Isolation and characterization of Campylobacter bacteriophages from retail poultry

The ability of phages to survive processing is an important aspect of their potential use in the biocontrol of Campylobacter in poultry production. To this end, we have developed a procedure to recover Campylobacter bacteriophages from chilled and frozen retail poultry and have validated the sensitivity of the method by using a characterized Campylobacter phage (i.e., NCTC 12674). By using this method, we have shown that Campylobacter phages can survive on retail chicken under commercial storage conditions. Retail chicken portions purchased in the United Kingdom were screened for the presence of endogenous Campylobacter phages. Thirty-four Campylobacter bacteriophages were isolated from 300 chilled retail chicken portions, but none could be recovered from 150 frozen chicken portions. The phage isolates were characterized according to their lytic profiles, morphology, and genome size. The free-range products were significantly more likely to harbor phages (P < 0.001 by single-factor analysis of variance) than were standard or economy products. This study demonstrates that Campylobacter bacteriophages, along with their hosts, can survive commercial poultry processing procedures and that the phages exhibited a wide range of recovery rates from chicken skin stored at 4 degrees C.     

Molecules and morphology: evidence for cryptic hybridization in African Hyalomma (Acari: Ixodidae)

The role of natural hybridization and introgression as part of the evolutionary process is of increasing interest to zoologists, particularly as more examples of gene exchange among species are identified. We present mitochondrial and nuclear sequence data for Hyalomma dromedarii, Hyalomma truncatum, and Hyalomma marginatum rufipes (Acari: Ixodidae) collected from one-humped camels in Ethiopia. These species are well differentiated morphologically and genetically; sequence data from the mitochondrial DNA (mtDNA) cytochrome oxidase I gene indicates 10-14% divergence between the species. However, incongruence between morphology and the mtDNA phylogeny was observed, with multiple individuals of H. dromedarii and H. truncatum present on the same mtDNA lineage as H. marginatum rufipes. Thus, individuals with morphology of H. dromedarii and H. truncatum are indistinguishable from H. marginatum rufipes on the basis of mtDNA. Multiple copies of ITS-2 were subsequently cloned and sequenced for a subset of individuals from the mtDNA phylogeny, representing both 'normal' and 'putative hybrid' individuals. Very low sequence divergence (0.3%) was observed within 'normal' individuals of both H. dromedarii and H. truncatum relative to the 'putative hybrid' individuals (6 and 2.7%, respectively). The pattern of intra-individual variation in ITS-2 within 'putative hybrid' individuals, particularly in H. dromedarii, strongly suggests that gene flow has occurred among these Hyalomma species, but no indication of this is given by the morphology of the individuals.     

Synthesis,structure-activity relationship and in vitro evaluation of coelenterazine and coelenteramine derivatives as inhibitors of lipid peroxidation

Coelenterazine (2-p -hydroxybenzyl-6-(3'-hydroxyphenyl)-8-benzyl-3,7-dihydroimidazolo[1,2-a]pyrazin-3-one, CLZn) and coelenteramine (2-amino-3-benzyl-5-(4'-hydroxyphenyl)-1,4-pyrazine CLM), first described as luciferin and etioluciferin, respectively, of bioluminescent systems in marine organisms are endowed with antioxidant properties. This study was aimed at understanding the structural basis of their chain-breaking properties and at designing new compounds with improved antioxidative properties. For this, a series of 2-amino-1,4-pyrazine derivatives and their related imidazolopyrazinones were synthesised and examined for their capacity to inhibit lipid peroxidation in linoleate micelles subjected to the peroxidizing action of AAPH. Structure-activity relationship studies indicated that the reduction of the peroxidation rate by CLM is mainly determined by the concomitant presence of 5-p-hydroxyphenyl and 2-amino groups in para position. The lipophilic character of substituents also affected this effect. All imidazolopyrazinones induced a lag-time before the onset of the peroxidation process. The hetero-bicyclic imidazolopyrazinone moiety appears as the main contributor to this activity while phenol groups play little role in it. On the other hand, phenol groups were required for the reduction of the peroxidation rate after the lag-phase. The introduction of a supplementary p-hydroxyphenyl substituent at C₈ position did not increase chain-breaking properties. The substitution of the C₅-p-hydroxyphenyl with a catechol moiety or the introduction of a second amino group on the pyrazine ring yielded the most active compounds, superior to imidazolopyrazinones and reference antioxidants like epigallocatechin gallate, vitamin E and trolox. The strong antioxidant properties of 2,6-diaminopyrazines are not dependent on the presence of hydroxyl groups indicating that their reaction mechanism differs from that of 2-amino-1,4-pyrazine derivatives.     

The generation and characterization of antagonist RNA aptamers to human oncostatin M

Oncostatin M (OSM) is a multifunctional member of the interleukin-6 cytokine family. OSM has been implicated as a powerful proinflammatory mediator and may represent a potentially important, novel therapeutic opportunity for treatment of established rheumatoid arthritis. To further investigate the role of OSM in inflammatory disorders, we have isolated a series of RNA aptamers that bind specifically to human OSM. The highest affinity aptamer, designated ADR58, has been characterized in a series of in vitro and cell based assays. ADR58 has an affinity of 7 nm for human OSM, and it can antagonize OSM binding to the gp130 receptor and specifically antagonize OSM mediated signaling. The aptamer has been truncated in length to 33 bases, all pyrimidine positions are substituted with 2' fluorine, and 14 of 18 purine positions have been substituted with 2' O-methyl to increase stability toward nucleases. This truncated, modified form of ADR58 retains complete affinity and functional activity for OSM. This aptamer may be used as a tool to further investigate the role of OSM in inflammatory disorders and may also have role as a therapeutic agent.     

Aspartyl tRNA-synthetase from Escherichia coli: flexibility and adaptability to the substrates

The crystal structure of aspartyl-tRNA synthetase from Escherichia coli has been determined to a resolution of 2.7 A. The structure is compared to the same enzyme co-crystallized with tRNA(Asp) and containing aspartyl adenylate or ATP. The asymmetric unit contains three monomers of the enzyme. While most parts of the protein show no significant differences in the three monomers, a few regions cannot be superimposed. Those regions are characterized by a high B-factor, and consist mostly of loops that make contacts with the tRNA in the complexes. The flexibility of the protein is seen at a global level, by the observation of a 10 to 15 degrees rotation of the N-terminal and insertion domains upon tRNA binding, and at the level of the individual amino acid residues, by main-chain and side-chain rearrangements. In contrast to these induced-fit conformational changes, a few residues essential for the tRNA anticodon or aspartyl-adenylate recognition exist in a predefined conformation, ensured by specific interactions within the protein.     

Identification of methyl farnesoate in the cypris larva of the barnacle, Balanus amphitrite, and its role as a juvenile hormone

Previous investigations have shown that insect juvenile hormone (JH) and its analogues induce precocious metamorphosis of barnacle cypris larvae. In the present study, methyl farnesoate (MF; structurally identical to JH III, except for the absence of an epoxide group) has been shown to have a concentration-dependent effect on the development of cyprids of the barnacle Balanus amphitrite. Analysis of cypris extracts by gas chromatography-mass spectrometry with selected ion monitoring (GC-MS-SIM) confirmed the presence of endogenous MF. These data provide evidence that MF functions as a juvenilizing hormone in barnacle cyprids, an effect that hitherto has not been noted.     

Use of a dual firefly and Renilla luciferase reporter gene assay to simultaneously determine drug selectivity at human corticotrophin releasing hormone 1 and 2 receptors

The aim of this study was to investigate and validate the use of a dual glow-signal luciferase reporter gene assay to simultaneously evaluate drug activity at two different seven-transmembrane receptor subtypes. Stable cell lines (CHO) transfected with either human corticotrophin releasing hormone 1 (hCRH1) receptors and a firefly luciferase reporter gene or hCRH2 and a Renilla luciferase reporter gene were created to provide different luciferase readouts for CRH1 and CRH2 receptors, respectively. Cells were combined for stimulation and measurement of luciferase luminescence in a 96-well plate format assay. The nonselective CRH agonists rat/human CRH and sauvagine caused concentration-dependent increases in luminescence via activation of CRH1 (firefly luciferase; pEC50 = 8.40 +/- 0.06 and 8.39 +/- 0.08, respectively, n = 8) and CRH2 (Renilla luciferase; pEC50 = 8.89 +/- 0.14 and 8.92 +/- 0.13, respectively, n = 8) receptors. The nonselective CRH antagonist astressin blocked these agonist-induced increases in luciferase at both CRH1 and CRH2 receptors. The selective CRH1 antagonist CP154,526 blocked r/hCRH- and sauvagine-induced increases in luciferase at CRH1 receptors only. These data report the expected pharmacology for CRH1 and CRH2 receptors. This assay enabled two receptor subtypes to be studied simultaneously in the same 96-well plate and generated robust data with low variability. It has the potential advantage of significant time and cost savings, with application to both basic research and compound screening.     

Structural effects of DNA sequence on T.A recognition by hydroxypyrrole/pyrrole pairs in the minor groove

Synthetic polyamides composed of three types of aromatic amino acids, N-methylimidazole (Im), N-methylpyrrole (Py) and N-methyl-3-hydroxypyrrole (Hp) bind specific DNA sequences as antiparallel dimers in the minor groove. The side-by-side pairings of aromatic rings in the dimer afford a general recognition code that allows all four base-pairs to be distinguished. To examine the structural consequences of changing the DNA sequence context on T.A recognition by Hp/Py pairs in the minor groove, crystal structures of polyamide dimers (ImPyHpPy)(2) and the pyrrole counterpart (ImPyPyPy)(2) bound to the six base-pair target site 5'-AGATCT-3' in a ten base-pair oligonucleotide have been determined to a resolution of 2.27 and 2.15 A, respectively. The structures demonstrate that the principles of Hp/Py recognition of T.A are consistent between different sequence contexts. However, a general structural explanation for the non-additive reduction in binding affinity due to introduction of the hydroxyl group is less clear. Comparison with other polyamide-DNA cocrystal structures reveals structural themes and differences that may relate to sequence preference.     

Low-molecular-weight organic acids in rhizosphere soils of durum wheat and their effect on cadmium bioaccumulation

Cadmium (Cd) accumulation has been found to vary between cultivars of durum wheat (Triticum turgidum var. durum), and it is hypothesized that low-molecular-weight organic acids (LMWOAs) produced at the soil-root interface (rhizosphere) may play an important role in the availability and uptake of Cd by these plants. The objective of this study, therefore, was to (1) investigate the nature and quantity of LMWOAs present in the rhizosphere of durum wheat cultivars Arcola (low Cd accumulator) and Kyle (high Cd accumulator) grown in three different soils: Yorkton, Sutherland and Waitville, and (2) determine the relationship between Cd accumulation in these plants and LMWOAs present in the rhizosphere. Plants were grown for two weeks in pot-cultures under growth chamber conditions. Oxalic, fumaric, succinic, L-malic, tartaric, citric, acetic, propionic and butyric acids were found and quantified in the water extracts of rhizosphere soil, with acetic and succinic acids being predominant. No water extractable LMWOAs were identified in the bulk soil. Total amount of LMWOAs in the rhizosphere soil of the high Cd accumulator (Kyle) was significantly higher than that for the low Cd accumulator (Arcola) in all three soils. Furthermore, large differences in amounts of LMWOAs were found in the rhizosphere soil for the same cultivars grown in different soils and followed the pattern: Sutherland > Waitville > Yorkton. Extractable soil Cd (M NH4Cl) and Cd accumulation in the plants also followed the same soil sequence as LMWOA production. Cadmium accumulation by the high and low Cd accumulating cultivars was proportional to the levels of LMWOAs found in the rhizosphere soil of each cultivar. These results suggest that the differing levels of LMWOAs present in the rhizosphere soil played an important role in the solubilization of particulate-bound Cd into soil solution and its subsequent phytoaccumulation by the high and low Cd accumulating cultivars.     

Crystal structure of rubredoxin from Pyrococcus furiosus at 0.95 Å resolution, and the structures of N-terminal methionine and formylmethionine variants of Pf Rd. Contributions of N-terminal interactions to thermostability

The high-resolution crystal structure of the small iron-sulfur protein rubredoxin (Rd) from the hyperthermophilic archeon Pyrococcus furiosus (Pf) is reported in this paper, together with those of its methionine ([_0M]Pf Rd) and formylmethionine (f[_0M]Pf Rd) variants. These studies were conducted to assess the consequences of the presence or absence of a salt bridge between the amino terminal nitrogen of Ala1 and the side chain of Glu14 to the structure and stability of this rubredoxin. The structure of wild-type Pf Rd was solved to a resolution of 0.95?Å and refined by full-matrix least-squares techniques to a crystallographic agreement factor of 12.8% [F>2σ(F) data, 25?617 reflections], while those of the [_0M]Pf and f[_0M]Pf Rd variants were solved at slightly lower resolutions (1.1?Å, R=11.5%, 17?213 reflections; 1.2?Å, R=13.7%, 12?478 reflections, respectively). The quality of the data was such that about half of the hydrogen atoms of the protein were clearly visible. All three structures were ultimately refined using the program SHELXL-93 with anisotropic atomic displacement parameters for all non-hydrogen protein atoms, and calculated hydrogen positions included but not refined. In this paper we also report thermostability data for all three forms of Pf Rd, and show that they follow the sequence wild-type >[_0M]Pf>formyl[_0M]Pf. Comparison of the three Pf Rd structures in the N-terminal region show that the structures of wild-type Pf Rd and f[_0M]Pf are rather similar, while that of [_0M]Pf Rd shows a number of additional hydrogen bonds involving the extra methionine group. While the salt bridge between the Ala1 amino group and the Glu14 carboxylate is not the primary determinant of the thermostability of Pf Rd, alterations to the amino terminus do have a moderate influence on the thermostability of this protein.