Nucleotide-Specific Recognition of Iron-Responsive Elements by Iron Regulatory Protein 1

IRP1 [iron regulatory protein (IRP) 1] is a bifunctional protein with mutually exclusive end-states. In one mode of operation, IRP1 binds iron-responsive element (IRE) stem–loops in messenger RNAs encoding proteins of iron metabolism to control their rate of translation. In its other mode, IRP1 serves as cytoplasmic aconitase to correlate iron availability with the energy and oxidative stress status of the cell. IRP1/IRE binding occurs through two separate interfaces, which together contribute about two-dozen hydrogen bonds. Five amino acids make base-specific contacts and are expected to contribute significantly to binding affinity and specificity of this protein:RNA interaction. In this mutagenesis study, each of the five base-specific amino acids was changed to alter binding at each site. Analysis of IRE binding affinity and translational repression activity of the resulting IRP1 mutants showed that four of the five contact points contribute uniquely to the overall binding affinity of the IRP1:IRE interaction, while one site was found to be unimportant. The stronger-than-expected effect on binding affinity of mutations at Lys379 and Ser681, residues that make contact with the conserved nucleotides G16 and C8, respectively, identified them as particularly critical for providing specificity and stability to IRP1:IRE complex formation. We also show that even though the base-specific RNA-binding residues are not part of the aconitase active site, their substitutions can affect the aconitase activity of holo-IRP1, positively or negatively.     

Wnt/β-Catenin Signaling Protects Mouse Liver against Oxidative Stress-induced Apoptosis through the Inhibition of Forkhead Transcription Factor FoxO3

Numerous liver diseases are associated with extensive oxidative tissue damage. It is well established that Wnt/β-catenin signaling directs multiple hepatocellular processes, including development, proliferation, regeneration, nutrient homeostasis, and carcinogenesis. It remains unexplored whether Wnt/β-catenin signaling provides hepatocyte protection against hepatotoxin-induced apoptosis. Conditional, liver-specific β-catenin knockdown (KD) mice and their wild-type littermates were challenged by feeding with a hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet to induce chronic oxidative liver injury. Following the DDC diet, mice with β-catenin-deficient hepatocytes demonstrate increased liver injury, indicating an important role of β-catenin signaling for liver protection against oxidative stress. This finding was further confirmed in AML12 hepatocytes with β-catenin signaling manipulation in vitro using paraquat, a known oxidative stress inducer. Immunofluorescence staining revealed an intense nuclear FoxO3 staining in β-catenin-deficient livers, suggesting active FoxO3 signaling in response to DDC-induced liver injury when compared with wild-type controls. Consistently, FoxO3 target genes p27 and Bim were significantly induced in β-catenin KD livers. Conversely, SGK1, a β-catenin target gene, was significantly impaired in β-catenin KD hepatocytes that failed to inactivate FoxO3. Furthermore, shRNA-mediated deletion of FoxO3 increased hepatocyte resistance to oxidative stress-induced apoptosis, confirming a proapoptotic role of FoxO3 in the stressed liver. Our findings suggest that Wnt/β-catenin signaling is required for hepatocyte protection against oxidative stress-induced apoptosis. The inhibition of FoxO through its phosphorylation by β-catenin-induced SGK1 expression reduces the apoptotic function of FoxO3, resulting in increased hepatocyte survival. These findings have relevance for future therapies directed at hepatocyte protection, regeneration, and anti-cancer treatment.     

Kindlin Binds Migfilin Tandem LIM Domains and Regulates Migfilin Focal Adhesion Localization and Recruitment Dynamics

Focal adhesions (FAs), sites of tight adhesion to the extracellular matrix, are composed of clusters of transmembrane integrin adhesion receptors and intracellular proteins that link integrins to the actin cytoskeleton and signaling pathways. Two integrin-binding proteins present in FAs, kindlin-1 and kindlin-2, are important for integrin activation, FA formation, and signaling. Migfilin, originally identified in a yeast two-hybrid screen for kindlin-2-interacting proteins, is a LIM domain-containing adaptor protein found in FAs and implicated in control of cell adhesion, spreading, and migration. By binding filamin, migfilin provides a link between kindlin and the actin cytoskeleton. Here, using a combination of kindlin knockdown, biochemical pulldown assays, fluorescence microscopy, fluorescence resonance energy transfer (FRET), and fluorescence recovery after photobleaching (FRAP), we have established that the C-terminal LIM domains of migfilin dictate its FA localization, shown that these domains mediate an interaction with kindlin in vitro and in cells, and demonstrated that kindlin is important for normal migfilin dynamics in cells. We also show that when the C-terminal LIM domain region is deleted, then the N-terminal filamin-binding region of the protein, which is capable of targeting migfilin to actin-rich stress fibers, is the predominant driver of migfilin localization. Our work details a correlation between migfilin domains that drive kindlin binding and those that drive FA localization as well as a kindlin dependence on migfilin FA recruitment and mobility. We therefore suggest that the kindlin interaction with migfilin LIM domains drives migfilin FA recruitment, localization, and mobility.     

Segregation in the African wintering ranges of English and Swiss Swallow Hirundo rustica populations: a stable isotope study

Capsule Stable isotope analysis of Swallow feathers, grown in Africa, revealed significant differences between populations breeding in Switzerland and England.

Aims To investigate the extent to which Swallow populations breeding in Switzerland and England are separated on their African wintering grounds.

Methods Swallows were caught at breeding colonies, biometric measurements were taken and feathers, grown in Africa, were collected. Feathers were combusted in a Carlo Erba C/N/S analyser and the δ¹³C and δ¹⁵N signatures were measured using a mass spectrometer.

Results The δ¹³C signatures of Swiss birds were significantly more depleted than those of birds from England. The δ¹⁵N signatures did not differ between the two populations.

Conclusion Birds from Switzerland and England probably winter in geographically distinct parts of Africa. The Swiss birds probably feed on prey that are more reliant on C₃ vegetation, from woodlands, than the prey of English birds, which are more reliant on C₄ vegetation, from grasslands.     

Latitudinal and seasonal patterns in clutch size of some single‐brooded British birds

Capsule Daylength, rather than latitude, was found to be an important determinant of variation in clutch size.

Aims To describe the nature of spatial and temporal variation in clutch size, and explore the ecological correlates of these patterns.

Methods We tested the prediction that seasonal declines in clutch size will be greater at higher latitudes. The environmental variables focused on were the influence of daylength, plant productivity, seasonality (i.e. Ashmole's hypothesis) and physiological mechanisms that relate clutch size to ambient temperature. We used data from 1980 to 2003 on spatial variation in clutch size across Britain for single‐brooded species, in which clutch size can be taken as a measure of annual reproductive investment. We included all seven species, from five families, with sufficient data in the British Trust for Ornithology's Nest Record Scheme.

Results There are strong seasonal declines in clutch size but little evidence for latitudinal gradients in clutch size or in latitudinal gradients in the rate of seasonal clutch size decline. Of the environmental variables investigated, daylength had the most marked effect on clutch size; this was positive in diurnal species and negative in the one nocturnal species.

Conclusions Although this study was confined to a relatively small latitudinal range of 8°, we found marked latitudinal gradients in a number of factors thought to drive spatial patterns in clutch size. Moreover, such variation is of sufficient magnitude to generate spatial patterns in other ecological variables in Britain. There is thus no simple explanation for the lack of a latitudinal gradient in clutch size. The results concerning daylength indicate that the time available for foraging is an important determinant of variation in clutch size.     

SEASONALITY MAINTAINS ALTERNATIVE LIFE‐HISTORY PHENOTYPES

Many organisms express discrete alternative phenotypes (polyphenisms) in relation to predictable environmental variation. However, the evolution of alternative life‐history phenotypes remains poorly understood. Here, we analyze the evolution of alternative life histories in seasonal environments by using temperate insects as a model system. Temperate insects express alternative developmental pathways of diapause and direct development, the induction of a certain pathway affecting fitness through its life‐history correlates. We develop a methodologically novel and holistic simulation model and optimize development time, growth rate, body size, reproductive effort, and adult life span simultaneously in both developmental pathways. The model predicts that direct development should be associated with shorter development time (duration of growth) and adult life span, higher growth rate and reproductive effort, smaller body size as well as lower fecundity compared to the diapause pathway, because the two generations divide the available time unequally. These predictions are consistent with many empirical data. Our analysis shows that seasonality alone can explain the evolution of alternative life histories.     

Germline Mutations in NFKB2 Implicate the Noncanonical NF-κB Pathway in the Pathogenesis of Common Variable Immunodeficiency

Common variable immunodeficiency (CVID) is a heterogeneous disorder characterized by antibody deficiency, poor humoral response to antigens, and recurrent infections. To investigate the molecular cause of CVID, we carried out exome sequence analysis of a family diagnosed with CVID and identified a heterozygous frameshift mutation, c.2564delA (p.Lys855Serfs^∗7), in NFKB2 affecting the C terminus of NF-κB2 (also known as p100/p52 or p100/p49). Subsequent screening of NFKB2 in 33 unrelated CVID-affected individuals uncovered a second heterozygous nonsense mutation, c.2557C>T (p.Arg853^∗), in one simplex case. Affected individuals in both families presented with an unusual combination of childhood-onset hypogammaglobulinemia with recurrent infections, autoimmune features, and adrenal insufficiency. NF-κB2 is the principal protein involved in the noncanonical NF-κB pathway, is evolutionarily conserved, and functions in peripheral lymphoid organ development, B cell development, and antibody production. In addition, Nfkb2 mouse models demonstrate a CVID-like phenotype with hypogammaglobulinemia and poor humoral response to antigens. Immunoblot analysis and immunofluorescence microscopy of transformed B cells from affected individuals show that the NFKB2 mutations affect phosphorylation and proteasomal processing of p100 and, ultimately, p52 nuclear translocation. These findings describe germline mutations in NFKB2 and establish the noncanonical NF-κB signaling pathway as a genetic etiology for this primary immunodeficiency syndrome.     

Electron Microscopy of the Megaspore Horstisporites Semireticulatus from Liassic Strata of Germany

Ultra-thin sections of paratypes of the megaspore Horstisporites semireticulatus Jung from Liassic strata of Germany have been investigated by the transmission electron microscope. By this it could be demonstrated that the fossil sporoderm consists of two distinct layers. The inner layer (exine) is very thin (about 0.5 μ) and reveals a lamellated structure. The outer layer (perine) is thicker by far (about 25 μ or more) and is composed of ramifying sporonin threads, which form a three-dimensional network. Proximally, in the region of the triradiate dehiscence commissure, both layers coalesce. Distally the exine separates from the perine, forming a cavity hitherto, erroneously, called mesosporoid. The structural similarity of the Horstisporites semireticulatus sporoderm and that of such megaspores of Selaginella which show a monozonal kind of perine formation e.g. Selaginella selaginoides, favours the idea that the fossil species in question belongs to the Selaginellaceae     

Microstructures of the Mesozoic Megaspore Tasmanitriletes N.G.

The Mesozoic megaspore Trileites pedinacron has been studied by transmission electron microscopy. The wall consists of one relatively thick layer (perine) which is perforated by radial tubes. This wall structure is compared with similar wall patterns of the zoosporangia of marine planctonic algae (Tasmanites, Pleurozonaria, Pachysphaera). Essential differences are pointed out. With T. pedinacron as type species a new form genus is established: Tasmanitriletes.