High nuclear genetic diversity, high levels of outcrossing and low differentiation among remnant populations of Quercus petraea at the margin of its range in Ireland

Background and Aims Quercus petraea colonized Ireland after the last glaciation from refugia on mainland Europe. Deforestation. however. beginning in Neolithic times, has resulted in small, scattered forest fragments, now covering less than 12 000 ha. Methods Plastid (three fragments) and microsatellite variation (13 loci) were characterized in seven Irish populations sampled along a north-south gradient. Using Bayesian approaches and Wright's F-statistics, the effects of colonization and fragmentation on the genetic structure and mating patterns of extant oak populations were investigated. Key-Results All Populations possessed cytotypes common to the Iberian Peninsula. Despite the distance from the refugial core and the extensive deforestation in Ireland, nuclear genetic variation was high and comparable to mainland Europe. Low population differentiation was observed within Ireland and populations showed no evidence for isolation by distance. As expected of a marker with an effective Population size of one-quarter relative to the nuclear genome, plastid variation indicated higher differentiation. Individual inbreeding coefficients indicated high levels of outcrossing. Conclusions Consistent with a large effective Population size in the historical migrant gene pool and/or with high gene flow among populations, high within-population diversity and low population differentiation was observred within Ireland. It is proposed that native Q. petraea populations in Ireland share a common phylogeographic history and that the present genetic structure does not reflect founder effects. (C) 2004 Annals of Botany Company.

Divergent environments and population bottlenecks fail to generate premating isolation in Drosophila pseudoobscura

While the feasibility of bottleneck-induced speciation is in doubt, population bottlenecks may still affect the speciation process by interacting with divergent selection. To explore this possibility, I conducted a laboratory speciation experiment using Drosophila pseudoobscura involving 78 replicate populations assigned in a two-way factorial design to both bottleneck (present vs. absent) and environment (ancestral vs. novel) treatments. Populations independently evolved under these treatments and were then tested for assortative mating and male mating success against their common ancestor. Bottlenecks alone did not generate any premating isolation, despite an experimental design that was conducive to bottleneck-induced speciation. Premating isolation also did not evolve in the novel environment treatment, neither in the presence nor absence of bottlenecks. However, male mating success was significantly reduced in the novel environment treatment, both as a plastic response to this environment and as a result of environment-dependent inbreeding effects in the bottlenecked populations. Reduced mating success of derived males will hamper speciation by enhancing the mating success of immigrant, ancestral males. Novel environments are generally thought to promote ecological speciation by generating divergent natural selection. In the current experiment, however, the novel environment did not cause the evolution of any premating isolation and it reduced the likelihood of speciation through its effects on male mating success.

Isolation and characterization of integron-containing bacteria without antibiotic selection

The emergence of antibiotic resistance among pathogenic and commensal bacteria has become a serious problem worldwide. The use and overuse of antibiotics in a number of settings are contributing to the development of antibiotic-resistant microorganisms. The class 1 and 2 integrase genes (intI1 and intI2, respectively) were identified in mixed bacterial cultures enriched from bovine feces by growth in buffered peptone water (BPW) followed by integrase-specific PCR. Integrase-positive bacterial colonies from the enrichment cultures were then isolated by using hydrophobic grid membrane filters and integrase-specific gene probes. Bacterial clones isolated by this technique were then confirmed to carry integrons by further testing by PCR and DNA sequencing. Integron-associated antibiotic resistance genes were detected in bacteria such as Escherichia coli, Aeromonas spp., Proteus spp., Morganella morganii, Shewanella spp., and urea-positive Providencia stuartii isolates from bovine fecal samples without the use of selective enrichment media containing antibiotics. Streptomycin and trimethoprim resistance were commonly associated with integrons. The advantages conferred by this methodology are that a wide variety of integron-containing bacteria may be simultaneously cultured in BPW enrichments and culture biases due to antibiotic selection can be avoided. Rapid and efficient identification, isolation, and characterization of antibiotic resistance-associated integrons are possible by this protocol. These methods will facilitate greater understanding of the factors that contribute to the presence and transfer of integron-associated antibiotic resistance genes in bacterial isolates from red meat production animals.

Isolation and characterization at cholinergic nicotinic receptors of a neurotoxin from the venom of the Acanthophis sp Seram death adder

The present study describes the isolation of the first neurotoxin (acantoxin IVa) from Acanthophis sp. Seram death adder venom and an examination of its activity at nicotinic acetylcholine receptor (naChR) subtypes. Acantoxin IVa (MW 6815; 0.1-1.0 muM) caused concentration-dependent inhibition of indirect twitches (0.1 Hz, 0.2 ms, supramaximal V) and inhibited contractile responses to exogenous nicotinic agonists in the chick biventer cervicis nerve-muscle, confirming that this toxin is a postsynaptic neurotoxin. Acantoxin IVa (1-10 nM) caused pseudo-irreversible antagonism at skeletal muscle nAChR with an estimated pA(2) Of 8.36 +/- 0.17. Acantoxin IVa was approximately two-fold less potent than the long-chain (Type 11) neurotoxin, alpha-bungarotoxin. With a pK(i) value of 4.48, acantoxin IVa was approximately 25,000 times less potent than a-bungarotoxin at alpha7-type neuronal nAChR. However, in contrast to alpha-bungarotoxin, acantoxin IVa completely inhibited specific [H-3]-methyllycaconitine (MLA) binding in rat hippocampus homogenate. Acantoxin IVa had no activity at ganglionic nAChR, alpha4beta2 subtype neuronal nAChR or cytisine-resistant [H-3]-epibatidine binding sites. While long-chain neurotoxin resistant [H-3]-MLA binding in hippocampus homogenate requires further investigation, we have shown that a short-chain (Type 1) neurotoxin is capable of fully inhibiting specific [H-3]-MLA binding. (C) 2004 Elsevier Inc. All rights reserved.

Parallel evolution of sexual isolation in sticklebacks

Mechanisms of speciation are not well understood, despite decades of study. Recent work has focused on how natural and sexual selection cause sexual isolation. Here, we investigate the roles of divergent natural and sexual selection in the evolution of sexual isolation between sympatric species of threespine sticklebacks. We test the importance of morphological and behavioral traits in conferring sexual isolation and examine to what extent these traits have diverged in parallel between multiple, independently evolved species pairs. We use the patterns of evolution in ecological and mating traits to infer the likely nature of selection on sexual isolation. Strong parallel evolution implicates ecologically based divergent natural and/or sexual selection, whereas arbitrary directionality implicates nonecological sexual selection or drift. In multiple pairs we find that sexual isolation arises in the same way: assortative mating on body size and asymmetric isolation due to male nuptial color. Body size and color have diverged in a strongly parallel manner, similar to ecological traits. The data implicate ecologically based divergent natural and sexual selection as engines of speciation in this group.

Enrichment, isolation and characterisation of ruminal bacteria that degrade non-protein amino acids from the tropical legume Acacia angustissima

Acacia angustissima has been proposed as a protein supplement in countries where low quality forages predominate. A number of non-protein amino acids have been identified in the leaves of A. angustissima and these have been linked to toxicity in ruminants. The non-protein amino acid 4-n-acetyl-2,4-diaminobutyric acid (ADAB) has been shown to be the major amino acid in the leaves of A. angustissima. The current study aimed to identify micro-organisms from the rumen environment capable of degrading ADAB by using a defined rumen-simulating media with an amino acid extract from A. angustissima. A mixed enrichment culture was obtained that exhibited substantial ADAB-degrading ability. Attempts to isolate an ADAB-degrading micro-organism were carried out, however no isolates were able to degrade ADAB in pure culture. This enrichment culture was also able to degrade the non-protein amino acids diaminobutyric acid (DABA) and diaminopropionic acid (DAPA) which have structural similarities to ADAB. Two isolates were obtained which could degrade DAPA. One isolate is a novel Grain-positive rod (strain LPLR3) which belongs to the Firmicutes and is not closely related to any previously isolated bacterium. The other isolate is strain LPSR1 which belongs to the Gammaproteobacteria and is closely related (99.93% similar) to Klebsiella pneumoniae subsp. ozaenae. The studies demonstrate that the rumen is a potential rich source of undiscovered micro-organisms which have novel capacities to degrade plant secondary compounds. (c) 2005 Elsevier B.V. All rights reserved.

Isolation of polymorphic tetranucleotide microsatellite markers in the satin bowerbird, Ptilonorhynchus violaceus

We isolated 13 polymorphic microsatellite markers from the satin bowerbird, Ptilonorhynchus violaceus from a genomic library enriched in (AAGG)(n) repetitive elements and characterized them in 20 individuals. The number of alleles ranged from two to 18 per locus with the observed heterozygosity ranging from 0.15 to 1.00. These markers will be useful for analysing questions concerning parentage, population genetic structure and models of speciation.

Isolation and characterization of novel cyclotides from Viola hederaceae - Solution structure and anti-HIV activity of vhl-1, a leaf-specific expressed cyclotide

Based on a newly established sequencing strategy featured by its efficiency, simplicity, and easy manipulation, the sequences of four novel cyclotides (macrocyclic knotted proteins) isolated from an Australian plant Viola hederaceae were determined. The three-dimensional solution structure of V. hederaceae leaf cyclotide-1 ( vhl-1), a leaf-specific expressed 31-residue cyclotide, has been determined using two-dimensional H-1 NMR spectroscopy. vhl-1 adopts a compact and well defined structure including a distorted triple-stranded β- sheet, a short 310 helical segment and several turns. It is stabilized by three disulfide bonds, which, together with backbone segments, form a cyclic cystine knot motif. The three-disulfide bonds are almost completely buried into the protein core, and the six cysteines contribute only 3.8% to the molecular surface. A pH titration experiment revealed that the folding of vhl-1 shows little pH dependence and allowed the pK(a) of 3.0 for Glu(3) and ∼ 5.0 for Glu(14) to be determined. Met(7) was found to be oxidized in the native form, consistent with the fact that its side chain protrudes into the solvent, occupying 7.5% of the molecular surface. vhl-1 shows anti-HIV activity with an EC50 value of 0.87 μ m.

Isolation of polymorphic tetranucleotide microsatellite for the large-billed scrubwren (Sericomis magnirostris)

We isolated 12 polymorphic microsatellite markers for the large-billed scrubwren Sericornis magnirostris from genomic libraries enriched for (AAGG)(n) and (AACC)(n) repetitive elements and characterized them in 11 individuals. The number of alleles ranged from four to 15 per locus with the observed heterozygosity ranging from 0.14 to 0.91. These markers will be useful to address questions concerning population genetic structure and models of speciation.

Isolation of polymorphic tetranucleotide microsatellite markers for the grey-headed robin (Poecilodryas albispecularis)

We isolated 12 polymorphic microsatellite markers for the grey-headed robin Poecilodryas albispecularis from genomic libraries enriched for (AAGG)n and (AACC)n repetitive elements and characterized them in 12 individuals. The number of alleles ranges from three to nine per locus with the observed heterozygosity ranging from 0.33 to 0.90. These markers will be useful for analysis of questions concerning population genetic structure and testing models of speciation.

(-)-echinobetaine A: Isolation, structure elucidation, synthesis, and SAR studies on a new nematocide from a southern Australian marine sponge, Echinodictyum sp.

A nematocidal agent present in a southern Australian marine sponge of the genus Echinodictyum has been isolated and identified by detailed spectroscopic analysis and total synthesis as the novel betaine (-)-echinobetaine A (6). Preliminary SAR investigations have been undertaken.

Isolation, solution structure, and insecticidal activity of Kalata B2, a circular protein with a twist: Do Mobius strips exist in nature?

A large number of macrocyclic miniproteins with diverse biological activities have been isolated from the Rubiaceae, Violaceae, and Cucurbitaceae plant families in recent years. Here we report the three-dimensional structure determined using H-1 NMR spectroscopy and demonstrate potent insecticidal activity for one of these peptides, kalata B2. This peptide is one of the major components of an extract from the leaves of the plant Oldenlandia affinis. The structure consists of a distorted triple-stranded beta-sheet and a cystine knot arrangement of the disulfide bonds and is similar to those described for other members of the cyclotide family. The unique cyclic and knotted nature of these molecules makes them a fascinating example of topologically complex proteins. Examination of the sequences reveals that they can be separated into two subfamilies, one of which contains a larger number of positively charged residues and has a bracelet-like circularization of the backbone. The second subfamily contains a backbone twist due to a cis-peptidyl-proline bond and may conceptually be regarded as a molecular Mobius strip. Kalata B2 is the second putative member of the Mobius cyclotide family to be structurally characterized and has a cis-peptidyl-proline bond, thus validating the suggested name for this subfamily of cyclotides. The observation that kalata B2 inhibits the growth and development of Helicoverpa armigera larvae suggests a role for the cyclotides in plant defense. A comparison of the sequences and structures of kalata B1 and B2 provides insight into the biological activity of these peptides.

(+)-Echinobetaine B: isolation, structure elucidation, synthesis and preliminary SAR studies on a new nematocidal betaine from a southern Australian marine sponge, Echinodictyum sp.

The principle nematocidal agent present in a southern Australian marine sponge of the genus Echinodictyum has been isolated and identfied as the novel betaine (+)-echinobetaine B (6), and the structure assigned by spectroscopic analysis has been confirmed by total synthesis. Preliminary SAR conclusions are drawn from analysis of synthetic intermediates and the known marine metabolites zooanemonin (12) and norzooanemonin (13), and the new sponge metabolite norzooanemonin methyl ester (14). The latter compound is reported for the first time from a selection of Australian sponges, including an Axinyssa sp., a Niphates sp., an Axinella sp. and a Ptilocaulis sp.

Rifampicin and sodium fusidate reduces the frequency of methicillin-resistant Staphylococcus aureus (MRSA) isolation in adults with cystic fibrosis and chronic MRSA infection

Nosocomial transmission of methicillin-resistant Staphylococcus aureus (MRSA) to patients with cystic fibrosis (CF) frequently results in chronic respiratory tract carriage. This is an increasing problem, adds to the burden of glycopeptide antibiotic use in hospitals, and represents a relative contraindication to lung transplantation. The aim of this study was to determine whether it is possible to eradicate MRSA with prolonged oral combination antibiotics, and whether this treatment is associated with improved clinical status. Adult CF patients (six mate, one female) with chronic MRSA infection were treated for six months with rifampicin and sodium fusidate. Outcome data were examined for six months before treatment, on treatment and after treatment. The patients had a mean age of 29.3 (standard deviation = 6.3) years and FEV1 of 36.1% (standard deviation = 12.7) predicted. The mean duration of MRSA isolation was 31 months. MRSA isolates identified in these patients was of the same lineage as the known endemic strain at the hospital when assessed by pulsed-field get electrophoresis. Five of the seven had no evidence of MRSA during and for at [east six months after rifampicin and sodium fusidate. The proportion of sputum samples positive for MRSA was lower during the six months of treatment (0.13) and after treatment (0.19) compared with before treatment (0.85) (P < 0.0001). There was a reduction in the number of days of intravenous antibiotics per six months with 20.3 +/- 17.6 on treatment compared with 50.7 before treatment and 33.0 after treatment (P = 0.02). There was no change in lung function. Gastrointestinal side effects occurred in three, but led to therapy cessation in only one patient. Despite the use of antibiotics with anti-staphylococcal activity for treatment of respiratory exacerbation, MRSA infection persists. MRSA can be eradicated from the sputum of patients with CF and chronic MRSA carriage by using rifampicin and sodium fusidate for six months. This finding was associated with a significant reduction in the duration of intravenous antibiotic treatment during therapy. (C) 2003 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.