Escherichia coli purine nucleoside phosphorylase II, the product of the xapA gene

Purine nucleoside phosphorylases (PNPs, E. C. 2.4.2.1) use orthophosphate to cleave the N-glycosidic bond of beta-(deoxy)ribonucleosides to yield alpha-(deoxy)ribose 1-phosphate and the free purine base. Escherichia coli PNP-II, the product of the xapA gene, is similar to trimeric PNPs in sequence, but has been reported to migrate as a hexamer and to accept xanthosine with comparable efficiency to guanosine and inosine, the usual physiological substrates for trimeric PNPs. Here, we present a detailed biochemical characterization and the crystal structure of E.coli PNP-II. In three different crystal forms, PNP-II trimers dimerize, leading to a subunit arrangement that is qualitatively different from the "trimer of dimers" arrangement of conventional high molecular mass PNPs. Crystal structures are compatible with similar binding modes for guanine and xanthine, with a preference for the neutral over the monoanionic form of xanthine. A single amino acid exchange, tyrosine 191 to leucine, is sufficient to convert E.coli PNP-II into an enzyme with the specificity of conventional trimeric PNPs, but the reciprocal mutation in human PNP, valine 195 to tyrosine, does not elicit xanthosine phosphorylase activity in the human enzyme.     

MCH released in a novel pheromone dispenser prevents spruce beetle,Dendroctonus rufipennis (Coleoptera: Scolytidae), attacks in south-central Alaska

Field tests of 3-methyl-2-cyclohexen-1-one (MCH), the antiaggregation pheromone of the spruce beetle, Dendroctonus rufipennis Kirby, were conducted in south-central Alaska in stands of Lutz spruce, Picea x lutzii Little, and Sitka spruce, P. sitchensis (Bong.) Carr. The deployment of MCH in a novel releaser significantly reduced trap catches and spruce beetle attacks on standing live spruce by 96 and 87%, respectively. The results of this study demonstrate the first successful field test of MCH in Alaska for the prevention of spruce beetle attacks on standing, live spruce.     

Ability of adenosine-2'(3')-deoxy-3'(2')-triphosphates and related analogues to replace ATP as phosphate donor for all human and Drosphila melanogaster deoxyribonucleoside kinases

Six non-conventional adenosine-2'- and 3'-triphosphate analogues of ATP were tested as potential phosphate donors for all four human, and D. melanogaster, deoxyribonucleoside kinases. With dCK (only dAdo as acceptor), TK1, TK2 and dNK only 3'-deoxyadenosine-2'-triphosphate was an effective donor (5-60% that for ATP). With dCK (dCyd as acceptor) and dGK (dGuo as acceptor), sharing 45% sequence identity, donor activities ranged from 13 to 119% that for ATP. Products were 5'-phosphates. In some instances, kinetics are dependent on the nature of the acceptor, and donor and acceptors properties are mutually interdependent. Results are highly relevant to studies on the modes of interaction with the enzymes, and to interpretations of reported crystal structures of dCK and dNK with bound ligands.     

An assessment of re-randomization methods in bark beetle (Scolytidae) trapping bioassays

1 Numerous studies have explored the role of semiochemicals in the behaviour of bark beetles (Scolytidae). 2 Multiple‐funnel traps are often used to elucidate these behavioural responses. Sufficient sample sizes are obtained by using large numbers of traps to which treatments are randomly assigned once, or by frequent collection of trap catches and subsequent re‐randomization of treatments. 3 Recently, there has been some debate about the potential for trap contamination to occur when semiochemical treatments (baits), and not trap‐treatment units (traps and baits), are re‐randomized among existing traps. Due to the volatility of many semiochemicals, small levels of contamination could potentially confound results. 4 A literature survey was conducted to determine the frequency of re‐randomizing semiochemical treatments (baits) vs. trap‐treatment units (traps and baits) in scolytid trapping bioassays. An experiment was then conducted to determine whether differences in the response of Dendroctonus brevicomis LeConte to attractant‐baited traps exist between the two methods. 5 The majority of papers examined reported use of a large number of fixed replicates (traps) rather than re‐randomization of treatments at frequent intervals. Seventy‐five percent of papers for which re‐randomization methods could be determined reported relocation of semiochemical treatments (baits) only. 6 No significant differences in trap catch were observed among multiple‐funnel traps aged with D. brevcomis baits (Phero Tech Inc., Canada) for 0, 30 and 90 days, suggesting that contamination did not influence the results. 7 It is concluded that re‐randomizing baits is a viable cost‐effective option to re‐randomizing trap and bait units.     

CRYSTAL STRUCTURE OF 1,3,5-TRIMETHYL-N4-HYDROXYCYTOSINE,AND ITS RELEVANCE TO THE MECHANISM OF HYDROXYLAMINE MUTAGENESIS

Abstract

1,3,5-Trimethyl-N⁴-hydroxycytosine, an analogue of the promutagenic N⁴-hydroxycytosine and 5-methyl-N⁴-hydroxycytosine nucleosides, crystallizes in the monoclinic space group P 2₁/n with cell dimensions at ?147°C: a = 7.1481(7), b = 9.2565(5), c = 13.3086(12) Å, β = 97.90(2)°, V = 872.24(13) ų, ρ_c = 1.426 Mg m^?3, Z = 4, F(000) = 401.39, μ = 0.91 mm^?1, λ(Cu) = 1.54056 Å, 20(max) = 139.3°. The crystal structure has been solved by X-ray difraction and refined to R = 3.7 % for 1457 reflections. Notwithstandin the steric hindrance imposed by methyl groups at both N(3) and C(5), the exocyclic N⁴-OH group is located essentially in the plane of the ring, giving rise to an “overcrowded” molecule, like that of 1,5,N⁴,N⁴-tetramethylcytosine. The conformational parameters have also been compared with those of a number of related and previously reported N(1)-substituted cytosines. In the present compound the N⁴-OH rotamer is in the anti conformation relative to the ring N(3), hence similar to that of one of the rotamers in N(1)-substituted N⁴-hydroxycytosine, which permits normal Watson-Crick base pairing of the latter, relevant to the mechanism of hydroxylamine mutagenesis.     

The PSEN1, p.E318G Variant Increases the Risk of Alzheimer's Disease in APOE-ε4 Carriers

The primary constituents of plaques (Aβ42/Aβ40) and neurofibrillary tangles (tau and phosphorylated forms of tau [ptau]) are the current leading diagnostic and prognostic cerebrospinal fluid (CSF) biomarkers for AD. In this study, we performed deep sequencing of APP, PSEN1, PSEN2, GRN, APOE and MAPT genes in individuals with extreme CSF Aβ42, tau, or ptau levels. One known pathogenic mutation (PSEN1 p.A426P), four high-risk variants for AD (APOE p.L46P, MAPT p.A152T, PSEN2 p.R62H and p.R71W) and nine novel variants were identified. Surprisingly, a coding variant in PSEN1, p.E318G (rs17125721-G) exhibited a significant association with high CSF tau (p = 9.2×10⁻⁴) and ptau (p = 1.8×10⁻³) levels. The association of the p.E318G variant with Aβ deposition was observed in APOE-ε4 allele carriers. Furthermore, we found that in a large case-control series (n = 5,161) individuals who are APOE-ε4 carriers and carry the p.E318G variant are at a risk of developing AD (OR = 10.7, 95% CI = 4.7–24.6) that is similar to APOE-ε4 homozygous (OR = 9.9, 95% CI = 7.2.9–13.6), and double the risk for APOE-ε4 carriers that do not carry p.E318G (OR = 3.9, 95% CI = 3.4–4.4). The p.E318G variant is present in 5.3% (n = 30) of the families from a large clinical series of LOAD families (n = 565) and exhibited a higher frequency in familial LOAD (MAF = 2.5%) than in sporadic LOAD (MAF = 1.6%) (p = 0.02). Additionally, we found that in the presence of at least one APOE-ε4 allele, p.E318G is associated with more Aβ plaques and faster cognitive decline. We demonstrate that the effect of PSEN1, p.E318G on AD susceptibility is largely dependent on an interaction with APOE-ε4 and mediated by an increased burden of Aβ deposition.     

Efficacy of Pneumococcal Nontypable Haemophilus influenzae Protein D Conjugate Vaccine (PHiD-CV) in Young Latin American Children: A Double-Blind Randomized Controlled Trial

BackgroundThe relationship between pneumococcal conjugate vaccine–induced antibody responses and protection against community-acquired pneumonia (CAP) and acute otitis media (AOM) is unclear. This study assessed the impact of the ten-valent pneumococcal nontypable Haemophilus influenzae protein D conjugate vaccine (PHiD-CV) on these end points. The primary objective was to demonstrate vaccine efficacy (VE) in a per-protocol analysis against likely bacterial CAP (B-CAP: radiologically confirmed CAP with alveolar consolidation/pleural effusion on chest X-ray, or non-alveolar infiltrates and C-reactive protein ≥ 40 µg/ml); other protocol-specified outcomes were also assessed.ConclusionsEfficacy was demonstrated against a broad range of pneumococcal diseases commonly encountered in young children in clinical practice.

Trial registration

www.ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00466947","term_id":"NCT00466947"}}NCT00466947Please see later in the article for the Editors'' Summary     

Agrobacterium tumefaciens-mediated genetic transformation of Digitalis purpurea L.

Genetic transformation is a tool of special interest for developing new biotechnological strategies for the production of bio-active compounds such as cardenolides, which are exclusively obtained from plants. To date, Digitalis plants are the main economically viable source of cardenolides for the pharmaceutical industry. This study describes the development of efficient plant regeneration and Agrobacterium-mediated genetic transformation protocols for Digitalis purpurea L. First, a plant regeneration procedure starting from leaf segments of in vitro-cultivated plants was established and the minimal inhibitory concentration of G-418 (geneticin) for callus induction was determined. Both leaf segments and callus tissue were sensitive to G-418 70 mg l^?1. Afterwards, two Agrobacterium strains were used to test their T-DNA transfer ability on D. purpurea leaf tissues, EHA105 and C58C1Rif^R (pMP90), both harboring the binary vector pTJK136. Strain C58C1Rif^R (pMP90) yielded a higher number of transformed plants than EHA105. Successful transformation was confirmed by histochemical β-glucuronidase (GUS) assays of the putative transgenic tissues and PCR analyses using β-glucuronidase (uidA)- and neomycin phosphotransferase II (nptII)-specific primers. Southern blot hybridization confirmed the stable integration of the nptII gene in the transgenic plants. In total, 518 independent transgenic lines were regenerated with an average of 6.91 transgenic lines per initial leaf segment infected with A. tumefaciens strain C58C1Rif^R (pMP90). To date, only a few studies have been published on the genetic transformation of Digitalis species. The protocols for plant regeneration and genetic transformation described in this paper will contribute to functional studies for a better understanding of cardenolide biosynthetic pathways and the metabolic engineering of cardenolides to develop high-yielding improved genotypes.     

Peptidyl Arginine Deiminase from Porphyromonas gingivalis Abolishes Anaphylatoxin C5a Activity

Evasion of killing by the complement system, a crucial part of innate immunity, is a key evolutionary strategy of many human pathogens. A major etiological agent of chronic periodontitis, the Gram-negative bacterium Porphyromonas gingivalis, produces a vast arsenal of virulence factors that compromise human defense mechanisms. One of these is peptidylarginine deiminase (PPAD), an enzyme unique to P. gingivalis among bacteria, which converts Arg residues in polypeptide chains into citrulline. Here, we report that PPAD citrullination of a critical C-terminal arginine of the anaphylatoxin C5a disabled the protein function. Treatment of C5a with PPAD in vitro resulted in decreased chemotaxis of human neutrophils and diminished calcium signaling in monocytic cell line U937 transfected with the C5a receptor (C5aR) and loaded with a fluorescent intracellular calcium probe: Fura-2 AM. Moreover, a low degree of citrullination of internal arginine residues by PPAD was also detected using mass spectrometry. Further, after treatment of C5 with outer membrane vesicles naturally shed by P. gingivalis, we observed generation of C5a totally citrullinated at the C-terminal Arg-74 residue (Arg74Cit). In stark contrast, only native C5a was detected after treatment with PPAD-null outer membrane vesicles. Our study suggests reduced antibacterial and proinflammatory capacity of citrullinated C5a, achieved via lower level of chemotactic potential of the modified molecule, and weaker cell activation. In the context of previous studies, which showed crosstalk between C5aR and Toll-like receptors, as well as enhanced arthritis development in mice infected with PPAD-expressing P. gingivalis, our findings support a crucial role of PPAD in the virulence of P. gingivalis.