Biotic indirect effects: a neglected concept in invasion biology

Indirect effects involve more than two species and are defined as how one species alters the effect that another species has on a third. These complex interactions are often overlooked in studies of interactions between alien and native species, and their role in influencing biological invasions has been rarely considered. Based on a comprehensive review of the invasion biology literature, we examine the evidence for the occurrence of four of the most commonly documented indirect effects (apparent competition, indirect mutualism/commensalism, exploitative competition, and trophic cascades) in the invasion process. Studies investigating indirect effects in the context of invasion biology are relatively rare, but have been increasing in recent years, and there are sufficient examples to indicate that this kind of interaction is likely to be more common than is currently recognized. Whether indirect interactions are mediated by an alien or a native species, and whether they occur between ecologically similar or dissimilar alien and native species, depends in part on the type of interaction considered and no predictable patterns were detected in the literature. Further empirical studies will help to elucidate such patterns. At this stage, the inherent unpredictability of indirect interactions means that their impacts in relation to invasions are particularly challenging for land managers to deal with, and their role in invasions is a complex, but is a valuable area of investigation for researchers.     

Crystal structure of ferric-yersiniabactin, a virulence factor of Yersinia pestis

Yersiniabactin (Ybt), the siderophore produced by Yersinia pestis, has been crystallized successfully in the ferric complex form and the crystal structure has been determined. The crystals are orthorhombic with a space group of P2(1)2(1)2(1) and four distinct molecules per unit cell with cell dimensions of a=11.3271(+/-0.0003)A, b=22.3556(+/-0.0006)A, and c=39.8991(+/-0.0011)A. The crystal structure of ferric Ybt shows that the ferric ion is coordinated as a 1:1 complex by three nitrogen electron pairs and three negatively charged oxygen atoms with a distorted octahedral coordination. The molecule displays a Delta absolute configuration with chiral centers at N2, C9, C10, C12, C13, and C19 in R, R, R, R, S, S configurations, respectively. Few of the crystal structures of siderophores have been solved, and those which have been are of simple hydroxamate and catechol types such as ferrioxamine B and agrobactin. To our knowledge this is the first report of the ferric crystal structure of 5-member heterocycle siderophore.     

Isolation and characterization of cholangiocyte primary cilia

Primary cilia are distinct organelles expressed by many vertebrate cells, including cholangiocytes; however, their functions remain obscure. To begin to explore the physiological role of these organelles in the liver, we described the morphology and structure of cholangiocyte cilia and developed new approaches for their isolation. Primary cilia were present only in bile ducts and were not observed in hepatocytes or in hepatic arterial or portal venous endothelial cells. Each cholangiocyte possesses a single cilium that extends from the apical membrane into the bile duct lumen. In addition, the length of the cilia was proportional to the bile duct diameter. We reproducibly isolated enriched fractions of cilia from normal rat and mouse cholangiocytes by two different approaches as assessed by scanning electron, transmission electron, and confocal microscopy. The purity of isolated ciliary fractions was further analyzed by Western blot analysis using acetylated tubulin as a ciliary marker and P2Y(2) as a nonciliary cell membrane marker. These novel techniques produced enriched ciliary fractions of sufficient purity and quantity for light and electron microscopy and for biochemical analyses. They will permit further assessment of the role of primary cilia in normal and pathological conditions.     

Transmembrane segment 7 of human P-glycoprotein forms part of the drug-binding pocket

P-gp (P-glycoprotein; ABCB1) protects us by transporting a broad range of structurally unrelated compounds out of the cell. Identifying the regions of P-gp that make up the drug-binding pocket is important for understanding the mechanism of transport. The common drug-binding pocket is at the interface between the transmembrane domains of the two homologous halves of P-gp. It has been shown in a previous study [Loo, Bartlett and Clarke (2006) Biochem. J. 396, 537-545] that the first transmembrane segment (TM1) contributed to the drug-binding pocket. In the present study, we used cysteine-scanning mutagenesis, reaction with an MTS (methanethiosulfonate) thiol-reactive analogue of verapamil (termed MTS-verapamil) and cross-linking analysis to test whether the equivalent transmembrane segment (TM7) in the C-terminal-half of P-gp also contributed to drug binding. Mutation of Phe728 to cysteine caused a 4-fold decrease in apparent affinity for the drug substrate verapamil. Mutant F728C also showed elevated ATPase activity (11.5-fold higher than untreated controls) after covalent modification with MTS-verapamil. The activity returned to basal levels after treatment with dithiothreitol. The substrates, verapamil and cyclosporin A, protected the mutant from labelling with MTS-verapamil. Mutant F728C could be cross-linked with a homobifunctional thiol-reactive cross-linker to cysteines I306C(TM5) and F343C(TM6) that are predicted to line the drug-binding pocket. Disulfide cross-linking was inhibited by some drug substrates such as Rhodamine B, calcein acetoxymethyl ester, cyclosporin, verapamil and vinblastine or by vanadate trapping of nucleotides. These results indicate that TM7 forms part of the drug-binding pocket of P-gp.     

A reassessment of copper(II) binding in the full-length prion protein

It has been shown previously that the unfolded N-terminal domain of the prion protein can bind up to six Cu2+ ions in vitro. This domain contains four tandem repeats of the octapeptide sequence PHGGGWGQ, which, alongside the two histidine residues at positions 96 and 111, contribute to its Cu2+ binding properties. At the maximum metal-ion occupancy each Cu2+ is co-ordinated by a single imidazole and deprotonated backbone amide groups. However two recent studies of peptides representing the octapeptide repeat region of the protein have shown, that at low Cu2+ availability, an alternative mode of co-ordination occurs where the metal ion is bound by multiple histidine imidazole groups. Both modes of binding are readily populated at pH 7.4, while mild acidification to pH 5.5 selects in favour of the low occupancy, multiple imidazole binding mode. We have used NMR to resolve how Cu2+ binds to the full-length prion protein under mildly acidic conditions where multiple histidine co-ordination is dominant. We show that at pH 5.5 the protein binds two Cu2+ ions, and that all six histidine residues of the unfolded N-terminal domain and the N-terminal amine act as ligands. These two sites are of sufficient affinity to be maintained in the presence of millimolar concentrations of competing exogenous histidine. A previously unknown interaction between the N-terminal domain and a site on the C-terminal domain becomes apparent when the protein is loaded with Cu2+. Furthermore, the data reveal that sub-stoichiometric quantities of Cu2+ will cause self-association of the prion protein in vitro, suggesting that Cu2+ may play a role in controlling oligomerization in vivo.     

Common chromatin structures at breakpoint cluster regions may lead to chromosomal translocations found in chronic and acute leukemias

The t(9;22) BCR/ABL fusion is associated with over 90% of chronic myelogenous and 25% of acute lymphocytic leukemia. Chromosome 11q23 translocations in acute myeloid and lymphoid leukemia cells demonstrate myeloid lymphoid leukemia (MLL) fusions with over 40 gene partners, like AF9 and AF4 on chromosomes 9 and 4, respectively. Therapy-related leukemia is associated with the above gene rearrangements following the treatment with topoisomerase II (topo II) inhibitors. BCR, ABL, MLL, AF9 and AF4 have defined patient breakpoint cluster regions. Chromatin structural elements including topo II and DNase I cleavage sites and scaffold attachment sites have previously been shown to closely associate with the MLL and AF9 breakpoint cluster regions, implicating these elements in non-homologous recombination (NHR). In this report, using cell lines and primary cells, chromatin structural elements were analyzed in BCR, ABL and AF4 and, for comparison, in MLL2, which is a homolog to MLL, but not associated with chromosome translocations. Topo II and DNase I cleavage sites associated with all breakpoint cluster regions, whereas SARs associated with ABL and AF4, but not with BCR. No close breakpoint clustering with the topo II/DNase I sites were observed; however, a statistically significant 5′ or 3′ distribution of patient breakpoints to the topo II DNase I sites was found, implicating DNA repair and exonucleases. Although MLL2 was expressed in all cell lines tested, except for the presence of one DNAse I site in the promoter, no other structural elements were found in MLL2. A NHR model presented demonstrates the importance of chromatin structure in chromosome translocations involved with leukemia.     

Humanization of a recombinant monoclonal antibody to produce a therapeutic HER dimerization inhibitor, pertuzumab

Dimerization is essential for activity of human epidermal growth factor receptors (HER1/EGFR, HER2/ErbB2, HER3/ErbB3, and ErbB4) and mediates intracellular signaling events leading to cancer cell proliferation, survival, and resistance to therapy. HER2 is the preferred dimerization partner. Activation of HER signaling pathways may be blocked by inhibition of dimer formation using a monoclonal antibody (MAb) directed against the dimerization domain of HER2. The murine MAb 2C4 that specifically binds the HER2 dimerization domain was cloned as a chimeric antibody, humanized using a computer-generated model to guide framework substitutions, and variants were tested as Fabs. Pharmacokinetics and toxicology were evaluated in rodents and cynomolgus monkeys. Cloning the variable domains of MAb 2C4 into a vector containing human kappa and CH1 domains allowed construction of a mouse-human chimeric Fab. DNA sequencing of the chimeric clone permitted identification of CDR residues. The full-length IgG1 of variant F-10 was equivalent in binding to chimeric IgG1 and was designated pertuzumab (rhuMAb 2C4; Omnitarg). Pertuzumab pharmacokinetics was best described by a two-compartment model with a distribution phase of <1 day, terminal half-life of ~10 days, and volume of distribution of ~40 mL/kg that approximates serum volume. With the exception of diarrhea, pertuzumab was generally well tolerated in cynomolgus monkeys. Pertuzumab, a recombinant humanized IgG1 MAb, is the first of a new class of agents known as HER dimerization inhibitors. Inhibition of HER dimerization may be an effective anticancer strategy in tumors with either normal or elevated expression of HER2.     

Non-linear chromosomal inversion response in prostate after low dose X-radiation exposure

Somatic intrachromosomal recombination can result in inversions and deletions in DNA, which are important mutations in cancer. The pKZ1 chromosomal inversion assay is a sensitive assay for studying the effects of DNA damaging agents using chromosomal inversion as a mutation end-point. We have previously demonstrated that the chromosomal inversion response in pKZ1 spleen after single low doses of X-radiation exposure does not follow the linear no-threshold dose–response model. Here, we optimised a chromosomal inversion screening method to study the effect of low dose X-radiation exposure in pKZ1 prostatic tissue. In the present study, a significant induction in inversions was observed after ultra-low doses of 0.005–0.01 mGy or after a high dose of 1000 mGy, whereas a reduction in inversions to below the sham-treated frequency was observed between 1 and 10 mGy exposure. This is the first report of a reduction to below endogenous frequency for any mutation end-point in prostate. In addition, the doses of radiation studied were at least three orders of magnitude lower than have been reported in other mutation assays in prostate in vivo or in vitro. In sham-treated pKZ1 controls and in pKZ1 mice treated with low doses of 1–10 mGy the number of inversions/gland cross-section rarely exceeded three. Up to 4 and 7 inversions were observed in individual prostatic gland cross-sections after doses ≤0.02 mGy and after 1000 mGy, respectively. The number of inversions identified in individual cross-sections of prostatic glands of untreated mice and all treated mice other than the 1000 mGy treatment group followed a Poisson distribution. The dose–response curves and fold changes observed after all radiation doses studied were similar in spleen and prostate. These results suggest that the pKZ1 assay is measuring a fundamental response to DNA damage after low dose X-radiation exposure which is independent of tissue type.     

Variations in soil N cycling and trace gas emissions in wet tropical forests

We used a previously described precipitation gradient in a tropical montane ecosystem of Hawai’i to evaluate how changes in mean annual precipitation (MAP) affect the processes resulting in the loss of N via trace gases. We evaluated three Hawaiian forests ranging from 2200 to 4050 mm year⁻¹ MAP with constant temperature, parent material, ecosystem age, and vegetation. In situ fluxes of N₂O and NO, soil inorganic nitrogen pools (NH₄⁺ and NO₃⁻), net nitrification, and net mineralization were quantified four times over 2 years. In addition, we performed ¹⁵N-labeling experiments to partition sources of N₂O between nitrification and denitrification, along with assays of nitrification potential and denitrification enzyme activity (DEA). Mean NO and N₂O emissions were highest at the mesic end of the gradient (8.7±4.6 and 1.1±0.3 ng N cm⁻² h⁻¹, respectively) and total oxidized N emitted decreased with increased MAP. At the wettest site, mean trace gas fluxes were at or below detection limit (≤0.2 ng N cm⁻² h⁻¹). Isotopic labeling showed that with increasing MAP, the source of N₂O changed from predominately nitrification to predominately denitrification. There was an increase in extractible NH₄⁺ and decline in NO₃⁻, while mean net mineralization and nitrification did not change from the mesic to intermediate sites but decreased dramatically at the wettest site. Nitrification potential and DEA were highest at the mesic site and lowest at the wet site. MAP exerts strong control N cycling processes and the magnitude and source of N trace gas flux from soil through soil redox conditions and the supply of electron donors and acceptors.