Zebrafish hoxd4a Acts Upstream of meis1.1 to Direct Vasculogenesis,Angiogenesis and Hematopoiesis

Mice lacking the 4^th-group paralog Hoxd4 display malformations of the anterior vertebral column, but are viable and fertile. Here, we report that zebrafish embryos having decreased function of the orthologous hoxd4a gene manifest striking perturbations in vasculogenesis, angiogenesis and primitive and definitive hematopoiesis. These defects are preceded by reduced expression of the hemangioblast markers scl1, lmo2 and fli1 within the posterior lateral plate mesoderm (PLM) at 13 hours post fertilization (hpf). Epistasis analysis revealed that hoxd4a acts upstream of meis1.1 but downstream of cdx4 as early as the shield stage in ventral-most mesoderm fated to give rise to hemangioblasts, leading us to propose that loss of hoxd4a function disrupts hemangioblast specification. These findings place hoxd4a high in a genetic hierarchy directing hemangioblast formation downstream of cdx1/cdx4 and upstream of meis1.1. An additional consequence of impaired hoxd4a and meis1.1 expression is the deregulation of multiple Hox genes implicated in vasculogenesis and hematopoiesis which may further contribute to the defects described here. Our results add to evidence implicating key roles for Hox genes in their initial phase of expression early in gastrulation.     

Dietary divergence in space and time – Lessons from the dwarf-goat Myotragus balearicus (Pleisto-Holocene,Mallorca, Spain)

Newly colonised, isolated habitats, like islands, provide diverse niches to be filled and are prone to facilitate ecological separation which might lead to an adaptive radiation. Examples of such radiations can be found in the Mediterranean for the genera Candiacervus (Crete), Nesogoral (Sardinia) and Hoplitomeryx (Gargano). A different strategy to cope with limited resources on islands is generalism. We test whether populations of the endemic bovid Myotragus balearicus from two sites and Pleistocene as well as Holocene levels on Mallorca island displays ecological separation indicated by diet, or whether the species shifted its dietary trait towards generalism. We expect to find either: (1) dietary divergence in space and time (between sites and stratigraphic levels), which would indicate niche partitioning and/or a shift in dietary traits due to environmental influences; or (2) dietary congruence in a less specialised, generalistic dietary strategy in space and time which would indicate a flexible trait to cope with instable resource availability. We compare individuals from a fossil assemblage at a northern site and one assemblage from the eastern coast in terms of their dietary traits. Traits are reconstructed using dental dietary proxies, complementary in time scale and resolution. (1) 3D-dental topometry and (2) enamel surface texture analysis. Data suggest that individuals from both assemblages of M. balearicus behaved as variable browse dominated intermediate feeders. We thus conclude that the observed variability relates to a shift towards generalism as a subsistence strategy. We consider hypsodonty the pre-adaptation for this life style that enabled M. balearicus to exploit almost any food source in its energetically restricted island habitat.     

Structural and Mechanistic Studies Reveal the Functional Role of Bicovalent Flavinylation in Berberine Bridge Enzyme

Berberine bridge enzyme (BBE) is a member of the recently discovered family of bicovalently flavinylated proteins. In this group of enzymes, the FAD cofactor is linked via its 8α-methyl group and the C-6 atom to conserved histidine and cysteine residues, His-104 and Cys-166 for BBE, respectively. 6-S-Cysteinylation has recently been shown to have a significant influence on the redox potential of the flavin cofactor; however, 8α-histidylation evaded a closer characterization due to extremely low expression levels upon substitution. Co-overexpression of protein disulfide isomerase improved expression levels and allowed isolation and purification of the H104A protein variant. To gain more insight into the functional role of the unusual dual mode of cofactor attachment, we solved the x-ray crystal structures of two mutant proteins, H104A and C166A BBE, each lacking one of the covalent linkages. Information from a structure of wild type enzyme in complex with the product of the catalyzed reaction is combined with the kinetic and structural characterization of the protein variants to demonstrate the importance of the bicovalent linkage for substrate binding and efficient oxidation. In addition, the redox potential of the flavin cofactor is enhanced additively by the dual mode of cofactor attachment. The reduced level of expression for the H104A mutant protein and the difficulty of isolating even small amounts of the protein variant with both linkages removed (H104A-C166A) also points toward a possible role of covalent flavinylation during protein folding.Since the discovery of the first known example of a covalent bond between a flavin cofactor and an amino acid side chain occurring in enzymes in the 1950s (1), a number of different types of linkages have been identified: 8α-histidylation (either to N1 or to N3), 8α-O-tyrosylation, 8α-S-cysteinylation, and 6-S-cysteinylation. For current reviews relating to these modes of flavin attachment, see Refs. 2 and 3. Recently, another way of covalent tethering of FAD to proteins was discovered in x-ray crystallographic studies on glucooligosaccharide oxidase (GOOX)⁴ from Acremonium strictum (4). The mode of flavin linkage observed in this case employs both 8α-histidylation and 6-S-cysteinylation to form a bicovalently attached cofactor. Representative members of all these groups have been studied in detail, and several explanations for the role of the covalent flavinylation have been put forward. Some of the suggestions tend to be rather specific for the system being studied, e.g. prevention of cofactor inactivation at the C-6 position for trimethylamine dehydrogenase (5) or facilitation of electron transfer from the flavin to the cytochrome subunit for p-cresol methylhydroxylase (6). Other explanations including the increase of the flavin redox potential due to the covalent linkage (7–9) and the prevention of cofactor dissociation (10, 11) were found for several enzymes also harboring different types of cofactor attachments. Taking into account that protein stability (12) and optimal binding of substrate molecules (11, 13) are also positively influenced by covalent tethering of the flavin, one might speculate that no generally applicable explanation for the covalent attachment of flavins to proteins exists. Therefore, it seems likely that the large variety of systems operating with one of the above mentioned modes of cofactor tethering might have evolved to also adapt to a diversity of enzymatic challenges.Berberine bridge enzyme (BBE) from Eschscholzia californica is a plant enzyme involved in alkaloid biosynthesis, catalyzing the challenging oxidative cyclization of (S)-reticuline to (S)-scoulerine (Scheme 1). This enzyme was recently shown to belong to the group of flavoenzymes with a bicovalently attached FAD (14). After the discovery of this unusual mode of linkage in the crystal structure of GOOX (4), several members of this group, all belonging to the vanillyl-alcohol oxidase family (15), were identified by biochemical methods (16–18) and also structural studies (19). Because some of the suggested benefits of a covalent cofactor attachment can easily be brought about by a single linkage, e.g. prevention of cofactor dissociation or stabilization of the tertiary structure, the two amino acids attached to FAD might have different and individual functions as well as an additive effect on physicochemical properties such as redox potentials or substrate binding and oxidation. To elucidate the relative importance for the overall enzymatic functioning of members of this group, more detailed studies have been performed on GOOX (11), chito-oligosaccharide oxidase (ChitO) from Fusarium graminearum (17), and BBE (20). Common results of these analyses show that the bicovalent FAD has a redox potential of about +130 mV, which is among the highest potentials reported for flavoenzymes. Replacement of one of the amino acids involved in anchoring of the cofactor generally reduces the rate of cofactor reduction and the steady-state turnover rate, but whether this can be directly linked to reduced redox potentials of these mutant proteins has been under debate (11).Open in a separate windowSCHEME 1.Overall reaction catalyzed by BBE.To address these issues further, we report the expression of the H104A mutant protein of BBE. A biochemical characterization of this protein variant with respect to the redox potential, transient kinetics, and steady-state analysis is combined with the structural analysis of both the H104A and the C166A mutant proteins. In addition, a structure of wild type (WT) BBE in complex with the product of the enzyme-catalyzed reaction is presented, which provides further insights toward the involvement of active site amino acids during the course of the reaction. Together with the recently reported x-ray crystal structure of WT BBE with and without substrate bound (21) and the biochemical characterization of the C166A mutant protein (20), these results provide interesting insights into the role of bicovalent FAD attachment in enzymes.     

Synthesis,binding and bioactivity of γ-methylene γ-lactam ecdysone receptor ligands: Advantages of QSAR models for flexible receptors

Nuclear hormone receptors, such as the ecdysone receptor, often display a large amount of induced fit to ligands. The size and shape of the binding pocket in the EcR subunit changes markedly on ligand binding, making modelling methods such as docking extremely challenging. It is, however, possible to generate excellent 3D QSAR models for a given type of ligand, suggesting that the receptor adopts a relatively restricted number of binding site configurations or ‘attractors’. We describe the synthesis, in vitro binding and selected in vivo toxicity data for γ-methylene γ-lactams, a new class of high-affinity ligands for ecdysone receptors from Bovicola ovis (Phthiraptera) and Lucilia cuprina (Diptera). The results of a 3D QSAR study of the binding of methylene lactams to recombinant ecdysone receptor protein suggest that this class of ligands is indeed recognised by a single conformation of the EcR binding pocket.     

Promoter- and attenuator-related metabolic regulation of the Salmonella typhimurium histidine operon.

Expression of the histidine (his) operon in Salmonella typhimurium was found to be positively correlated with the intracellular level of guanosine tetraphosphate (ppGpp). Limitation for amino acids other than histidine elicited a histidine-independent metabolic regulation of the operon. In bacteria grown at decreased growth rates, his operon expression was metabolically regulated up to a point, after which further decreases in growth rate no longer resulted in further enhancement of operon expression. Studies using strains carrying various regulatory and deletion mutations indicated that metabolic regulation is achieved predominantly by increased RNA chain initiations at the primary (P1) and internal (P2) promoters. Metabolic regulation ordinarly did not involve changes in RNA chain terminations at the attenuator site of the his operon. A model is proposed that involves ppGpp-induced changes in RNA polymerase initiation specificity at particular promoters. A second, special form of metabolic regulation may operate which also is histidine independent, but does involve relief of attenuation.     

Tufted ducks Aythya fuligula do not control buoyancy during diving

Work against buoyancy during submergence is a large component of the energy costs for shallow diving ducks. For penguins, buoyancy is less of a problem, however they still seem to trade‐off levels of oxygen stores against the costs and benefits of buoyant force during descent and ascent. This trade‐off is presumably achieved by increasing air sac volume and hence pre‐dive buoyancy (B_pre) when diving deeper. Tufted ducks, Aythya fuligula, almost always dive with nearly full oxygen stores so these cannot be increased. However, the high natural buoyancy of tufted ducks guarantees a passive ascent, so they might be expected to decrease B_pre before particularly deep, long dives to reduce the energy costs of diving. Body heat lost to the water can also be a cause of substantial energy expenditure during a dive, both through dissipation to the ambient environment and through the heating of ingested food and water. Thus dive depth (d_d), duration and food type can influence how much heat energy is lost during a dive. The present study investigated the relationship between certain physiological and behavioural adjustments by tufted ducks to d_d and food type. Changes in B_pre, deep body temperature (T_b) and dive time budgeting of four ducks were measured when diving to two different depths (1.5 and 5.7 m), and for two types of food (mussels and mealworms). The hypothesis was that in tufted ducks, B_pre decreases as d_d increases. The ducks did not change B_pre in response to different diving depths, and thus the hypothesis was rejected. T_b was largely unaffected by dives to either depth. However, diving behaviour changed at the greater d_d, including an increase in dive duration and vertical descent speed. Behaviour also changed depending on the food type, including an increase in foraging duration and vertical descent speed when mussels were present. Behavioural changes seem to represent the major adjustment made by tufted ducks in response to changes in their diving environment.     

Visualization of the Two-Step Fusion Process of the Retrovirus Avian Sarcoma/Leukosis Virus by Cryo-Electron Tomography

Retrovirus infection starts with the binding of envelope glycoproteins to host cell receptors. Subsequently, conformational changes in the glycoproteins trigger fusion of the viral and cellular membranes. Some retroviruses, such as avian sarcoma/leukosis virus (ASLV), employ a two-step mechanism in which receptor binding precedes low-pH activation and fusion. We used cryo-electron tomography to study virion/receptor/liposome complexes that simulate the interactions of ASLV virions with cells. Binding the soluble receptor at neutral pH resulted in virions capable of binding liposomes tightly enough to alter their curvature. At virion-liposome interfaces, the glycoproteins are ∼3-fold more concentrated than elsewhere in the viral envelope, indicating specific recruitment to these sites. Subtomogram averaging showed that the oblate globular domain in the prehairpin intermediate (presumably the receptor-binding domain) is connected to both the target and the viral membrane by 2.5-nm-long stalks and is partially disordered, compared with its native conformation. Upon lowering the pH, fusion took place. Fusion is a stochastic process that, once initiated, must be rapid, as only final (postfusion) products were observed. These fusion products showed glycoprotein spikes on their surface, with their interiors occupied by patches of dense material but without capsids, implying their disassembly. In addition, some of the products presented a density layer underlying and resolved from the viral membrane, which may represent detachment of the matrix protein to facilitate the fusion process.     

Computational lipidology: predicting lipoprotein density profiles in human blood plasma

Monitoring cholesterol levels is strongly recommended to identify patients at risk for myocardial infarction. However, clinical markers beyond "bad" and "good" cholesterol are needed to precisely predict individual lipid disorders. Our work contributes to this aim by bringing together experiment and theory. We developed a novel computer-based model of the human plasma lipoprotein metabolism in order to simulate the blood lipid levels in high resolution. Instead of focusing on a few conventionally used predefined lipoprotein density classes (LDL, HDL), we consider the entire protein and lipid composition spectrum of individual lipoprotein complexes. Subsequently, their distribution over density (which equals the lipoprotein profile) is calculated. As our main results, we (i) successfully reproduced clinically measured lipoprotein profiles of healthy subjects; (ii) assigned lipoproteins to narrow density classes, named high-resolution density sub-fractions (hrDS), revealing heterogeneous lipoprotein distributions within the major lipoprotein classes; and (iii) present model-based predictions of changes in the lipoprotein distribution elicited by disorders in underlying molecular processes. In its present state, the model offers a platform for many future applications aimed at understanding the reasons for inter-individual variability, identifying new sub-fractions of potential clinical relevance and a patient-oriented diagnosis of the potential molecular causes for individual dyslipidemia.