Deriving how far structural information is transmitted through parallel homodimeric coiled‐coils: A correlation analysis of helical staggers

How local conformation is affected by local sequence is fairly well understood for alpha‐helical coiled‐coils, but less is known about how local conformation is influenced by distant features. Here, I describe an approach to detect such an effect, based on computing correlation coefficients of local out‐of‐register alignments, or so‐called “staggers” between the helices, as a function of the axial distance between the staggers. This approach requires parallel homodimers, in which each stagger can occur with two “signs,” where either one helix or the other is shifted towards the N terminus. The signs of such staggers separated by up to 12 residues are strongly correlated, indicating that the conformations of the ends of coiled‐coils are commonly influenced by attached structures. Thus, the structures of coiled‐coil residues aberrantly attached to alternative proteins, such as those resulting from leukemogenic chromosomal rearrangements, may be distinguishable from those in normal tissues, and in turn serve as targets of selective drug design. The signs of helical staggers separated by between 13 and 30 residues are moderately yet significantly correlated, indicating that some of the coiled‐coils transmit this conformational feature axially for at least 45 Å. A positive, albeit noisy, correlation also exists among tropomyosin coiled‐coils for signed staggers separated by the 40‐residue actin repeat distance, consistent with the semi‐flexible tropomyosin filament binding F‐actin and regulating skeletal muscle contraction in a partially cooperative manner. Communication of the signs of axial staggers is explained in part by minimization of main‐chain hydrogen bond deformations. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Theoretical Analysis of the Footprinting Experiment

Abstract

In the footprinting experiment, an end-radiolabeled DNA restriction fragment is subjected to digest by an endonuclease in the presence and absence of a ligand which alters the endonuclease cleavage rate at sites of ligand-DNA contact. The location of these sites, and the strength of the ligand binding, are then deduced from the measured concentrations of the different oligonucleotides produced by the digest. We analyze the experiment in terms of coupled kinetic equations which take into account the cutting rates of endonuclease for sites with ligand present and absent, and the rates of binding and dissociation of the ligand to a site. As long as the ligand concentration remains essentially constant (which occurs, for example, if digest is terminated early enough to assure that all fragments result from single cuts by the endonuclease), the oligonucleotide concentrations reflect only the ligand binding equilibrium constant (ratio of rate constants) and the cutting rates in the presence and absence of ligand. We also show how the measured oligonucleotide concentrations (from, e.g. an autoradiogram) can be used to deduce the ligand equilibrium binding constants for the various sites on the polymer.     

Studies on Bleomycin-DNA and Bleomycin-Iron Interactions

Abstract

The bleomycins, a group of antitumor antibiotics (Figure 1), cause the degradation of DNA by a process requiring iron(II) and dioxygen (1,2). DNA degradation appears to involve two steps: association of the drug with the nucleic acid and degradation of the DNA. As part of studies directed toward achieving an understanding of how the bleomycins degrade DNA, we have examined various properties of the drug using a variety of chemical and physico- chemical techniques, including NMR and Mössbauer spectroscopy. We have studied both the interaction of the antibiotic with its target (DNA) as well as its association with its metal ion cofactor. This work has been performed on the intact drug and its derivatives as well as on synthetic models of the parent drug. This paper reviews and updates the recent work from this laboratory on the bleomycins.     

Hydrated mucilage reduces post-dispersal seed removal of a sand desert shrub by ants in a semiarid ecosystem

Post-dispersal seed removal by animals can lead to extensive seed loss and thus is an important factor in structuring plant communities. However, we know much less about post-dispersal seed predation than about other forms of herbivory. Mucilage plays many ecological roles in adaptation of plants to diverse environments; nevertheless, until now the role of mucilage in ant-mediated seed movement remains largely hypothetical. We studied the role of mucilage in seed removal of Artemisia sphaerocephala by ants in Mu Us Sandland in Inner Mongolia, China. Messor aciculatus was the most active seed predator of Artemisia sphaerocephala. Time to first ant collecting (T _1st) of wet intact seeds was longest and significantly different from that for dry intact seeds, wet demucilaged seeds, and dry demucilaged seeds; number of seeds removed to ant nests was lowest for wet intact seeds. After they were collected by ants, 5 % of wet intact seeds were dropped during transport. Our results indicate that seed mucilage of Artemisia sphaerocephala may play a significant role in post-dispersal seed removal by (1) making seeds less attractive to ants, thus resulting in a delay of collection time; (2) forming a strong bond to soil particles, making it difficult for ants to remove seeds; and (3) making seeds more likely to be dropped during transport, thereby allowing them to escape from predation even after collection by ants. This study demonstrates the importance of mucilage in reducing seed removal by ants and thus in anchoring seeds of desert plants in the vicinity of mother plants.     

Origin and evolution of the Pseudorhyncocyonidae,a European Paleogene family of insectivorous placental mammals

Two new species of pseudorhyncocyonid, Fordonia lawsoni sp. nov. and Leptictidium prouti sp. nov. from the UK earliest Eocene, described here, are older than any previously recorded member of the family. They are represented by teeth from numerous loci, which allow a better understanding of the sparsely known dentitions of currently known pseudorhyncocyonids. This facilitates the recognition of two further species of Leptictidium, L. listeri sp. nov. from the Middle Eocene of Germany and L. storchi sp. nov. from the Late Eocene of France. Study of occlusal relationships also helps to fill gaps in our knowledge of missing tooth loci. Cladistic analysis of pseudorhyncocyonids with their previously judged closest relatives, the Leptictidae, Pantolesta and Palaeanodonta, shows that two European species, Diaphyodectes prolatus and Palaeictops? levei, formerly thought to be leptictids, are instead primitive pseudorhyncocyonids, extending the range of the family further back in time to the Middle Paleocene. P? levei is placed in the new genus Phakodon gen. nov. The analysis also shows that the Pseudorhyncocyonidae are sister group to the other three groups combined and that family‐level differentiation in this probable clade took place as early as the earliest Paleocene.     

Characterization of the complex formed between a potent neutralizing ovine-derived polyclonal anti-TNFα Fab fragment and human TNFα

TNFα (tumour necrosis factor α) is an early mediator in the systemic inflammatory response to infection and is therefore a therapeutic target in sepsis. AZD9773 is an ovine-derived, polyclonal anti-TNFα Fab fragment derived from a pool of serum and currently being developed as a treatment for severe sepsis and septic shock. In the present study, we show that although AZD9773 has a modest affinity for TNFα in a binding assay, the K_i in a cell-based assay is approximately four orders of magnitude lower. We show using SEC (size exclusion chromatography) that the maximum size of the complex between AZD9773 and TNFα is consistent with approximately 12 Fabs binding to one TNFα trimer. A number of approaches were taken to map the epitopes recognized by AZD9773. These revealed that a number of different regions on TNFα are involved in binding to the polyclonal Fab. The data suggest that there are probably three epitopes per monomer that are responsible for most of the inhibition by AZD9773 and that all three can be occupied at the same time in the complex. We conclude that AZD9773 is clearly demonstrated to bind to multiple epitopes on TNFα and suggest that the polyclonal nature may account, at least in part, for the very high potency observed in cell-based assays.     

Formation of Covellite (CuS) Under Biological Sulfate-Reducing Conditions

Sulfate-reducing bacteria (SRB) play a major role in the precipitation of metal sulfides in the environment. In this work, biogenic copper sulfide formation was examined in cultures of SRB and compared to chemically initiated Cu sulfide precipitation as a reference system. Mixed cultures of SRB were incubated at 22, 45, and 60°C in nutrient solutions that contained copper sulfate. Abiotic reference samples were produced by reacting uninoculated liquid media with Na₂S solutions under otherwise identical conditions. Precipitates were collected anaerobically by centrifugation, frozen in liquid N₂, and freeze-dried, followed by analysis using X-ray diffraction (XRD), X-ray fluorescence, and scanning electron microscopy. Covellite (CuS) was the only mineral found in the precipitates. Covellite was less crystalline in the biogenic precipitates than in the abiotic samples based on XRD peak widths and peak to background ratios. Poor crystallinity may be the result of slower precipitation rates in bacterial cultures as compared to the abiotic reference systems. Furthermore, bacterial cells may inhibit the nucleation steps that lead to crystal formation. Incubation at elevated temperatures improved the crystallinity of the biotic specimens.     

Effect of increasing GnRH and PGF2α dose during Double-Ovsynch on ovulatory response,luteal regression,and fertility of lactating dairy cows

Ovsynch-type synchronization of ovulation protocols have suboptimal synchronization rates due to reduced ovulation to the first GnRH treatment and inadequate luteolysis to the prostaglandin F_2α (PGF_2α) treatment before timed artificial insemination (TAI). Our objective was to determine whether increasing the dose of the first GnRH or the PGF_2α treatment during the Breeding-Ovsynch portion of Double-Ovsynch could improve the rates of ovulation and luteolysis and therefore increase pregnancies per artificial insemination (P/AI). In experiment 1, cows were randomly assigned to a two-by-two factorial design to receive either a low (L) or high (H) doses of GnRH (Gonadorelin; 100 vs. 200 μg) and a PGF_2α analogue (cloprostenol; 500 vs. 750 μg) resulting in the following treatments: LL (n = 263), HL (n = 277), LH (n = 270), and HH (n = 274). Transrectal ultrasonography and serum progesterone (P4) were used to assess ovulation to GnRH1, GnRH2, and luteal regression after PGF_2α during Breeding-Ovsynch in a subgroup of cows (n = 651 at each evaluation). Pregnancy status was assessed 29, 39, and 74 days after TAI. In experiment 2, cows were randomly assigned to LL (n = 220) or HH (n = 226) treatment as described for experiment 1. For experiment 1, ovulation to GnRH1 was greater (P = 0.01) for cows receiving H versus L GnRH (66.6% [217/326] vs. 57.5% [187/325]) treatment, but only for cows with elevated P4 at GnRH1. Cows that ovulated to GnRH1 had increased (P < 0.001) fertility compared with cows that did not ovulate (52.2% vs. 38.5%); however, no effect of higher dose of GnRH on fertility was detected. The greater PGF_2α dose increased luteal regression primarily in multiparous cows (P = 0.03) and tended to increase fertility (P = 0.05) only at the pregnancy diagnosis 39 days after TAI. Overall, P/AI was 47.0% at 29 days and 39.7% at 74 days after TAI; P/AI did not differ (P = 0.10) among treatments at 74 days (LL, 34.6%; HL, 40.8%; LH, 42.2%; HH, 40.9%) and was greater (P < 0.001) for primiparous cows than for multiparous cows (46.1% vs. 33.8%). For experiment 2, P/AI did not differ (P = 0.21) between H versus L treatments (44.2% [100/226] vs. 40.5% [89/220]). Thus, despite an increase in ovulatory response to GnRH1 and luteal regression to PGF_2α, there were only marginal effects of increasing dose of GnRH or PGF_2α on fertility to TAI after Double-Ovsynch.     

A novel combinatorial biocatalytic approach for producing antibacterial compounds effective against Mycobacterium tuberculosis (TB)

Two bacterial hosts expressing cloned aromatic oxygenases were used to catalyze the oxidation and polymerization of indole and related substrates, creating mixtures of indigoid compounds comprised of novel dimers and trimers. Crude extracts and purified compounds were tested for their ability to inhibit the growth of Gram-positive organisms, in general, and Mycobacterium tuberculosis (TB), in particular. Of the 74 compounds tested against M. tuberculosis, ~66 % had minimum inhibitory concentrations (MIC) of 5 μg/ml or less. The most effective antibiotic found was designated SAB-P1, a heterodimer of indole and anthranil, which had a MIC of 0.16 μg/ml, and did not inhibit kidney cells (IC⁵⁰) at concentrations of >8 μg/ml. Combinatorial biocatalysis was used to create a series of halogenated derivatives of SAB-P1 with a wider therapeutic window. None of the derivatives had MIC values that were superior to SAB-P1, but some had a wider therapeutic window because of decreased kidney cell toxicity. Generally, the indigoid dimers that were effective against TB appeared to be specific for TB. Some of the trimers generated, however, had a broader spectrum of activity inhibiting not only TB (MIC?=?1.1 μg/ml) but also the growth of Mycobacterium smegmatis MC2 155, Bacillus cereus, Enterococcus faecalis, Staphylococcus epidermidis, Bacillus subtilis 168, and Clostridium acetobutylicum. The structure of two of the novel dimers (SAB-C4 and SAB-P1) and a trimer (SAB-R1) were solved using X-ray crystallography.