Peptide substrates for G protein-coupled receptor kinase 2

G protein-coupled receptor kinases (GRKs) control the signaling and activation of G protein-coupled receptors through phosphorylation. In this study, consensus substrate motifs for GRK2 were identified from the sequences of GRK2 protein substrates, and 17 candidate peptides were synthesized to identify peptide substrates with high affinity for GRK2. GRK2 appears to require an acidic amino acid at the −2, −3, or −4 positions and its consensus phosphorylation site motifs were identified as (D/E)X_1–3(S/T), (D/E)X_1–3(S/T)(D/E), or (D/E)X_0–2(D/E)(S/T). Among the 17 peptide substrates examined, a 13-amino-acid peptide fragment of β-tubulin (DEMEFTEAESNMN) showed the highest affinity for GRK2 (K_m, 33.9 μM; V_max, 0.35 pmol min⁻¹ mg⁻¹), but very low affinity for GRK5. This peptide may be a useful tool for investigating cellular signaling pathways regulated by GRK2.     

Carbon Monoxide Promotes Lateral Root Formation in Rapeseed

Carbon monoxide (CO), an odorless, tasteless and colorless gas, has recently proved to be an important bioactive or signalmolecule in mammalian cells, with its effects mediated mainly by nitric oxide (NO). In the present report, we show thatexogenous CO induces lateral root (LR) formation, an NO-dependent process. Administration of the CO donor hematin torapeseed (Brassica napus L. Yangyou 6) seedlings for 3 days, dose-dependently promoted the total length and number ofLRs. These responses were also seen following the application of gaseous CO aqueous solutions of different saturatedconcentrations. Furthermore, the actions of CO on seedlings were fully reversed when the CO scavenger hemoglobin (Hb)or the CO-specific synthetic inhibitor zinc protoporphyrin-IX (ZnPPIX) were added. Interestingly, depletion of endogenousNO using its specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO)or the nitric oxide synthase (NOS) inhibitor N~G-nitro-L-arginine methyl ester (L-NAME),led to the complete abolition ofLR development, illustrating an important role for endogenous NO in the action of CO on LR formation. However, theinduction of LR development by 200 umol/L sodium nitroprusside (SNP),an NO donor, was not affected by the presenceor absence of ZnPPIX. Furthermore, using an anatomical approach combined with laser scanning confocal microscopywith the NO-specific fluorophore 4,5-diaminofluorescein diacetate, we observed that both hematin and SNP increased NOrelease compared with control samples and that the NO signal was mainly distributed in the LR primordia (LRP), especiallyafter 36 h treatment. The LRP were found to have similar morphology in control, SNP-and hematin-treated seedlings.Similarly, the enhancement of the NO signal by CO at 36 h was differentially quenched by the addition of cPTIO, L-NAME,ZnPPIX and Hb. In contrast, the induction of NO caused by SNP was not affected by the application of ZnPPIX. Therefore,we further deduced that CO induces LR formation probably mediated by the NO/NOS pathway and NO may act downstreamof CO signaling, which has also been shown to occur in animals.     

Evaluation of Systematic Position of Helicoprorodontids and Chaeneids (Ciliophora,Litostomatea): An Attempt to Break Long Branches in 18S rRNA Gene Phylogenies

Phylogenetic position of some free‐living litostomatean taxa has not been correctly determined because of long‐branch artifacts in 18S rRNA gene trees. The main aim of this study was to test the effectiveness of various masking algorithms, tree‐building techniques, binarization of DNA data as well as combining morphological and molecular data to eliminate long‐branch attraction of two problematic groups, helicoprorodontids and chaeneids. Guidance and SlowFaster masking in a combination with PhyloBayesian tree construction erased the artifactual positions of helicoprorodontids and chaeneids. On the other hand, binarization of DNA sequences and the strategy of combining morphological and molecular data eliminated only the artifactual position of chaeneids but not that of helicoprorodontids which were still being attracted by out‐group taxa. According to statistical tree topology tests and comparative morphological studies, helicoprorodontids are classified as a distinct order while chaeneids are considered to be fast evolving members of the order Lacrymariida. The high body contractility, “cephalization” of the anterior body end, and helicalization of the anterior portion of some or all somatic ciliary rows indicate relatedness of helicoprorodontids, chaeneids, and lacrymariids. On the other hand, the dorsal brush separated from the circumoral kinety by dense ciliary files supports kinships of chaeneids, lacrymariids, and didiniids.     

Application of repeated aspartate tags to improving extracellular production of Escherichia coli l-asparaginase isozyme II

Asparaginase isozyme II from Escherichia coli is a popular enzyme that has been used as a therapeutic agent against acute lymphoblastic leukemia. Here, fusion tag systems consisting of the pelB signal sequence and various lengths of repeated aspartate tags were devised to highly express and to release active asparaginase isozyme II extracellularly in E. coli. Among several constructs, recombinant asparaginase isozyme II fused with the pelB signal sequence and five aspartate tag was secreted efficiently into culture medium at 34.6 U/mg cell of specific activity. By batch fermentation, recombinant E. coli produced 40.8 U/ml asparaginase isozyme II in the medium. In addition, deletion of the gspDE gene reduced extracellular production of asparaginase isozyme II, indicating that secretion of recombinant asparaginase isozyme II was partially ascribed to the recognition by the general secretion machinery. This tag system composed of the pelB signal peptide, and repeated aspartates can be applied to extracellular production of other recombinant proteins.     

The synthetic precursor specific region of pre-pro-parathyroid hormone forms ion channels in lipid bilayers

We have used the chemically synthesized sequence of pre-pro-parathyroid hormone and several of its analogues to test the notion that the capacity of amphipathic peptides to aggregate in membranes and form ion-permeable channels correlates with their ability to function as signal sequences for secreted proteins. We found that pre-pro-parathyroid hormone (the signal sequence and pro-region of parathyroid hormone (M)), as well as some of its analogues, forms aggregates of monomers which are ion-permeable. The ion-permeable aggregates (2–3 monomers) formed by (M) are voltage-dependent and are more permeable for cations than for anions. The compounds which formed ion channels in bilayers also acted as potential signal sequences. We conclude that the ability of peptides to form ion-permeable pathways in bilayers may be correlated to their ability to function as signal peptides.     

植物细胞中的前纤维蛋白

肌动蛋白组成的微丝骨架是真核细胞中的重要结构,在体内处于高度动态变化之中,受多种肌动蛋白结合蛋白（actin-binding proteins）的调节。前纤维蛋白（profilin）是一种单体肌动蛋白结合蛋白,存在于所有的真核细胞中,在植物细胞中也得到较多的研究。前纤维蛋白除可以结合单体肌动蛋白之外,还可以与磷脂酰肌醇及富含多聚脯氨酸的蛋白质等多种分子结合,在细胞信号转导中行使着重要的功能。本文结合本实验室的研究结果,概述了前纤维蛋白的最新研究进展。     

Wie V?gel ihr Auge schützen: Zur Arbeitsteilung von Oberlid, Unterlid und Nickhaut

Zusammenfassung Vögel schließen ihre Augen im Schlaf in einer für die großen Taxa typischen Weise. Entweder geht das Unterlid hoch wie bei der Mehrzahl der Arten, oder das Oberlid bewegt sich abwärts (Psittaciformes, Trochili), oder aber beide Lider schließen die Lidspalte (Strigiformes, Caprimulgi). Solche Kenntnis fehlt von den meisten Ordnungen, oder die Handbücher geben falsche oder widersprüchliche Information. Neben dem tonischen, schlafbegleitenden Augenschluss bewegen Vögel im Wachzustand eines oder beide Lider phasisch und meist schnell. Dieser häufige Lidschlag ist durch ein anderes Bewegungsmuster und durch eine andere Funktion gekennzeichnet. Photodokumente und genaue Beobachtungen führen erstmals zu einer funktionellen Deutung, der zufolge der Lidschlag das Auge mechanisch schützt. Droht dem Auge von vorn oder von oben eine potentielle Schädigung, so schließt das Oberlid bei Tauben, Eulen und Singvögeln, im Sprühwasser gleichzeitig auch das Unterlid (Cinclus). Der unabweisbarste Beleg stammt aus dem Vergleich des Aufpickens dorniger, sperriger Beuteinsekten mit Oberlidschluss gegenüber dem Aufnehmen harmloser Beeren ohne jede Lidbewegung (Gallicolumba). Weiter ist die Antwort des Oberlids, anders als beim Unterlid, öfter seitengerecht reizorientiert, so dass die Bewegung einseitig sein kann. Zudem kann der Schluss des Oberlids auch bei stationärem (Feind-)reiz seitenweise alternieren (Otus). Ausnahmsweise tritt eine adaptive Asymmetrie auch während kurzer Zeiten der Augenöffnung zum Spähen nach Feinden im Schlaf auf, und zwar hier beim Unterlid der bedrohten Seite (Anas).Eine neue Funktion wird auch dem Schlag der Nickhaut (Membrana nictitans) zugeschrieben. Traditionell als die Cornea reinhaltendes Organ gesehen, dient auch sie dem mechanischen Schutz des Auges. Auch sie kann seitenrichtig reizorientiert schlagen, doch ist hierüber wenig bekannt. Dieselben Reize, die den Lidschlag auslösen, können bei anderen Arten die Nickhaut schlagen lassen. Ihre Schlagrate ist schwierig zu messen, da viele Schläge (nur?) mit denen des Oberlides zusammenfallen und so verborgen bleiben (Otus). Diese Synchronie ist mit keiner der bisher vorgeschlagenen Funktionen erklärbar, ebenso wenig wie die verborgenen Schläge bei tonischem Augenschluss (Passer).Die Annahme einer Ausschaltung störender Sinnesinformation, z.B. während rascher Kopfbewegungen, durch die Nickhaut lässt sich aus vier Gründen verwerfen. Die Zunahme der Schlagrate während des Feindalarms (Ficedula) bleibt funktionell unerklärt.In einer bei Vögeln einzigartigen Weise schützt der Samtkleiber (Sitta azurea) sein Auge durch Zusammenziehen des nackten Augenrings (Lidblende), wenn er rücklings an der Unterseite von Ästen nahrungssuchend einem ständigen Regen von losgelösten Rindenteilchen u. ä. ausgesetzt ist.Sekundär haben sich die Bewegungen eines oder beider Lider oder aber der Nickhaut zu optischen Signalen entwickelt, und zwar durch kontrastierende Feder- oder Nickhautfärbung. Die betreffenden blitzschnell aufleuchtenden Signale sind an den Paarpartner (Cinclus, Corvidae,Cepphus), an mögliche Feinde (Anas) oder an bisher unbekannte Empfänger gesichtet (Ficedula).

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On how birds protect their eyes: division of labour between the upper lid, lower lid and the nictitating membrane

Summary Birds close their eyes during sleep in various taxon-specific ways. Either the lower lid moves up as in the majority of species including the Anseres, Accipitres, Falconiformes, Galli, Charadrioidea, Columbiformes, and Oscines; or the upper lid moves down (Psittaciformes, Trochili), or both lids close the eye as in Strigiformes and Caprimulgi. Such information is absent for most orders, or the handbooks provide wrong or conflicting information. Beside the tonic, sleep-related eye closure, birds move one or both lids in a phasic, usually swift mode when awake. These frequent lid movements are typified by their different co-ordination and function. Photographic and observational evidence strongly suggests mechanical protection of the eye as a novel function (where this had not been proposed previously). When an impact from any object is imminent from in front of or above the head, the upper lid shuts in pigeons, owls and oscines, and with water splashing, the lower lid as well (Cinclus). The most convincing evidence for mechanical protection comes from the deployment of the upper lid during the picking up of spiny insect prey as compared to easy-to-swallow berries, when both lids stay at rest (Gallicolumba).Further, the response of the upper lid is more stimulus-oriented so that both upper lids move asymmetrically. But there is also a unilateral, alternating winking of the upper lids when causative (predator) stimuli remain stationary. This never occurs with the lower lids (Otus). As an exception, an adaptive asymmetry occurs during brief phases of unilateral scanning interrupting sleep, designed to detect approaching predators. This scanning involves the lower lid (Anas).A new function is also attributed to the beating of the nictitating membrane (Membrana nictitans). Traditionally viewed as a cleaning device it also serves to protect the eye from mechanical impact, and it also can be tuned to the side from where danger is threatening, though by and large there is a dearth of information from avian taxa. The non-visually elicited action of the membrane seems always to be bilateral (Falco, Harpia). The very stimuli eliciting the blinking of a lid can, in different species, trigger the beat of the membrane, and can cause it to move tonically (Falco). The membrane beats at a rate difficult to measure since many of its beats coincide with the blinking of the upper lid and thus remain hidden (Otus). This coincidence is difficult to account for by any function discussed so far, as are the many hidden beats during tonic eye closure with the lids (Passer).The hypothesis according to which the action of the membrane is filtering out undesirable retinal stimulation during e.g. rapid head movements is dismissed on four different grounds. The increase of the membrane activity during predator alarm (Ficedula) is functionally unaccounted for.In a fashion unique among birds, the Blue Nuthatch (Sitta azurea) protects its eyes by contracting the naked skin surrounding the eye, thereby minimizing the exposure of the cornea; during foraging along the underside of branches, a continual rain of bark particles and debris jeopardizes unimpeded vision.Secondarily, one or both lids or the nictitating membrane have taken on the function of optic signals by virtue of contrasting feather colour or coloration. The phasic (flashing) signal movements involved are directed at the pair mate (Cinclus, Corvidae,Cepphus), predators (Anas) or at unknown parties (Ficedula).

Dies ist Veröffentlichung Nr. 29 des Philippine Endemic Species Conservation Project der Zoologischen Gesellschaft Frankfurt.