An assessment of the breeding range,colony sizes and population of the Westland petrel (Procellaria westlandica)

Abstract

The Westland petrel (Procellaria westlandica) is an endemic New Zealand species and one of the very few burrowing seabird species still breeding on mainland New Zealand. It nests only on a series of coastal ridgelines near to Punakaiki on the West Coast of the South Island. Between 2002 and 2005, surveys were undertaken at 28 of the 29 known colonies. The area occupied by the colonies was 73 ha; most colonies had fewer than 50 burrows, but six colonies had 201–500 burrows and four colonies had more than 1000 burrows. We find that the current breeding range of Westland petrel and the location of individual colonies are similar to those reported in both the 1950s and 1970s. Based on total burrow counts at 28 colonies and burrow occupancy rates determined by annual monitoring, the annual breeding population is estimated to be between 2954 and 5137 breeding pairs.     

Bronchoabsorption; a novel bronchoscopic technique to improve biomarker sampling of the airway

Background

Current techniques used to obtain lung samples have significant limitations and do not provide reproducible biomarkers of inflammation. We have developed a novel technique that allows multiple sampling methods from the same area (or multiple areas) of the lung under direct bronchoscopic vision. It allows collection of mucosal lining fluid and bronchial brushing from the same site; biopsy samples may also be taken. The novel technique takes the same time as standard procedures and can be conducted safely.

Methods

Eight healthy smokers aged 40–65 years were included in this study. An absorptive filter paper was applied to the bronchial mucosa under direct vision using standard bronchoscopic techniques. Further samples were obtained from the same site using bronchial brushings. Bronchoalveolar lavage (BAL) was obtained using standard techniques. Chemokine (C-C Motif) Ligand 20 (CCL20), CCL4, CCL5, Chemokine (C-X-C Motif) Ligand 1 (CXCL1), CXCL8, CXCL9, CXCL10, CXCL11, Interleukin 1 beta (IL-1β), IL-6, Vascular endothelial growth factor (VEGF), Matrix metalloproteinase 8 (MMP-8) and MMP-9 were measured in exudate and BAL. mRNA was collected from the bronchial brushings for gene expression analysis.

Results

A greater than 10 fold concentration of all the biomarkers was detected in lung exudate in comparison to BAL. High yield of good quality RNA with RNA integrity numbers (RIN) between 7.6 and 9.3 were extracted from the bronchial brushings. The subset of genes measured were reproducible across the samples and corresponded to the inflammatory markers measured in exudate and BAL.

Conclusions

The bronchoabsorption technique as described offers the ability to sample lung fluid direct from the site of interest without the dilution effects caused by BAL. Using this method we were able to successfully measure the concentrations of biomarkers present in the lungs as well as collect high yield mRNA samples for gene expression analysis from the same site. This technique demonstrates superior sensitivity to standard BAL for the measurement of biomarkers of inflammation. It could replace BAL as the method of choice for these measurements. This method provides a systems biology approach to studying the inflammatory markers of respiratory disease progression.

Trial registration

NHS Health Research Authority (13/LO/0256).     

G protein‐coupled estrogen receptor‐mediated effects on cytosolic calcium and nanomechanics in brain microvascular endothelial cells

G protein‐coupled estrogen receptor (GPER) is a relatively recently identified non‐nuclear estrogen receptor, expressed in several tissues, including brain and blood vessels. The mechanisms elicited by GPER activation in brain microvascular endothelial cells are incompletely understood. The purpose of this work was to assess the effects of GPER activation on cytosolic Ca²⁺ concentration, [Ca²⁺]_i, nitric oxide production, membrane potential and cell nanomechanics in rat brain microvascular endothelial cells (RBMVEC). Extracellular but not intracellular administration of G‐1, a selective GPER agonist, or extracellular administration of 17‐β‐estradiol and tamoxifen, increased [Ca²⁺]_i in RBMVEC. The effect of G‐1 on [Ca²⁺]_i was abolished in Ca²⁺‐free saline or in the presence of a L‐type Ca²⁺ channel blocker. G‐1 increased nitric oxide production in RBMVEC; the effect was prevented by NG‐nitro‐l ‐arginine methyl ester. G‐1 elicited membrane hyperpolarization that was abolished by the antagonists of small and intermediate‐conductance Ca²⁺‐activated K⁺ channels, apamin, and charibdotoxin. GPER‐mediated responses were sensitive to G‐36, a GPER antagonist. In addition, atomic force microscopy studies revealed that G‐1 increased the modulus of elasticity, indicative of cytoskeletal changes and increase in RBMVEC stiffness. Our results unravel the mechanisms underlying GPER‐mediated effects in RBMVEC with implications for the effect of estrogen on cerebral microvasculature.

     

Interdomain salt‐bridges in the Ebola virus protein VP40 and their role in domain association and plasma membrane localization

The Ebola virus protein VP40 is a transformer protein that possesses an extraordinary ability to accomplish multiple functions by transforming into various oligomeric conformations. The disengagement of the C‐terminal domain (CTD) from the N‐terminal domain (NTD) is a crucial step in the conformational transformations of VP40 from the dimeric form to the hexameric form or octameric ring structure. Here, we use various molecular dynamics (MD) simulations to investigate the dynamics of the VP40 protein and the roles of interdomain interactions that are important for the domain–domain association and dissociation, and report on experimental results of the behavior of mutant variants of VP40. The MD studies find that various salt‐bridge interactions modulate the VP40 domain dynamics by providing conformational specificity through interdomain interactions. The MD simulations reveal a novel salt‐bridge between D45‐K326 when the CTD participates in a latch‐like interaction with the NTD. The D45‐K326 salt‐bridge interaction is proposed to help domain–domain association, whereas the E76‐K291 interaction is important for stabilizing the closed‐form structure. The effects of the removal of important VP40 salt‐bridges on plasma membrane (PM) localization, VP40 oligomerization, and virus like particle (VLP) budding assays were investigated experimentally by live cell imaging using an EGFP‐tagged VP40 system. It is found that the mutations K291E and D45K show enhanced PM localization but D45K significantly reduced VLP formation.     

Membrane topology of NAADP-sensitive two-pore channels and their regulation by N-linked glycosylation

Two-pore channels (TPCs) localize to the endolysosomal system and have recently emerged as targets for the Ca(2+)-mobilizing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). However, their membrane topology is unknown. Using fluorescence protease protection assays, we show that human TPC1 and TPC2 possess cytosolic N and C termini and therefore an even number of transmembrane regions. Fluorophores placed at position 225 or 347 in TPC1, or 339 in TPC2 were also cytosolic, whereas a fluorophore at position 628 in TPC1 was luminal. These data together with sequence similarity to voltage-gated Ca(2+) and Na(+) channels, and unbiased in silico predictions are consistent with a topology in which two homologous domains are present, each comprising 6 transmembrane regions and a re-entrant pore loop. Immunocytochemical analysis of selectively permeabilized cells using antipeptide antibodies confirmed that the C-terminal tails of recombinant TPCs are cytosolic and that residues 240-254 of TPC2 prior to putative pore 1 are luminal. Both TPC1 and TPC2 are N-glycosylated with residues 599, 611, and 616 contributing to glycosylation of TPC1. This confirms the luminal position of these residues, which immediately precede the putative pore loop of the second domain. Mutation of all three glycosylation sites in TPC1 enhances NAADP-evoked cytosolic Ca(2+) signals. Our data establish essential features of the topology of two-pore channels.     

An NAADP-gated two-pore channel targeted to the plasma membrane uncouples triggering from amplifying Ca2+ signals

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a ubiquitous messenger proposed to stimulate Ca(2+) release from acidic organelles via two-pore channels (TPCs). It has been difficult to resolve this trigger event from its amplification via endoplasmic reticulum Ca(2+) stores, fuelling speculation that archetypal intracellular Ca(2+) channels are the primary targets of NAADP. Here, we redirect TPC2 from lysosomes to the plasma membrane and show that NAADP evokes Ca(2+) influx independent of ryanodine receptors and that it activates a Ca(2+)-permeable channel whose conductance is reduced by mutation of a residue within a putative pore. We therefore uncouple TPC2 from amplification pathways and prove that it is a pore-forming subunit of an NAADP-gated Ca(2+) channel.     

Presynaptic mechanism of action induced by 5-HT in nerve terminals: possible involvement of ryanodine and IP3 sensitive 2+ stores

Although modulation of transmitter release by serotonin (5-HT) at crayfish neuromuscular junctions has been known since 1965, the mechanisms of action have not been established in this classical synaptic preparation. We show that injections of adenophostin-A (an IP3 analog) in the nerve terminals greatly enhances synaptic transmission. Exposure to ryanodine (Ry) produces a biphasic response: at low concentration it is excitatory and high concentration it is inhibitory. Likewise, a low concentration (1 microM) of caffeine enhances synaptic transmission, whereas a high concentration (10 mM) has little effect on transmission. The varied responses and sensitivity to Ry and caffeine suggest a Ca(2+)-induced Ca(2+)-release mechanism and/or the presence of an IP3-receptor within the terminal. Thus, it is likely 5-HT's response is due to activation of intracellular pathways, which subsequently release Ca2+ from internal stores.