Stable isotope and pen feeding trial studies confirm the value of horseshoe crab Limulus polyphemus eggs to spring migrant shorebirds in Delaware Bay

We used stable isotope (SI) methods in combination with pen feeding trials to determine the importance of eggs of the Atlantic horseshoe crab Limulus polyphemus to migratory fattening of red knots Calidris canutus rufa and ruddy turnstones Arenaria interpres morinella during spring stopover in Delaware Bay. By manifesting measurable fractionation (ca +3‰) and rapid turnover, blood plasma δ¹⁵ nitrogen proved a functional marker for SI diet tracking during the short 3-week stopover. Blood samples from free-ranging knots (3 data sets) and turnstones (1 data set) produced similar convergence of plasma δ¹⁵N signatures with increasing body mass that indicated highly similar diets. Asymptotes deviated slightly (0.3‰ to 0.7‰) from that of captive shorebirds fed a diet of only crab eggs during stopover, thus confirming a strong crab egg-shorebird linkage. The plasma δ¹⁵N crab-egg diet asymptote was enriched ca +4.5‰ and therefore readily discriminated from that of either blue mussels Mytilus edulis or coquina clams Donax variabilis , the most likely alternative prey of knots in Delaware Bay. Crab eggs were highly palatable to captive knots and turnstones which achieved rates of mass gain (3–11 g/d) comparable to that of free-ranging birds. Peak consumption rates during hyperphagic events were 23,940 and 19,360 eggs/bird/d, respectively. The empirical conversions of eggs consumed to body mass gained (5,017 eggs/g for knots and 4,320 eggs/g for turnstones) indicate the large quantities of crab eggs required for the maintenance of these shorebird populations during stopover.     

Studying polyglutamine aggregation in Caenorhabditis elegans using an analytical ultracentrifuge equipped with fluorescence detection

This work explores the heterogeneity of aggregation of polyglutamine fusion constructs in crude extracts of transgenic Caenorhabditis elegans animals. The work takes advantage of the recent technical advances in fluorescence detection for the analytical ultracentrifuge. Further, new sedimentation velocity methods, such as the multi‐speed method for data capture and wide distribution analysis for data analysis, are applied to improve the resolution of the measures of heterogeneity over a wide range of sizes. The focus here is to test the ability to measure sedimentation of polyglutamine aggregates in complex mixtures as a prelude to future studies that will explore the effects of genetic manipulation and environment on aggregation and toxicity. Using sedimentation velocity methods, we can detect a wide range of aggregates, ranging from robust analysis of the monomer species through an intermediate and quite heterogeneous population of oligomeric species, and all the way up to detecting species that likely represent intact inclusion bodies based on comparison to an analysis of fluorescent puncta in living worms by confocal microscopy. Our results support the hypothesis that misfolding of expanded polyglutamine tracts into insoluble aggregates involves transitions through a number of stable intermediate structures, a model that accounts for how an aggregation pathway can lead to intermediates that can have varying toxic or protective attributes. An understanding of the details of intermediate and large‐scale aggregation for polyglutamine sequences, as found in neurodegenerative diseases such as Huntington's Disease, will help to more precisely identify which aggregated species may be involved in toxicity and disease.     

A trans‐Atlantic examination of haddock Melanogrammus aeglefinus food habits

The food habits of Melanogrammus aeglefinus were explored and contrasted across multiple north‐eastern and north‐western Atlantic Ocean ecosystems, using databases that span multiple decades. The results show that among all ecosystems, echinoderms are a consistent part of M. aeglefinus diet, but patterns emerge regarding where and when M. aeglefinus primarily eat fishes v. echinoderms. Melanogrammus aeglefinus does not regularly exhibit the increase in piscivory with ontogeny that other gadoids often show, and in several ecosystems there is a lower occurrence of piscivory. There is an apparent inverse relationship between the consumption of fishes and echinoderms in M. aeglefinus over time, where certain years show high levels of one prey item and low levels of the other. This apparent binary choice can be viewed as part of a gradient of prey options, contingent upon a suite of factors external to M. aeglefinus dynamics. The energetic consequences of this prey choice are discussed, noting that in some instances it may not be a choice at all.     

Structural Basis of Cyclic Nucleotide Selectivity in cGMP-dependent Protein Kinase II

Membrane-bound cGMP-dependent protein kinase (PKG) II is a key regulator of bone growth, renin secretion, and memory formation. Despite its crucial physiological roles, little is known about its cyclic nucleotide selectivity mechanism due to a lack of structural information. Here, we find that the C-terminal cyclic nucleotide binding (CNB-B) domain of PKG II binds cGMP with higher affinity and selectivity when compared with its N-terminal CNB (CNB-A) domain. To understand the structural basis of cGMP selectivity, we solved co-crystal structures of the CNB domains with cyclic nucleotides. Our structures combined with mutagenesis demonstrate that the guanine-specific contacts at Asp-412 and Arg-415 of the αC-helix of CNB-B are crucial for cGMP selectivity and activation of PKG II. Structural comparison with the cGMP selective CNB domains of human PKG I and Plasmodium falciparum PKG (PfPKG) shows different contacts with the guanine moiety, revealing a unique cGMP selectivity mechanism for PKG II.     

Analysis of Peg Formation in Cucumber Seedlings Grown on Clinostats and in a Microgravity (Space) Environment

Cucumis sativus L. cv Burpee Hybrid II) grown under conditions of normal gravity, microgravity, and simulated microgravity (clinostat rotation). Seeds were germinated on the ground, in clinostats and on board the space shuttle (STS-95) for 1–2 days, frozen and subsequently examined for their stage of development, degree of hook formation, number of pegs formed, and peg morphology. The frequency of peg formation in space-grown seedlings was found to be nearly identical to that of clinostat-grown seedlings and to differ from that of seedlings germinated under normal gravity only in a minority of cases; ˜6% of the seedlings formed two pegs and nearly 2% of the seedlings lacked pegs, whereas such abnormalities did not occur in ground controls. The degree of hook formation was found to be less pronounced for space-grown seedlings, compared to clinostat-grown seedlings, indicating a greater degree of decoupling between peg formation and hook formation in space. Nonetheless, in all seedlings having single pegs and a hook, the peg was found to be positioned correctly on the inside of the hook, showing that there is coordinate development even in microgravity environments. Peg morphologies were altered in space-grown samples, with the pegs having a blunt appearance and many pegs showing alterations in expansion, with the peg extending out over the edges of the seed coat and downwards. These phenotypes were not observed in clinostat or ground-grown seedlings. Received 12 October 1999/ Accepted in revised form 18 October 1999     

Deciphering the physiological response of Escherichia coli under high ATP demand

One long‐standing question in microbiology is how microbes buffer perturbations in energy metabolism. In this study, we systematically analyzed the impact of different levels of ATP demand in Escherichia coli under various conditions (aerobic and anaerobic, with and without cell growth). One key finding is that, under all conditions tested, the glucose uptake increases with rising ATP demand, but only to a critical level beyond which it drops markedly, even below wild‐type levels. Focusing on anaerobic growth and using metabolomics and proteomics data in combination with a kinetic model, we show that this biphasic behavior is induced by the dual dependency of the phosphofructokinase on ATP (substrate) and ADP (allosteric activator). This mechanism buffers increased ATP demands by a higher glycolytic flux but, as shown herein, it collapses under very low ATP concentrations. Model analysis also revealed two major rate‐controlling steps in the glycolysis under high ATP demand, which could be confirmed experimentally. Our results provide new insights on fundamental mechanisms of bacterial energy metabolism and guide the rational engineering of highly productive cell factories.     

Cationic (tripyrrinato)palladium(II) complexes

The formation of cationic palladium(II)complexes [TrpyPd^II]⁺X⁻ by salt metathesis of the respective trifluoroacetates with different salts of weakly coordinating anions X⁻ was investigated. With non-hydrolizable counterions, cationic mono- and dinuclear complexes are observed depending on the nature of the anion X⁻ and the solvent. The mononuclear cations, which are only formed with X = BAr^F, most probably carry a weakly bound molecule of dichloromethane at the fourth coordination site of Pd^II. When treated with diazoalkanes, only these are sufficiently reactive to form carbene complexes. Four- and five coordinate Lewis base adducts [TrpyPd^IIL]⁺ with L = CH₃NC, tBuNH₂, PMe₃, PEt₃ and PiPr₃ and [TrpyPd^IIL₂]⁺ with L = PMe₃ were prepared from the mononuclear cations [TrpyPd^II]⁺BAr^F−. From structural studies it becomes apparent, that the formation of stable five coordinate Pd^II species is restricted to medium size ligands and depends on the delicate balance between the steric influence of L and the strain, which is induced on the TrpyPd^II unit.     

Identification of host plants of the avocado borer, Stenoma catenifer Walsingham (Lepidoptera: Elachistidae) in Rio Grande do Sul

Fruits of Nectandra megapotamica Mez and Cinnamomum camphora (L.) (Lauraceae) were collected in Rio Grande do Sul, Brazil, in 2004. Species of five families of insects were found inside the fruits: two fly species (Diptera: Drosophilidae: Drosophila spp.), six beetle species, Heilipus sp., Conotrachelus sp. (Curculionidae), Hypothenemus sp. (Scolytidae) and three species of Carpophilus (Nitidulidae), and moths (Lepidoptera: Elachistidae). The moth especimes were identified as Stenoma catenifer Walsingham, the avocado borer. The occurrence of the moth was predominant from early April until middle May. The natural larval infestation level was low. Two new host plant of the pest were identified.     

Efficient production of membrane-integrated and detergent-soluble G protein-coupled receptors in Escherichia coli

G protein-coupled receptors (GPCRs) are notoriously difficult to express, particularly in microbial systems. Using GPCR fusions with the green fluorescent protein (GFP), we conducted studies to identify bacterial host effector genes that result in a general and significant enhancement in the amount of membrane-integrated human GPCRs that can be produced in Escherichia coli. We show that coexpression of the membrane-bound AAA+ protease FtsH greatly enhances the expression yield of four different class I GPCRs, irrespective of the presence of GFP. Using this new expression system, we produced 0.5 and 2 mg/L of detergent-solubilized and purified full-length central cannabinoid receptor (CB1) and bradykinin receptor 2 (BR2) in shake flask cultures, respectively, two proteins that had previously eluded expression in microbial systems.     

Synthesis of beta- and gamma-oxa isosteres of fosmidomycin and FR900098 as antimalarial candidates

To expand the structure-activity relationships of fosmidomycin and FR900098, two potent antimalarials interfering with the MEP-pathway, we decided to replace a methylene group in beta-position of the phosphonate moiety of these leads by an oxygen atom. beta-oxa-FR900098 (11) proved equally active as the parent compound. When applied to 4-[hydroxyl(methyl)amino]-4-oxobutyl phosphonic acid, featuring a hydroxamate instead of the retrohydroxamate moiety, a beta-oxa modification yielded a derivative (13) with superior activity against the Plasmodium falciparum 3D7 strain than fosmidomycin, while a gamma-oxa modification resulted in less active derivatives. A bis(pivaloyloxymethyl)ester of phosphonate 13 proved twice as active in inhibiting cultured parasites as a similar prodrug of FR900098.