Re-establishing a saltmarsh vegetation structure in a changing climate

Summary A major management decision in an ecological restoration or rehabilitation project is whether supplementary planting or natural vegetation regeneration is the better alternative or if a combination can be applied. Management decisions are further complicated when the project involves saltmarsh as tidal cycles, the effects of salinity and sea level rise add to the complexity of decisions. The ecological values of the saltmarsh community in Australia were only recognized relatively recently but the endangered ecological community listing in 1994 (under the New South Wales Threatened Species Conservation Act 1995) highlighted the need to protect, rehabilitate and restore saltmarsh. This project measured vegetation change after soil profile reconstruction in saltmarsh surrounding Sponsors Lagoon, Fingal Peninsula in north coastal New South Wales, Australia. Restored sites (planted and non‐planted) were compared with nearby disturbed (control) and reference sites. The dominant species in the community to be restored were Saltwater Couch (Sporobolus virginicus), Suaeda (Suaeda australis), Sarcocornia (Sarcocornia quinqueflora ssp. quinqueflora and Sea Rush (Juncus kraussii ssp. australiensis). Changes in percentage cover of the species in the first three years after soil reconstruction work showed that the dominant Saltwater Couch established only from vegetative growth arising from remnant vegetation but there was strong seedling regeneration of several other species. It was concluded that planting is important for species that are less vagile, in larger, denuded parts of this site where their resilience is lower and natural regeneration potential is limited. An understanding of the biology of the individual species is therefore likely to be essential in saltmarsh restoration projects. A combination of techniques, incorporating planting of slower establishing species and encouragement of seed‐germinated colonizers is useful for initial site stabilization and progressive ecological recovery at this site. The inclusion of migration zones in the planning phase will allow for the long‐term viability of this restoration project.     

PYR/PYL/RCAR family members are major in‐vivo ABI1 protein phosphatase 2C‐interacting proteins in Arabidopsis

Abscisic acid (ABA) mediates resistance to abiotic stress and controls developmental processes in plants. The group‐A PP2Cs, of which ABI1 is the prototypical member, are protein phosphatases that play critical roles as negative regulators very early in ABA signal transduction. Because redundancy is thought to limit the genetic dissection of early ABA signalling, to identify redundant and early ABA signalling proteins, we pursued a proteomics approach. We generated YFP‐tagged ABI1 Arabidopsis expression lines and identified in vivo ABI1‐interacting proteins by mass‐spectrometric analyses of ABI1 complexes. Known ABA signalling components were isolated including SnRK2 protein kinases. We confirm previous studies in yeast and now show that ABI1 interacts with the ABA‐signalling kinases OST1, SnRK2.2 and SnRK2.3 in plants. Interestingly, the most robust in planta ABI1‐interacting proteins in all LC‐MS/MS experiments were nine of the 14 PYR/PYL/RCAR proteins, which were recently reported as ABA‐binding signal transduction proteins, providing evidence for in vivo PYR/PYL/RCAR interactions with ABI1 in Arabidopsis. ABI1–PYR1 interaction was stimulated within 5 min of ABA treatment in Arabidopsis. Interestingly, in contrast, PYR1 and SnRK2.3 co‐immunoprecipitated equally well in the presence and absence of ABA. To investigate the biological relevance of the PYR/PYLs, we analysed pyr1/pyl1/pyl2/pyl4 quadruple mutant plants and found strong insensitivities in ABA‐induced stomatal closure and ABA‐inhibition of stomatal opening. These findings demonstrate that ABI1 can interact with several PYR/PYL/RCAR family members in Arabidopsis, that PYR1–ABI1 interaction is rapidly stimulated by ABA in Arabidopsis and indicate new SnRK2 kinase‐PYR/PYL/RCAR interactions in an emerging model for PYR/PYL/RCAR‐mediated ABA signalling.     

Identification of a sulfonamide series of CCR2 antagonists

A series of sulfonamide CCR2 antagonists was identified by high-throughput screening. Management of molecular weight and physical properties, in particular moderation of lipophilicity and study of pK_a, yielded highly potent CCR2 antagonists exhibiting good pharmacokinetic properties and improved potency in the presence of human plasma.     

Actin-Based Motility of Burkholderia thailandensis Requires a Central Acidic Domain of BimA That Recruits and Activates the Cellular Arp2/3 Complex

Burkholderia species use BimA for intracellular actin-based motility. Uniquely, Burkholderia thailandensis BimA harbors a central and acidic (CA) domain. The CA domain was required for actin-based motility, binding to the cellular Arp2/3 complex, and Arp2/3-dependent polymerization of actin monomers. Our data reveal distinct strategies for actin-based motility among Burkholderia species.In common with selected species of Listeria, Shigella, Rickettsia, and Mycobacterium, some members of the genus Burkholderia are capable of intracellular actin-based motility (reviewed in reference 9). Such motility promotes cell-to-cell spread in the absence of immune surveillance and, in some cases, escape from autophagy. The melioidosis pathogen Burkholderia pseudomallei forms actin-rich bacterium-containing membrane protrusions (5), in a manner that requires BimA (Burkholderia intracellular motility A) (11). B. pseudomallei BimA exhibits C-terminal homology to the Yersinia autosecreted adhesin YadA and possesses motifs associated with actin binding, including WASP homology 2 (WH2) domains and proline-rich motifs (11). Actin-based motility is also a feature of infection by the glanders pathogen Burkholderia mallei and the avirulent B. pseudomallei-like species Burkholderia thailandensis (10). BimA homologs exist in these species that can compensate for the actin-based motility defect of a B. pseudomallei bimA mutant (10), and mutation of B. mallei bimA results in loss of function (7). BimA homologs from B. mallei and B. thailandensis (BimA_th) differ markedly in primary sequence from the B. pseudomallei protein (BimA_ps) and each other (10), raising the possibility that they may act in distinct ways. Intraspecies conservation of BimA is high in natural populations of B. pseudomallei, with the exception of a geographically restricted B. mallei-like BimA variant (8).Mechanisms of bacterial actin-based motility converge on activation of the Arp2/3 (actin-related protein 2/3) complex. Activation of Arp2/3 requires cellular nucleation-promoting factors (NPFs) such as Wiskott-Aldrich syndrome protein (WASP) family members, and pathogens capable of actin-based motility often mimic the activity of NPFs or recruit and activate them at the bacterial pole (reviewed in references 3 and 9). Arp2/3 is localized throughout B. pseudomallei-induced actin tails (2); however, the role that it plays in actin-based motility is unclear, as expression of an inhibitory domain of the cellular NPF Scar1 does not interfere with actin-based motility of B. pseudomallei (2). Moreover, BimA_ps can polymerize actin in vitro in an Arp2/3-independent manner (11).Recruitment and activation of the Arp2/3 complex by cellular and pathogen-associated NPFs require one or more WH2 domains and an amphipathic central and acidic (CA) domain (3, 6). Analysis of the primary sequence of BimA homologs revealed a CA domain in B. thailandensis BimA that was absent in the BimA proteins of other Burkholderia species and conserved relative to WASP family members, Listeria ActA, and Rickettsia RickA (10). Here we surveyed the conservation of the CA domain and probed its role in actin-based motility and the binding and activation of the Arp2/3 complex.Primers were designed to amplify an 87-bp region of the CA domain (corresponding to amino acid residues 102 to 130 inclusive) of the bimA gene of the sequenced B. thailandensis strain E264 (5′-AGGCGGGTAATCGACTCA-3′ and 5′-TTCGTCGTCCGACCATGA-3′). These primers, and universal bimA-specific primers (8), were used to screen 203 Burkholderia isolates for bimA and the CA domain by PCR using Platinum Taq DNA polymerase (Invitrogen, Paisley, United Kingdom) and boiled lysates of single colonies as template. The strain collection was described previously (8), supplemented by an additional 52 B. pseudomallei, 23 B. thailandensis, and 19 B. mallei isolates and 10 other isolates representing 5 other Burkholderia species. A bimA amplicon was detected for all isolates of species B. pseudomallei, B. thailandensis, B. mallei, and Burkholderia oklahomensis. Consistent with analysis of sequenced genomes (8), the CA domain was restricted to, and always found in, B. thailandensis isolates. Such a test may prove useful to rapidly discriminate between avirulent B. thailandensis and the closely related biothreat agents B. pseudomallei and B. mallei.To investigate the function of the CA domain, we deleted the region corresponding to residues 96 to 130 of the strain E264 BimA by PCR-ligation-PCR (1) using the cloned bimA gene of B. thailandensis strain E30 as a template (10). Pfu proofreading DNA polymerase was used to separately amplify the region 5′ of the CA domain with primers B_th-comp forward (5′-CATGAATTCCCATGCGTGCAACAGTTGCT-3′) and 5′-CGAGCCGCCCGCGCCTCGCGTGTT-3′ and the region 3′ of the CA domain with B_th-comp reverse (5′-CTTCTCGAGTCACCATTGCCAGCTCATGCCTACGC-3′) and 5′-TCCCCTCCGCCGACGCCGATCGCAA-3′ from pME6032- bimA_th (10). The PCR amplicons were ligated, and the desired recombinant was amplified by a further round of PCR with primers B_th-comp forward and B_th-comp reverse. The product was subcloned under the Ptac promoter in pME6032 (4) via EcoRI and XhoI sites incorporated in the primers (underlined), yielding pME6032-bimA_th-ΔCA. Faithful amplification and deletion of the CA domain were confirmed by nucleotide sequencing (data not shown).To evaluate the role of the CA domain in actin-based motility, pME6032-bimA_th and the ΔCA variant were introduced into a B. pseudomallei strain 10276 bimA::pDM4 mutant (11) by electroporation with selection for tetracycline resistance. Strains were amplified in Luria-Bertani (LB) medium, and J774.2 murine macrophage-like cells were infected at a multiplicity of 10 in RPMI medium containing 10% (vol/vol) fetal calf serum at 37°C in a 5% CO₂ atmosphere. After 30 min cells were washed and overlaid with medium containing 250 μg/ml kanamycin to kill extracellular bacteria. During bacterial culture and cell infection, expression of BimA proteins was induced with 0.25 mM isopropyl-β-d-thiogalactoside (IPTG). Eight hours postinfection cells were washed, fixed, permeabilized, and stained for bacteria, polymerized F-actin, and nuclei essentially as described previously (10). Images were captured using a Leica confocal laser scanning microscope with LAS AF v.2.0 software. B. pseudomallei 10276 and 10276 bimA::pDM4 were included as positive and negative controls, respectively (Fig. ​(Fig.11 A and B). As expected B. thailandensis E30 BimA restored the ability of the 10276 bimA::pDM4 mutant to form actin tails (10) (Fig. ​(Fig.1C).1C). Deletion of the CA domain of B. thailandensis BimA abolished this activity (Fig. ​(Fig.1D),1D), indicating that it is required for actin-based motility.Open in a separate windowFIG. 1.Representative confocal laser scanning micrographs of J774.2 cells infected with B. pseudomallei strain 10276 (A), an isogenic bimA::pDM4 mutant (B), or 10276 bimA::pDM4 trans complemented with pME6032-bimA_th (C) or pME6032-bimA_th-ΔCA (D) and induced to express the proteins under IPTG induction. Bacteria (red) were stained using mouse monoclonal anti-B. pseudomallei lipopolysaccharide antibody (Camlab, Cambridge, United Kingdom) detected with anti-mouse Ig-Alexa Fluor⁵⁶⁸ (Molecular Probes, Leiden, Netherlands). F-actin (green) was stained with Alexa Fluor⁴⁸⁸-conjugated phalloidin. DNA (blue) was stained with 4′,6-diamidino-2-phenylindole. Bars, 5 μm. Bacteria forming actin tails are marked with arrows.Monoclonal antibodies raised against BimA_ps (11) failed to recognize BimA_th on the bacterial pole (data not shown); therefore, we were unable to conclude that loss of actin-based motility upon deletion of the CA domain may be a consequence of failed secretion or polar localization. To investigate the role of the CA domain in actin binding and polymerization, the ΔCA variant of B. thailandensis BimA was PCR amplified from pME6032-bimA_th-ΔCA with primers 5′-GGGCCCGGATCCGCCGCTGACGAGACG-3′ and 5′-GGGCCCGAATTCTCACGCTCGCGCGTCG-3′. The product was first cloned by a topoisomerase-mediated process into pCR2.1-TOPO (Invitrogen, Paisley, United Kingdom) and then subcloned as a BamHI and EcoRI fragment into similarly digested pGEX-2T-1 (Amersham Biosciences, Buckinghamshire, United Kingdom), creating a fusion to the carboxyl terminus of glutathione S-transferase (GST). The subcloned region corresponds to amino acids 47 to 386 of BimA_th and was confirmed by sequencing to be identical to the pGEX-BimA_th plasmid described previously (10), except for the deleted CA region. The region encoding B. pseudomallei BimA residues 54 to 455 (lacking the signal peptide and membrane anchor) was amplified from pME6032-bimA_ps (10) with primers 5′-GCGCGCGGATCCATGAATCCCCCCGAACCGCCGGGC-3′ and 5′-GCGCGCGAATTCTTAGCGCGCGGTGTCGGTG-3′ and fused to GST in pGEX-4T-1 as described above. Plasmids encoding GST or GST fusions to BimA_ps, BimA_th, or BimA_th-ΔCA domain were separately introduced into Escherichia coli K-12 Rosetta2(DE3)pLysS cells expressing rare aminoacyl tRNAs (Merck Biosciences, Nottingham, United Kingdom). LB cultures were induced to express the proteins at late logarithmic phase for 3 h at ambient temperature, which proved optimal for recovery of intact proteins using glutathione Sepharose 4B beads (Fig. ​(Fig.2A2A).Open in a separate windowFIG. 2.SDS-PAGE analysis of fusions of BimA_ps (residues 54 to 455), BimA_th (residues 47 to 386), or BimA_th lacking the CA domain to the carboxyl terminus of GST (A). Sepharose beads coated with these proteins, or GST alone, were examined for their ability to sequester actin (B), p34-Arc/ARPC2 (C), or Arp3 (D) from murine splenic extracts by Western blotting of bead-associated proteins using specific antibodies. Coated beads were also examined for their ability to bind highly purified rhodamine-labeled actin in PBS (red) by confocal laser scanning microscopy (E). Mw, molecular weights in thousands.Beads coated with GST, GST-BimA_ps, GST-BimA_th, or GST-BimA_th-ΔCA were incubated with murine splenic cell lysate as described previously (10). After 15 min of incubation at ambient temperature, beads were washed with ice-cold Tris-buffered saline and analyzed by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis and Western blotting with antiactin antibody (10). As reported previously, beads coated with GST-BimA_ps or GST-BimA_th, but not GST, sequestered actin from the splenic extract (Fig. ​(Fig.2B)2B) (10). Deletion of the CA domain did not impair actin binding, consistent with the fact that a predicted WH2 domain remains intact (10). It may be inferred that such binding is direct, since coated beads also bound highly purified rhodamine-labeled actin in phosphate-buffered saline (PBS) as assessed by confocal microscopy of beads treated as described previously (Fig. ​(Fig.2E)2E) (10). The ability of coated beads to sequester the Arp2/3 complex from murine splenic extracts was examined using rabbit anti-p34-Arc/ARPC2 (Millipore, Watford, United Kingdom) and goat anti-Arp3 (Autogen Bioclear, Wiltshire, United Kingdom) detected with species-specific Ig-horseradish peroxidase conjugates by a chemoluminescence method (Amersham Biosciences, Buckinghamshire, United Kingdom). GST-BimA_th, but not GST-BimA_ps, sequestered p34-Arc/ARPC2 (Fig. ​(Fig.2C)2C) and Arp3 (Fig. ​(Fig.2D).2D). Deletion of the CA domain abolished the ability to sequester Arp2/3 components (Fig. 2C and D), indicating that it plays a role in recruitment of the complex.The requirement for Arp2/3 and the CA domain in actin polymerization by B. thailandensis BimA was evaluated in vitro using pyrene-labeled actin. Polymerization of such monomers leads to an emission of fluorescence that can be sensitively recorded over time. Lyophilized pyrene-actin, Arp2/3, and the verprolin-like central and acidic (VCA) domain of WASP were obtained from Cytoskeleton (Universal Biologicals, Cambridge, United Kingdom) and prepared per the manufacturer''s instructions. Assay conditions were essentially as described previously (12). Briefly, 90-μl reaction mixtures were assembled in black opaque 96-well plates containing 100 nM GST or GST fusion protein, 2 μM pyrene-actin, and 30 nM Arp2/3 as required. Reactions were initiated by the addition of 10 μl of 10× polymerization buffer (100 mM Tris, pH 7.5, 500 mM KCl, 20 mM MgCl₂, 10 mM ATP), and the emission of fluorescence at 407 nm, after excitation at 365 nm, was followed every 30 s for 90 min using a Tecan Infinite M200 fluorescent plate reader with i-control software. The GST, GST-BimA_ps, GST-BimA_th, and GST-BimA_th-ΔCA proteins were prepared as described above but eluted from beads with 10 mM reduced glutathione. Triplicate determinations were performed with two independently purified sets of protein. Data from a representative assay are shown in Fig. ​Fig.3.3. Rates of polymerization were calculated as the rise in fluorescence units per second during the linear phase of polymerization, and the means of six values per protein ± standards of the means are recorded in Table ​Table1.1. Results were analyzed by pairwise Student t tests using R software (version 2.11), and P values of ≤0.05 were taken as significant.Open in a separate windowFIG. 3.Polymerization of pyrene-labeled actin monomers by GST (green lines), GST-BimA_ps (blue lines), GST-BimA_th (red lines), and GST-BimA_th-ΔCA (yellow lines) in the absence (solid lines) or presence (dotted lines) of the Arp2/3 complex. The Arp2/3 complex was confirmed to be active in the presence of the purified VCA domain of WASP (black line). The graph shows the increase in fluorescence units (at 407 nm) over time during a single representative experiment. Triplicate determinations were performed with two sets of independently purified proteins, and the mean rates of fluorescence are recorded in Table ​Table11.

TABLE 1.

Rates of polymerization of pyrene-labeled actin monomers by GST and GST-BimA fusion proteins in the absence or presence of the Arp2/3 complex

Quantification of six herbicide metabolites in human urine

We developed a sensitive, selective and precise method for measuring herbicide metabolites in human urine. Our method uses automated liquid delivery of internal standards and acetate buffer and a mixed polarity polymeric phase solid phase extraction of a 2 mL urine sample. The concentrated eluate is analyzed using high-performance liquid chromatography-tandem mass spectrometry. Isotope dilution calibration is used for quantification of all analytes. The limits of detection of our method range from 0.036 to 0.075 ng/mL. The within- and between-day variation in pooled quality control samples range from 2.5 to 9.0% and from 3.2 to 16%, respectively, for all analytes at concentrations ranging from 0.6 to 12 ng/mL. Precision was similar with samples fortified with 0.1 and 0.25 ng/mL that were analyzed in each run. We validated our selective method against a less selective method used previously in our laboratory by analyzing human specimens using both methods. The methods produced results that were in agreement, with no significant bias observed.     

Analysis of the Salmonella typhimurium proteome through environmental response toward infectious conditions

Salmonella enterica serovar Typhimurium (also known as Salmonella typhimurium) is a facultative intracellular pathogen that causes approximately 8,000 reported cases of acute gastroenteritis and diarrhea each year in the United States. Although many successful physiological, biochemical, and genetic approaches have been taken to determine the key virulence determinants encoded by this organism, the sheer number of uncharacterized reading frames observed within the S. enterica genome suggests that many more virulence factors remain to be discovered. We used a liquid chromatography-mass spectrometry-based "bottom-up" proteomic approach to generate a more complete picture of the gene products that S. typhimurium synthesizes under typical laboratory conditions as well as in culture media that are known to induce expression of virulence genes. When grown to logarithmic phase in rich medium, S. typhimurium is known to express many genes that are required for invasion of epithelial cells. Conversely stationary phase cultures of S. typhimurium express genes that are needed for both systemic infection and growth within infected macrophages. Lastly bacteria grown in an acidic, magnesium-depleted minimal medium (MgM) designed to mimic the phagocytic vacuole have been shown to up-regulate virulence gene expression. Initial comparisons of protein abundances from bacteria grown under each of these conditions indicated that the majority of proteins do not change significantly. However, we observed subsets of proteins whose expression was largely restricted to one of the three culture conditions. For example, cells grown in MgM had a higher abundance of Mg(2+) transport proteins than found in other growth conditions. A second more virulent S. typhimurium strain (14028) was also cultured under these same growth conditions, and the results were directly compared with those obtained for strain LT2. This comparison offered a unique opportunity to contrast protein populations in these closely related bacteria. Among a number of proteins displaying a higher abundance in strain 14028 were the products of the pdu operon, which encodes enzymes required for propanediol utilization. These pdu operon proteins were validated in culture and during macrophage infection. Our work provides further support for earlier observations that suggest pdu gene expression contributes to S. typhimurium pathogenesis.     

In utero-initiated cancer: the role of reactive oxygen species

It is becoming more evident that not only can drugs and environmental chemicals interfere with normal fetal development by causing structural malformations, such as limb defects, but that xenobiotic exposure during development can also cause biochemical and functional abnormalities that may ultimately lead to cancer later on in life. Fetal toxicity may be partly mediated by the embryonic bioactivation of xenobiotics to free radical intermediates that can lead to oxidative stress and potentially lead, in some cases, to carcinogenesis. Using a number of examples, this review will focus on the role of reactive oxygen species (ROS) in the mechanisms pertaining to in utero initiated cancers.     

Mutation analysis of the natriuretic peptide precursor B (NPPB) gene in patients with hypertrophic cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a genetically heterogenous disease caused by mutations in genes that primarily encode sarcomeric proteins. No mutation is identified in up to 40% of HCM patients, suggesting other causative genes exist. Natriuretic peptide precursor B (NPPB; also known as "BNP") is a cardiac hormone involved in body fluid homeostasis and cardiac myocyte growth. NPPB concentrations are markedly increased in patients with ventricular hypertrophy, and it is therefore possible mutations in the NPPB gene could cause HCM. METHODS: Genomic DNA was extracted from peripheral blood in 238 consecutive probands with HCM. The coding regions and intron/exon boundaries in the NPPB gene were amplified by PCR, and products were screened for sequence variants using high-performance liquid chromatography, followed by direct DNA sequencing. RESULTS: Four sequence variants in the NPPB gene were identified in 9 of the 238 probands screened. Two of the variants were intronic, one was a synonymous variant at codon 79, and the final variant resulted in an amino acid substitution from arginine to histidine at codon 47 (Arg47His). The Arg47His variant was identified in a control population consisting of 204 chromosomes at an allelic frequency of 0.5%, and is therefore unlikely to cause disease. CONCLUSION: No disease causing mutations were identified in the NPPB gene in this cohort, indicating that mutations in this gene are unlikely to be responsible for HCM.