing amounts of native or oxidized PAPC (oxPAPC, 10-20 microg/ml). Cells incubated with oxPAPC showed a dose-dependent inhibition of inducible nitric oxide synthesis, as well as reduced iNOS protein expression and mRNA levels. Additionally, chromatin immunoprecipitation assay revealed that oxPAPC reduced the interaction of the active NF-kappaB subunit p65 with the iNOS promoter region when compared to native PAPC.     

Orientation of birds in total darkness

Magnetic compass orientation of migratory birds is known to be light dependent, and radical-pair processes have been identified as the underlying mechanism. Here we report for the first time results of tests with European robins, Erithacus rubecula, in total darkness and, as a control, under 565 nm green light. Under green light, the robins oriented in their normal migratory direction, with southerly headings in autumn and northerly headings in spring. By contrast, in darkness they significantly preferred westerly directions in spring as well as autumn. This failure to show the normal seasonal change characterizes the orientation in total darkness as a "fixed direction" response. Tests in magnetic fields with the vertical or the horizontal component inverted showed that the preferred direction depended on the magnetic field but did not involve the avian inclination compass. A high-frequency field of 1.315 MHz did not affect the behavior, whereas local anesthesia of the upper beak resulted in disorientation. The behavior in darkness is thus fundamentally different from normal compass orientation and relies on another source of magnetic information: It does not involve the radical-pair mechanism but rather originates in the iron-containing receptors in the upper beak.     

Pedigree and marker information requirements to monitor genetic variability

There are several measures available to describe the genetic variability of populations. The average inbreeding coefficient of a population based on pedigree information is a frequently chosen option. Due to the developments in molecular genetics it is also possible to calculate inbreeding coefficients based on genetic marker information. A simulation study was carried out involving ten sires and 50 dams. The animals were mated over a period of 20 discrete generations. The population size was kept constant. Different situations with regard to the level of polymorphism and initial allele frequencies and mating scheme (random mating, avoidance of full sib mating, avoidance of full sib and half sib mating) were considered. Pedigree inbreeding coefficients of the last generation using full pedigree or 10, 5 and 2 generations of the pedigree were calculated. Marker inbreeding coefficients based on different sets of microsatellite loci were also investigated. Under random mating, pedigree-inbreeding coefficients are clearly more closely related to true autozygosity (i.e., the actual proportion of loci with alleles identical by descent) than marker-inbreeding coefficients. If mating is not random, the demands on the quality and quantity of pedigree records increase. Greater attention must be paid to the correct parentage of the animals.     

Validation of the human T-lymphocyte cloning assay — ring test report from the EU concerted action on HPRT mutation (EUCAHM)

The T-cell cloning assay, which enables the enumeration and molecular analysis of 6-thioguanine resistant (HPRT-negative) mutant T-cells, has been extensively used for studying human somatic gene mutation in vivo. However, large inter-laboratory variations in the HPRT mutant frequency (MF) call for further investigation of inter-laboratory differences in the experimental methodology, and development of an optimal but easy uniform cloning protocol. As part of the EU Concerted Action on HPRT Mutation (EUCAHM), we have carried out two Ring tests for the T-cell cloning assay. For each test, duplicate and coded samples from three buffy coats were distributed to five laboratories for determination of MF using six different protocols. The results indicated a good agreement between split samples within each laboratory. However, both the cloning efficiencies (CEs) and MFs measured for the same blood donors showed substantial inter-laboratory variations. Also, different medium compositions used in one and the same laboratory resulted in a remarkable difference in the level of MF. A uniform operating protocol (UOP) was proposed and compared with the traditional protocols in the second Ring test. The UOP (preincubation) increased the CE in laboratories traditionally using preincubation, but decreased the CE in laboratories traditionally using priming. Adjusted for donor, use of different protocols contributed significantly to the overall variation in lnCE (P=0.0004) and lnMF (P=0.03), but there was no significant laboratory effect on the lnCE (P=0.38) or lnMF (P=0.14) produced by the UOP alone. Finally, a simplified version of the UOP using the serum-free medium X-Vivo 10 and PMA was tested in one laboratory, and found to produce a considerable increase in CE. This modified UOP needs to be further evaluated in order to be used for future databases on HPRT MFs in various populations.     

Das Orientierungssystem der V?gel I. Kompa?mechanismen

Zusammenfassung V?gel stellen den Bezug zum Ziel indirekt über ein externes Referenzsystem her. Der Navigationsproze? besteht deshalb aus zwei Schritten: zun?chst wird die Richtung zum Ziel als Kompa?kurs festgelegt, dann wird dieser Kurs mit Hilfe eines Kompa?mechanismus aufgesucht. Das Magnetfeld der Erde und Himmelsfaktoren werden von den V?gel als Kompa? benutzt. In der vorliegenden Arbeit werden der Magnetkompa?, der Sonnenkompa? und der Sternkompa? der V?gel in ihrer Funktionsweise, ihrer Entstehung und ihrer biologischen Bedeutung vorgestellt. Der Magnetkompa? erwies sich als Inklinationskompa?, der nicht auf der Polarit?t, sondern auf der Neigung der Feldlinien im Raum beruht; er unterscheidet „polw?rts“ und „?quatorw?rts“ statt Nord und Süd. Er ist ein angeborener Mechanismus und wird beim Vogelzug und beim Heimfinden benutzt. Seine eigentliche Bedeutung liegt jedoch darin, da? er ein Referenzsystem bereitstellt, mit dessen Hilfe andere Orientierungsfaktoren zueinander in Beziehung gesetzt werden k?nnen. Der Sonnenkompa? beruht auf Erfahrung; Sonnenazimut, Tageszeit und Richtung werden durch Lernprozesse miteinander verknüpft, wobei der Magnetkompa? als Richtungsreferenzsystem dient. Sobald er verfügbar ist, wird der Sonnenkompa? bei der Orientierung im Heimbereich und beim Heimfinden bevorzugt benutzt; beim Vogelzug spielt er, wahrscheinlich wegen seiner Abh?ngigkeit von der geographischen Breite, kaum eine Rolle. Der Sternkompa? arbeitet ohne Beteiligung der Inneren Uhr; die V?gel leiten Richtungen aus den Konfigurationen der Sterne zueinander ab. Lernprozesse erstellen den Sternkompa? in der Phase vor dem ersten Zug; dabei fungiert die Himmelsrotation als Referenzsystem. Sp?ter, w?hrend des Zuges, übernimmt der Magnetkompa? diese Rolle. Die relative Bedeutung der verschiedenen Kompa?systeme wurde in Versuchen untersucht, bei denen Magnetfeld und Himmelsfaktoren einander widersprechende Richtungs-information gaben. Die erste Reaktion der V?gel war von Art zu Art verschieden; langfristig scheinen sich die V?gel jedoch nach dem Magnetkompa? zu richten. Dabei werden die Himmelsfaktoren umgeeicht, so da? magnetische Information und Himmelsinformation wieder im Einklang stehen. Der Magnetkompa? und die Himmelsfaktoren erg?nzen einander: der Magnetkompa? ersetzt Sonnen- und Sternkompa? bei bedecktem Himmel; die Himmelsfaktoren erleichtern den V?geln das Richtungseinhalten, zu dem der Magnetkompa? offenbar wenig geeignet ist. Magnetfeld und Himmelsfaktoren sollten deshalb als integrierte Komponenten eines multifaktoriellen Systems zur Richtungsorientierung betrachtet werden.

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The orientation system of birds — I. Compass mechanisms

Summary Because of the large distances involved, birds establish contact with their goal indirectly via an external reference. Hence any navigation is a two-step process: in the first step, the direction to the goal is determined as a compass course; in the second step, this course is located with a compass. The geomagnetic field and celestial cues provide birds with compass information. The magnetic compass of birds, the sun compass the star compass and the interactions between the compass mechanisms are described in the present paper. Magnetic compass orientation was first demonstrated by testing night-migrating birds in experimentally altered magnetic fields: the birds changed their directional tendencies according to the deflected North direction. The avian magnetic compass proved to be an inclination compass: it does not use polarity; instead it is based on the axial course of the field lines and their inclination in space, distinguishing “poleward” and “equatorward” rather than North and South. Its functional range is limited to intensities around the local field strength, but this biological window is flexible and can be adjusted to other intensities. The magnetic compass is an innate mechanism that is widely used in bird migration and in homing. Its most important role, however, is that of a basic reference system for calibrating other kinds of orientation cues. Sun compass orientation is demonstrated by clock-shift experiments: Shifting the birds' internal clock causes them to misjudge the position of the sun, thus leading to typical deflections which indicate sun compass use. The analysis of the avian sun compass revealed that it is based only on sun azimuth and the internal clock; the sun's altitude is not involved. The role of the pattern of polarized light associated with the sun is unclear; only at sunset has it been shown to be an important cue for nocturnal migrants, being part of the sun compass. The sun compass is based on experience; sun azimuth, time of day and direction are combined by learning processes during a sensitive period, with the magnetic compass serving as directional reference. When established, the sun compass becomes the preferred compass mechanism for orientation tasks within the home region and homing: in migration, however, its role is minimal, probably because of the changes of the sun's arc with geographic latitude. The star compass was demonstrated in night-migrating birds by projecting the northern stars in different directions in a planetarium. The analysis of the mechanism revealed that the internal clock is not involved; birds derive directions from the spatial relationship of the star configurations. The star compass is also established by experience; the directional reference is first provided by celestial rotation, later, during migration, by the magnetic compass. The relative importance of the various compass mechanisms has been tested in experiments in which celestial and magnetic cues gave conflicting information. The first response of birds to conflicting cues differs considerably between species; after repeated exposures, however, the birds oriented according to magnetic North, indicating a long-term dominance of the magnetic compass. Later tests in the absence of magnetic information showed that celestial cues were not simply ignored, but recalibrated so that they were again in agreement with magnetic cues. The magnetic compass and celestial cues complement each other: the magnetic field ensures orientation under overcast sky; celestial cues facilitate maintaining directions, for which the magnetic compass appears to be ill suited. In view of this, the magnetic field and celestial cues should be regarded as integrated components of a multifactorial system for directional orientation.

     

De Novo Mutation and Rapid Protein (Co-)evolution during Meiotic Adaptation in Arabidopsis arenosa

A sudden shift in environment or cellular context necessitates rapid adaptation. A dramatic example is genome duplication, which leads to polyploidy. In such situations, the waiting time for new mutations might be prohibitive; theoretical and empirical studies suggest that rapid adaptation will largely rely on standing variation already present in source populations. Here, we investigate the evolution of meiosis proteins in Arabidopsis arenosa, some of which were previously implicated in adaptation to polyploidy, and in a diploid, habitat. A striking and unexplained feature of prior results was the large number of amino acid changes in multiple interacting proteins, especially in the relatively young tetraploid. Here, we investigate whether selection on meiosis genes is found in other lineages, how the polyploid may have accumulated so many differences, and whether derived variants were selected from standing variation. We use a range-wide sample of 145 resequenced genomes of diploid and tetraploid A. arenosa, with new genome assemblies. We confirmed signals of positive selection in the polyploid and diploid lineages they were previously reported in and find additional meiosis genes with evidence of selection. We show that the polyploid lineage stands out both qualitatively and quantitatively. Compared with diploids, meiosis proteins in the polyploid have more amino acid changes and a higher proportion affecting more strongly conserved sites. We find evidence that in tetraploids, positive selection may have commonly acted on de novo mutations. Several tests provide hints that coevolution, and in some cases, multinucleotide mutations, might contribute to rapid accumulation of changes in meiotic proteins.     

Das Orientierungssystem der V?gel IV. Evolution

Zusammenfassung Ein erster Versuch von Bellrose, die Evolution des Orientierungssystems der Vögel zu beschreiben, ging von der Annahme aus, Kompaßorientierung und die Fähigkeit zur Navigation habe sich im Zusammenhang mit dem Vogelzug entwickelt. Kompaßmechanismen sowie die Mosaik- und die Navigationskarte spielen jedoch bereits bei der Orientierung im Heimbereich entscheidende Rollen, müssen sich also dort entwickelt haben unter dem Selektionsdruck, die täglichen Flugwege zu optimieren, vielleicht schon bei den Vorfahren der Vögel.Magnetkompaßorientierung erscheint als der einfachste Orientierungsmechanismus und müßte deshalb an den ältesten Orientierungsstrategien beteiligt gewesen sein. Ein Magnetkompaß ist bei Wirbeltieren weit verbreitet, doch gibt es Hinweise auf unterschiedliche Funktionsprinzipien. Es ist deshalb offen, ob die Vögel ihn von ihren Vorfahren übernommen oder eigenständig entwickelt haben. Das gleiche gilt für den Sonnenkompaß. Die entscheidende Rolle des Magnetkompaß bei der ontogenetischen Entwicklung des Sonnenkompaß läßt eine ähnliche Beziehung bei der phylogenetischen Entwicklung vermuten.Über kurze Entfernungen kann man sich Orientierung durch Wegumkehr allein mit Kompaßmechanismen vorstellen, wobei Umwege integriert werden müssen. Bei dieser Strategie akkumulieren sich jedoch die Fehler; die bei größeren Entfernungen resultierende Ungenauigkeit erzeugte einen Selektionsdruck, der das Benutzen von Ortsinformation begünstigte. Dies führte zur Entstehung der Mosaikkarte, die auf Kompaßorientierung und Landmarken beruht. Sie ist heute als eigenständiger Mechanismus anzusehen, der nach angeborenen Regeln aufgebaut wird. Die Navigationskarte entsteht, indem die gleichen Regeln auf Faktoren mit Gradienten-Charakter angewandt werden; sie hat sich offenbar aus der Mosaikkarte entwickelt. Ob sie eine Sonderentwicklung der Vögel infolge ihrer Flugfähigkeit ist, muß offen bleiben. Da die Vögel die Grundelemente ihres Orientierungssystems wahrscheinlich von ihren Vorfahren übernommen haben, würden wir erwarten, daß diese Mechanismen bei allen Vögel gleich sind bzw. nach den gleichen Regeln erstellt werden.Vorstufen des Vogelzugs waren zunächst ungerichtete Flüge auf der Suche nach günstigeren Bedingungen; in diesem Stadium reichten die vorhandenen Navigationsmechanismen zur Orientierung zwischen den verschiedenen Gebieten aus. Als aus diesen ersten Ortsbewegungen ein regelmäßiger Zug zwischen zwei Regionen wurde, begann sich das Zugprogramm zu entwickeln, wobei sich zunächst eine spontane Richtungstendenz herausbildete. Der Magnetkompaß konnte als erstes Referenzsystem für diese Zugrichtung dienen. Später erhielt die Himmelsrotation ihre entscheidende Bedeutung, wobei die Vögel die Referenzrichtung Süd zunächst aus dem Polarisationsmuster am Tage ableiteten. Im Laufe der Zeit entstanden die differenzierten Zugprogramme mit Richtungsfolgen, steuernden Zeitprogrammen und Triggermechanismen. Die Zugrichtung und Länge der Zugstrecke unterliegen auch weiterhin einer ständigen Selektion, die für optimale Anpassung an die jeweiligen Umweltbedingungen sorgt. Der Übergang vom Tag- zum Nachtzug bereitete keine Probleme, denn die Vögel mußten zunächst keine neuen Orientierungsmechanismen entwickeln, da sich der Magnetkompaß zu jeder Tageszeit einsetzen läßt. Später entstand der Sternkompaß, der in seinen Funktionseigenschaften hervorragend auf die Bedürfnisse von Zugvögeln angepaßt ist und als eigenständige Entwicklung der Nachtzieher angesehen werden muß. Dazu erwarben die Nachtzieher die Fähigkeit, die Information der Himmelsrotation aus der Bewegung der Sterne abzuleiten und direkt auf den Sternkompaß zu übertragen. Da das Zugverhalten bei Vögeln mehrfach unabhängig voneinander entstanden ist, muß man Entsprechendes auch von den Mechanismen der Zugorientierung annehmen. Das bedeutet, daß sich die betreffenden Mechanismen bei den verschiedenen Arten unterschiedlich entwickelt haben könnten, doch ist mit konvergenten Entwicklungen zu rechnen.

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The orientation system of birds — IV. Evolution

Summary In a first attempt to explain the evolution of the avian navigational system, Bellrose suggested that compass mechanisms and the ability for true navigation had developed in connection with migration across increasing distances. Yet birds use compasses, the mosaic and the navigational maps even close to home and for homing. This means that those mechanisms must have developed for orientation within the home range, with the necessity to optimize the everyday flights acting as selective pressure. In view of this, any attempt to reconstruct the evolution of the avian navigational system must start out with the non-flying ancestors of birds.Considering the requirements of orientation by landmarks and by using a compass, compass orientation with the help of the magnetic field appears to be the simplest mechanism; consequently, it must be assumed to belong to the most ancient orientation strategies. The magnetic compass is wide-spread among animals, but it appears to function according to different principles among the various groups of vertebrates so that it is unclear whether birds inherited their magnetic compass from their reptilian ancestors or developed a mechanism of their own. The same is true for the sun compass. The crucial role of the magnetic compass in the ontogenetic development of the sun compass might indicate a similar relationship for the phylogenetic development.Over short distances within the home range, orientation based solely on compass orientation appears possible, using the strategy of route reversal, with non-straight routes being integrated. Since this strategy accumulates errors, it becomes inaccurate over longer distances, thus causing selective pressure to use local site-specific information. This leads to the formation of the mosaic map, a mechanism that includes landmarks as well as compass orientation. Today, the mosaic map of landmarks is a mechanism by itself, established according to innate learning principles that associate information on path integration with site-specific information, thus forming a directionally oriented mental representation of the distribution of landmarks. The navigational map is formed by applying the same principles to factors of the nature of gradients; it thus appears to have developed from the mosaic map. Whether or not it is a special development of birds associated with their flying ability is unclear. Because the birds probably inherited the basic mechanisms of orientation from their ancestors, one would expect these mechanisms to be similar in all birds. For the mechanisms involving learned components, this means that they are established following common rules. Birds improved those mechanisms and adapted them to their specific needs.Migration is assumed to have begun with non-directed search movements for regions offering better conditions. At this stage, the already existing mechanisms of homing were sufficient for navigation between the various areas. When these first movements turned into regular migration between two regions, the migratory program began to evolve, starting out with spontaneous tendencies in a preferred direction. The magnetic compass may have served as first reference system for the migratory direction; later, celestial rotation, indicated by the changing pattern of polarized light during the day, obtained its important role in indicating the reference direction geographic South. In the course of time, sophisticated migration programs with changes in direction, controlling time programs, responses to trigger mechanisms etc. developed. The migratory direction and distance, i.e. the amount of migratory activity, continue to be subject to selective pressure so that birds can respond to the environmental conditions in an optimal way. The transition from daytime migration to night migration did not require new mechanisms, as the magnetic compass can be used at any time of the day. Later, however, the star compass evolved, which is to be considered a special development of night-migrating birds, with its way of functioning well adapted to the specific needs of migrants. Birds also developed the ability to derive information on celestial rotation from the rotating stars at night and to transfer this information directly to the star compass. Since migratory habits evolved many times independently among birds, the same has to be assumed for the specific mechanisms of migratory orientation. This means that they need not necessarily be identical in all bird migrants. We are to expect convergent developments, however, leading to mechanisms of the most suitable type.

     

Avian magnetic compass can be tuned to anomalously low magnetic intensities

The avian magnetic compass works in a fairly narrow functional window around the intensity of the local geomagnetic field, but adjusts to intensities outside this range when birds experience these new intensities for a certain time. In the past, the geomagnetic field has often been much weaker than at present. To find out whether birds can obtain directional information from a weak magnetic field, we studied spontaneous orientation preferences of migratory robins in a 4 µT field (i.e. a field of less than 10 per cent of the local intensity of 47 µT). Birds can adjust to this low intensity: they turned out to be disoriented under 4 µT after a pre-exposure time of 8 h to 4 µT, but were able to orient in this field after a total exposure time of 17 h. This demonstrates a considerable plasticity of the avian magnetic compass. Orientation in the 4 µT field was not affected by local anaesthesia of the upper beak, but was disrupted by a radiofrequency magnetic field of 1.315 MHz, 480 nT, suggesting that a radical-pair mechanism still provides the directional information in the low magnetic field. This is in agreement with the idea that the avian magnetic compass may have developed already in the Mesozoic in the common ancestor of modern birds.