The Taiwan Firecrest ( Regulus goodfellowi ) belongs to the Goldcrest assemblage ( Regulus regulus s. l.): evidence from mitochondrial DNA and the territorial song of the Regulidae

We evaluated the phylogenetic relationships of the Taiwan Firecrest or Flamecrest, Regulus goodfellowi, on the basis of two mitochondrial markers (cytochrome b, 16S rRNA) and territorial song. Genetic samples from eighteen subspecies of all currently accepted crest and kinglet species were available for comparison. In all molecular tree reconstructions it was clearly apparent the Taiwan endemic species was the sister species of all the palearctic Goldcrests (Regulus regulus) from Bayesian inference of phylogeny, although with weak bootstrap support and conflicting position in the ML and NJ trees. Genetic distances based on cyt-b sequences between R. goodfellowi and subspecies of R. regulus ranged between 6.1 and 8.2%. Two separate divergence time estimates dated the colonization of Taiwan to the mid to late Pliocene from 5–2 Mya. The high-pitched territorial songs of R. goodfellowi strongly resemble those of Sino-Himalayan Goldcrests (ssp. himalayensis, sikkimensis, and yunnanensis), but the terminal flourish typical of Goldcrests is invariable and only rarely included in songs of R. goodfellowi. Discriminant analysis of spectral and temporal characteristics separated the songs of the Southeast Asian populations from those of two other large clusters, the Canarian and Northwest Palearctic. Songs of R. goodfellowi were 100% correctly assigned and well distinguishable from Sino–Himalayan songs. Cluster analysis of Regulus songs strongly corroborated the sister group relationship of R. goodfellowi and R. regulus as reconstructed from concatenated mitochondrial sequence data. All results from molecular and acoustic analysis justify the species rank of the Taiwan endemic species and suggest that it is only distantly related to the firecrest clade (R. ignicapillus, R. madeirensis).     

Cdc50p Plays a Vital Role in the ATPase Reaction Cycle of the Putative Aminophospholipid Transporter Drs2p

Members of the P₄ subfamily of P-type ATPases are believed to catalyze transport of phospholipids across cellular bilayers. However, most P-type ATPases pump small cations or metal ions, and atomic structures revealed a transport mechanism that is conserved throughout the family. Hence, a challenging problem is to understand how this mechanism is adapted in P₄-ATPases to flip phospholipids. P₄-ATPases form heteromeric complexes with Cdc50 proteins. The primary role of these additional polypeptides is unknown. Here, we show that the affinity of yeast P₄-ATPase Drs2p for its Cdc50-binding partner fluctuates during the transport cycle, with the strongest interaction occurring at a point where the enzyme is loaded with phospholipid ligand. We also find that specific interactions with Cdc50p are required to render the ATPase competent for phosphorylation at the catalytically important aspartate residue. Our data indicate that Cdc50 proteins are integral components of the P₄-ATPase transport machinery. Thus, acquisition of these subunits may have been a crucial step in the evolution of flippases from a family of cation pumps.P-type ATPases form a large family of membrane pumps that are transiently autophosphorylated at a conserved aspartate residue, hence the designation P-type. Prominent examples include the Ca²⁺-ATPase SERCA,⁴ which pumps Ca²⁺ from the cytosol into the lumen of the sarcoplasmic reticulum of skeletal muscle cells (1), and the Na⁺/K⁺-ATPase, which generates the electrochemical gradients for sodium and potassium that are vital to animal cells (2). Transport is accomplished by cyclic changes between two main enzyme conformations, E₁ and E₂, during which the ATPase is phosphorylated by ATP at the aspartate residue and subsequently dephosphorylated. These processes are coupled to vectorial transport and counter-transport by a controlled opening and closing of cytoplasmic and exoplasmic pathways, which give access to the ion-binding sites that are buried inside the membrane-spanning region of the pump (3). A host of crystal structures of the Ca²⁺ pump SERCA in well defined states of the reaction cycle revealed important aspects of the transport mechanism (4, 5). Sequence homology and structures of other ATPases show that this mechanism rests on principles and structural elements that apply to all P-type ATPases (6–8).Although P-type ATPases usually pump small cations or metal ions, members of the P₄ subfamily form a notable exception. A growing body of evidence indicates that P₄-ATPases catalyze phospholipid transport and create membrane lipid asymmetry (9–11). This process contributes to a multitude of cellular functions, including membrane vesiculation, cell division, and life span. The yeast Saccharomyces cerevisiae contains five P₄-ATPases, namely Dnf1p and Dnf2p at the plasma membrane, Drs2p and Dnf3p in the trans-Golgi network, and Neo1p in an endosomal compartment (12–14). Removal of Dnf1p and Dnf2p abolishes inward translocation of 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))-labeled analogs of phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylcholine (PC) and causes an aberrant exposure of endogenous aminophospholipids at the cell surface (13, 15). Trans-Golgi membranes isolated from a yeast strain that lacks the Dnf proteins and contains a temperature-sensitive drs2 allele display a defect in 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))-PS translocation when shifted to the non-permissive temperature (16). The latter finding provides strong evidence that Drs2p is directly coupled to flippase activity, and subsequent studies showed that Drs2p, together with Dnf3p, are required for maintaining PE asymmetry in post-Golgi secretory vesicles (17).Although no P₄-ATPase has been shown to display flippase activity in reconstitution experiments with purified enzyme, the relationship of P₄-ATPases to flippase activity and lipid asymmetry has gained further support from functional studies in various other organisms, including parasites (18), plants (19), worms (20), and mice (21). Besides a common domain organization, P₄-ATPases display a clear sequence homology with cation-transporting P-type pumps. Shared sequence motifs include the canonical phosphorylation site in the P domain, the nucleotide-binding site in the N domain, and a TGES-related sequence in the A domain (22). This implies that P₄-ATPases and cation pumps use the same mechanism to couple ATP hydrolysis to ligand transport. Phospholipid transport by P₄-ATPases would correspond to counter-transport of H⁺ ions by the Ca²⁺ pump and of K⁺ ions by the Na⁺/K⁺-ATPase as the direction of movement is from the exoplasmic to the cytoplasmic leaflet. During the reaction cycle of cation pumps, access to the ion-binding pocket alternates between the two sides of the membrane, with the ions becoming temporarily occluded after each ion binding event (23). How this mechanism is adapted in P₄-ATPases to translocate phospholipids is unclear. Flippases must provide a sizeable hydrophilic pathway for the polar headgroup to pass through the membrane as well as accommodate the hydrophobic nature of the lipid backbone. Whether P₄-ATPases alone are sufficient to accomplish this task is not known.Recent studies revealed that P₄-ATPases form complexes with members of the Cdc50 protein family (24). Cdc50 proteins consist of two membrane spans and a large, N-glycosylated ectodomain with one or more conserved disulfide bonds (25). The yeast family members Cdc50p, Lem3p, and Crf1p can be co-immunoprecipitated with Drs2p, Dnf1p/Dnf2p, and Dnf3p, respectively. Formation of these complexes is required for proper expression and endoplasmic reticulum (ER) export of either partner (24, 26) so that mutation of one member of the complex phenocopies mutations in the other (15, 25). This behavior in yeast is mirrored in other organisms; Ld Ros3, a Lem3p homolog in Leishmania parasites, is needed for proper trafficking of the P₄-ATPase Ld MT (18), whereas the human P₄-ATPase ATP8B1 requires a Cdc50p homolog, CDC50A, for ER exit and delivery to the plasma membrane (27). Moreover, the Arabidopsis P₄-ATPase ALA3 requires its Cdc50-binding partner ALIS1 to complement the lipid transport defect at the plasma membrane in a Δdnf1Δdnf2Δdrs2 yeast mutant (19).Together, the above findings indicate that Cdc50 subunits are indispensable for a proper functioning of P₄-ATPases and that it is the combination of the two that yields a physiologically active transporter. However, these studies have not clarified the primary function of the Cdc50 polypeptide in the complex. Here, we provide the first evidence that Cdc50 subunits play a crucial role in the P₄-ATPase reaction cycle. Using a genetic reporter system, we find that P₄-ATPase-Cdc50 interactions are dynamic and tightly coupled to the ATPase reaction cycle. Moreover, by characterizing the enzymatic properties of a purified P₄-ATPase-Cdc50 complex, we show that catalytic activity relies on direct and specific interactions between the subunit and transporter.     

Estimating maize genetic erosion in modernized smallholder agriculture

Replacement of crop landraces by modern varieties is thought to cause diversity loss. We studied genetic erosion in maize within a model system; modernized smallholder agriculture in southern Mexico. The local seed supply was described through interviews and in situ seed collection. In spite of the dominance of commercial seed, the informal seed system was found to persist. True landraces were rare and most informal seed was derived from modern varieties (creolized). Seed lots were characterized for agronomical traits and molecular markers. We avoided the problem of non-consistent nomenclature by taking individual seed lots as the basis for diversity inference. We defined diversity as the weighted average distance between seed lots. Diversity was calculated for subsets of the seed supply to assess the impact of replacing traditional landraces with any of these subsets. Results were different for molecular markers, ear- and vegetative/flowering traits. Nonetheless, creolized varieties showed low diversity for all traits. These varieties were distinct from traditional landraces and little differentiated from their ancestral stocks. Although adoption of creolized maize into the informal seed system has lowered diversity as compared to traditional landraces, genetic erosion was moderated by the distinct features offered by modern varieties.     

Biomimetic approach to tissue engineering

The overall goal of tissue engineering is to create functional tissue grafts that can regenerate or replace our defective or worn out tissues and organs. Examples of grafts that are now in pre-clinical studies or clinical use include engineered skin, cartilage, bone, blood vessels, skeletal muscle, bladder, trachea, and myocardium. Engineered tissues are also finding applications as platforms for pharmacological and physiological studies in vitro. To fully mobilize the cell's biological potential, a new generation of tissue engineering systems is now being developed to more closely recapitulate the native developmental milieu, and mimic the physiologic mechanisms of transport and signaling. We discuss the interactions between regenerative biology and engineering, in the context of (i) creation of functional tissue grafts for regenerative medicine (where biological input is critical), and (ii) studies of stem cells, development and disease (where engineered tissues can serve as advanced 3D models).     

Distracting the mind improves performance: an ERP Study

Background

When a second target (T2) is presented in close succession of a first target (T1), people often fail to identify T2, a phenomenon known as the attentional blink (AB). However, the AB can be reduced substantially when participants are distracted during the task, for instance by a concurrent task, without a cost for T1 performance. The goal of the current study was to investigate the electrophysiological correlates of this paradoxical effect.

Methodology/Principal Findings

Participants successively performed three tasks, while EEG was recorded. The first task (standard AB) consisted of identifying two target letters in a sequential stream of distractor digits. The second task (grey dots task) was similar to the first task with the addition of an irrelevant grey dot moving in the periphery, concurrent with the central stimulus stream. The third task (red dot task) was similar to the second task, except that detection of an occasional brief color change in the moving grey dot was required. AB magnitude in the latter task was significantly smaller, whereas behavioral performance in the standard and grey dots tasks did not differ. Using mixed effects models, electrophysiological activity was compared during trials in the grey dots and red dot tasks that differed in task instruction but not in perceptual input. In the red dot task, both target-related parietal brain activity associated with working memory updating (P3) as well as distractor-related occipital activity was significantly reduced.

Conclusions/Significance

The results support the idea that the AB might (at least partly) arise from an overinvestment of attentional resources or an overexertion of attentional control, which is reduced when a distracting secondary task is carried out. The present findings bring us a step closer in understanding why and how an AB occurs, and how these temporal restrictions in selective attention can be overcome.     

A sandwich enzyme-linked immunosorbent assay for the quantification of insoluble membrane and scaffold proteins

Enzyme-linked immunosorbent assays (ELISAs) are applied for the quantification of a vast diversity of small molecules. However, ELISAs require that the antigen is present in a soluble form in the sample. Accordingly, the few ELISAs described so far targeting insoluble proteins such as integral membrane and scaffold proteins have been restricted by limited extraction efficiencies and the need to establish an individual solubilization protocol for each protein. Here we describe a sandwich ELISA that allows the quantification of a diverse array of synaptic membrane and scaffold proteins such as munc13-1, gephyrin, NMDA R1 (N-methyl-d-aspartate receptor subunit 1), synaptic vesicle membrane proteins, and SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). The assay is based on initial solubilization by the denaturing detergent sodium dodecyl sulfate (SDS), followed by partial SDS removal using the detergent Triton X-100, which restores antigenicity while keeping the proteins in solution. Using recombinant standard proteins, we determined assay sensitivities of 78 ng/ml to 77 pg/ml (or 74-0.1 fmol). Calibration of the assay using both immunoblotting and mass spectroscopy revealed that in some cases correction factors need to be included for absolute quantification. The assay is versatile, allows parallel processing and automation, and should be applicable to a wide range of hitherto inaccessible proteins.     

Multifunctional flavonoid dioxygenases: Flavonol and anthocyanin biosynthesis in Arabidopsis thaliana L.

Flavonols and conditionally also anthocyanins, aside from flavonols, are the predominant polyphenols accumulated in various tissues of the model plant Arabidopsis thaliana L. In vitro experiments suggested that the dioxygenases involved in their biosynthesis, flavonol synthase and anthocyanidin synthase, are “multifunctional” enzymes showing distinct side activities. The in vivo relevance of the additional activities attributed to these enzymes, however, has remained obscure. In this review we summarize the most recent results and present final proof of the complementing activities of these synthases for flavonol and anthocyanidin formation in the model plant A. thaliana. The impact of their modification on the biosynthetic pathway and the pattern of flavonoids in different plant tissues are discussed.     

OLS Dialog: An open-source front end to the Ontology Lookup Service

Background  

With the growing amount of biomedical data available in public databases it has become increasingly important to annotate data in a consistent way in order to allow easy access to this rich source of information. Annotating the data using controlled vocabulary terms and ontologies makes it much easier to compare and analyze data from different sources. However, finding the correct controlled vocabulary terms can sometimes be a difficult task for the end user annotating these data.