We here study a general problem in cellular Ca2+-sensing, namely how similar Ca2+-binding proteins achieve functional selectivity to control finely adjusted cellular responses. We investigated two parameters of critical importance for the trigger and switch function of guanylate cyclase-activating proteins: the myristoylation status and the occupation of Ca2+-binding sites with Mg2+. All zGCAPs can be myristoylated in living cells using click chemistry. Myristoylation does not facilitate membrane binding of zGCAPs, but it significantly modified the regulatory properties of zGCAP2 and zGCAP5. We further determined for all zGCAPs at least two binding sites exhibiting high affinities for Ca2+ with KD values in the submicromolar range, whereas for other zGCAPs (except zGCAP3) the affinity of the third binding site was in the micromolar range. Mg2+ either occupied the low affinity Ca2+-binding site or it shifted the affinities for Ca2+-binding. Hydrodynamic properties of zGCAPs are more influenced by Ca2+ than by Mg2+, although to a different extent for each zGCAP. Posttranslational modification and competing ion-binding can tailor the properties of similar Ca2+-sensors.Zebrafish photoreceptor cells express six guanylate cyclase-activating proteins (zGCAPs) that share a high degree of amino acid sequence homology, but differ in Ca One family of Ca2+-binding proteins named neuronal calcium sensor (NCS) proteins are predominantly expressed in neuronal tissue and are involved in diverse intracellular processes22+-binding motifs, of which in most cases three (sometimes only two) motifs can bind micromolar to submicromolar Ca2+. One group of the NCS proteins is expressed in sensory cells and among them the guanylate cyclase-activating proteins (GCAPs) perform an important function in controlling the membrane bound guanylate cyclases (GCs) in retinal rod and cone cells45Calcium sensor proteins mediate signaling processes that respond to changing concentrations of Ca2+-free, Mg2+-bound form GCAPs activate GCs, but they switch to an inhibitory mode, when all Ca2+-binding sites are filled with Ca2+72+ in rod and cone outer segments are linked to changing levels of the intracellular messenger cGMP. After light activation of the photoreceptor cell the intracellular cGMP level is depleted, leading to a shutdown of cyclic nucleotide gated (CNG) channels in the outer segment of the cell. This stops the influx of Ca2+, which is however still extruded by the continuous operation of a Na+/ Ca2+, K+ exchanger leading to a net decrease of cytoplasmic Ca2+. This decrease is sensed by GCAPs which in turn increase the GC activity, leading to re-opening of the CNG-channels and is a necessary step for the recovery of the photoreceptor to the dark-adapted state45678In their Ca2+-sensitivity, impact on catalytic efficiency of the target GC and different structural implications of the N-terminally attached myristoyl group102+-relay mode fashion, where GCAP1 is activated at higher free Ca2+, followed by GCAP2, which becomes active, when Ca2+-levels have fallen to lower levels9102+-relay system seems also to work in zebrafish rod and cone cells, where, however, the system is more complex due to the larger number of GCAP forms132+-binding properties, Ca2+-sensitive GC regulation and spatial-temporal transcription/ expression profiles. Four zGCAPs, namely isoforms 3, 4, 5 and 7 are cone specific1214Bovine and mice photoreceptor cells express two GCAP forms, GCAP1 and GCAP2, which bind to distant regions in the target GC and have different properties with respect to Ca2+-binding sites with Mg2+781016N-myristoyltransferase (NMT) in E.coli15in vivo and in vitro click chemistry in combination with fluorescence microscopy. Revealing that all zGCAPs can exist in a myristoylated and non-myristoylated form we investigated its impact for target regulation and Ca2+-dependent membrane interaction. We further asked whether the presence of physiological Mg2+ can influence the binding of Ca2+ to zGCAPs and how Ca2+-induced conformational transitions in zGCAPs are influenced.Two parameters are of critical importance for the trigger and switch function of NCS proteins in general and GCAPs in particular: the myristoylation status and the occupation of EF-hand Ca2+-dependent membrane binding for all zGCAPs. Further Mg2+ ions control the Ca2+-affinity as well as the Ca2+-induced conformational changes in zGCAPs.Our results indicate that myristoylation has a strong impact on the regulatory properties of two zGCAPs (2 and 5), but it does not facilitate CaGreen-fluorescent protein (GFP) constructs of NCS proteins including all zGCAPs, bovine GCAP2 and recoverin were used to transfect HEK 293 cells, which were also supplemented with azido-dodecyl acid (a myristoyl substitute). This allowed us to incorporate an acyl moiety into an NCS protein in a living cell. Successful attachment of the acyl group was monitored in a subsequent copper (I)-catalyzed azide-alkyne cycloaddition using a biotin alkyne derivative forming a triazole ring. Thus, NCS proteins that were labeled with biotin could be detected via streptavidin coupled to peroxidase. Each transfected cell sample was analyzed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis, blot transfer and presence of biotin in the protein band was visualized by peroxidase staining . Since GN-terminal attached fatty acyl chain and the C-terminal attached GFP to the protein in transfected cells. 192+-concentration, which had been observed in previous studies and is mainly due to hydrophobic/ electrostatic interactions2122In a second alternative approach we employed a copperless cycloaddition suitable for introducing a fluorescent dye (DIBO-TAMRA-dye) to the fatty acyl group of the NCS proteins in living cells. Thus we were able to colocalize the putative 2+-myristoyl switch, which is typically observed in other NCS proteins like recoverin, neurocalcin δ, VILIP or hippocalcin242526E.coli and purified them afterwards. Principal attachment of the myristoyl group was verified by a click chemistry reaction involving azido-dodecanoic acid and the alkyne derivative of biotin as described above. We then incubated myristoylated zGCAPs and recoverin with isolated photoreceptor outer segment membranes in the absence and presence of Ca2+ , which was tested in living and in disrupted cells. This prompted us to ask, whether zGCAPs can perform a Ca of Ca2+ . No zGCA control .2+] at which the GC activity in the presence of a GCAP molecule is halfmaximal denoted as IC50 value and listed in 2+-sensitivity decreased about 4.7-fold for zGCAP2 and increased 5.7-fold for zGCAP5.Nonmyristoylated zGCAPs exhibit different activity profiles when targeting membrane bound GCs2+ under physiological conditions as it was observed for mammalian GCAP1 and 28. So far no precise values of Ca2+-binding to myristoylated zGCAPs are available and furthermore it is not known, whether Ca2+-binding is affected by physiological concentrations of Mg2+. We used a calorimetric approach (ITC) to analyze the energetics of Ca2+-binding to zGCAPs in the presence and absence of 1 mM Mg2+, which allows us to determine precise values of apparent dissociation constants (KD) for multiple binding sites and the associated changes in binding enthalpy (ΔH). For each titration Ca2+-free zGCAP was kept in a temperature controlled compartment, in which a series of small volumes of CaCl2 was injected. Representative examples for all zGCAPs are shown in 2 (zGCAP3) and −17 kcal per mol of CaCl2 (zGCAP7). Small endothermic responses were only observed with zGCAP1  showed high affinity for Ca2+, whereas for other zGCAPs the affinity of KD3 was in the micromolar range.All zGCAPs contain four EF-hand motifs in their primary structure, where the first one is suggested to bind no Cah zGCAP1 a,5a. Howccessful . We inteplicable . In all 2+the multiphasic binding isotherm for Ca2+-binding gave a best fit with a two site model for zGCAP1, 2, and 3. This result can be best interpreted as having two high affinity sites filled with Ca2+, but the lower affinity site being occupied by Mg2+ .Size exclusion chromatography of myristoylated zGCAPs revealed that the monomeric form was the dominant species for all zGCAPs except for zGCAP5 and 7. A Ca2+-myristoyl switch operation in zGCAPs  differed significantly among the proteins exhibiting the following order: zGCAP5 > zGCAP2 > zGCAP7 > zGCAP3 = zGCAP4. No signals were observed for zGCAP1, although the purified protein was functional shown by the GC activation assay.Finally, the maximal amplitudes that were reached at the end of the Ca2+-induced conformational changes, which however have different consequences for the protein hydrodynamic properties indicating differences in the extent of conformational transitions.We conclude from these results that all zGCAPs undergo Ca2+-signaling that result in specific cellular responses can be mediated by rather similar Ca2+-sensor proteins, which are very often expressed in the same cell type. In order to work on these issues we chose to compare zGCAPs representing one subfamily of NCS proteins. These NCS proteins are well suited for a comparative analysis, since previous work has shown 1) that all zGCAPs are expressed in photoreceptor cells in the larval and adult stages of the zebrafish retina1314152+-binding proteins12152+-dependent manner12131516Calcium sensor proteins like the group of NCS proteins are involved in unique patterns of cellular regulatory pathways and therefore mediate various physiological responses including ion channel function, enzyme activity control and cellular trafficking22+-sensitivity of mammalian GCAP1, but less influence on the regulatory properties of mammalian GCAP21029in vitro conditions. We here demonstrate using complementary approaches that the six zGCAPs are myristoylated in cell lysates and in living cells. Further, myristoylation is not involved in reversible Ca2+-dependent membrane association  leading to one group of zGCAPs with high Ca2+-sensitivity  and one with low Ca2+-sensitivities . Myristoylation is catalyzed by a retinal NMT, which is not active before 7 dpf due the myristoylation pattern we reported previously for zGCAP3Our previous work on the six nonmyristoylated zGCAPs revealed that zGCAP1, 2 and 3 display GC-activating response curves that are halfmaximal around 30 nM free  was done exactly as described121515Analysis of purified zGCAP samples by sodium dodecyl-sulfate polyacrylamide gel electrophoresis and determination of GC activity (three to five repetitions) in the presence of zGCAPs as a function of the free [Ca2 stock solution at T = 25 °C. The titration buffer was decalcified using a self-packed gravity flow Chelex 100 column (Bio-Rad). The remaining Ca2+concentration was determined by a BAPTA absorption assay and was found to range between 30 and 100 nM. All buffers were filtered (0.22 μm) and degassed at least 1h before use. At least three independent repetitions were made for each titration set, if not stated otherwise.ITC experiments with zGCAP-isoforms were performed on a VP-ITC from MicroCal  exactly as described previously for mammalian GCAP1 variants2+ into decalcified buffer without any zGCAP was performed, but did not show significant heat changes in the recording cell. Each titration was analyzed by a model implemented in the software Origin  assuming either three or two Ca2+ binding sites. The best fitting results out of these models were used to obtain dissociation constants KDapp and enthalpy changes (ΔH).Reference injections of Ca2+-titration experiments and data analysis were performed as outlined elsewhere30312 of the highest grade available was dissolved in the decalcified buffer to a final concentration of 46 mM. This stock solution was used to obtain Ca2+ samples of 0.4 μM, 0.7 μM, 0.9 μM, 1.1 μM, 1.6 μM, 2.5 μM, 4.8 μM, 14 μM, 37 μM and 46.2 μM. All buffers were filtered (0.22 μm) and degassed for at least 1h before use. Immobilization of protein samples was achieved by attaching them to the carboxy-methyl dextran matrix of CM5 sensorchips  via the terminal amino group or via internal accessible lysine residues. Typical immobilization densities ranged from 3.5 ng to 10.5 ng/mm2.For SPR experiments, we used exactly the same decalcified buffer conditions as for the ITC experiments, except that Tween20 was added to a final concentration of 0.005% (v/v).The CaHow to cite this article: Sulmann, S. et al. Retina specific GCAPs in zebrafish acquire functional selectivity in Ca2+-sensing by myristoylation and Mg2+-binding. Sci. Rep.5, 11228; doi: 10.1038/srep11228 (2015).


Circulating urotensin II (UII) is involved in the development of atherosclerosis. However, the role of autocrine UII in the development of atherosclerosis remains unclear. Here, we tested the hypothesis that autocrine UII would promote atherosclerosis. Transgenic rabbits were created as a model to study macrophage-specific expressing human UII (hUII) and used to investigate the role of autocrine UII in the development of atherosclerosis. Transgenic rabbits and their nontransgenic littermates were fed a high cholesterol diet to induce atherosclerosis. Comparing the transgenic rabbits with their nontransgenic littermates, it was observed that hUII expression increased the macrophage-positive area in the atherosclerotic lesions by 45% and the positive area ratio by 56% in the transgenic rabbits. Autocrine hUII significantly decreased the smooth muscle cell-positive area ratio in transgenic rabbits (by 54%), without affecting the plasma levels of total cholesterol, triglycerides, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and glucose and adipose tissue contents. These results elucidated for the first time that autocrine UII plays an important role in the development of atherosclerosis by increasing the accumulation of macrophage-derived foam cell. Atherosclerotic cardiovascular diseases, including heart attack, stroke, and peripheral vascular insufficiency, continue to be the principal cause of death and disability in the world. Vasoactive peptides play an important role in the development of atherosclerosis . UrotensJapanese white rabbits were supplied by the Laboratory Animal Center of Xi'an Jiaotong University. The generation of the transgenic rabbits expressing human UII (hUII) was conducted in our laboratory by microinjection, as described in a previous study . For thePeritoneal macrophages were harvested from the peritoneal cavity 4 days after injection of 4% Fluid's thioglycolate medium. Alveolar macrophages were collected by subjecting the lungs to lavage with 100 mL of phosphate buffered saline . Peritoneal and alveolar macrophages were confirmed based on the criteria of morphology and nonspecific esterase reactivity. The total RNA from various tissues and isolated peritoneal and alveolar macrophages was rapidly isolated using TRIzol reagent  and reverse-transcribed into cDNA using a reverse transcription kit . The expression of hUII in the macrophages, heart, lung, liver, spleen, kidney, and small intestine was evaluated using quantitative real-time PCR. The following sets of primers were used: forward 5′ TTCATCTTATGCTCTGCGTCACTT 3′, reverse 5′ATGTTGGTACTTGAGTCTGCTTTCC3′, 259 bp; rabbit GAPDH forward 5′ATCACTGCCACCCAGAAGAC3′, reverse 5′ GTGAGTTTCCCGTTCAGCTC 3′, 146 bp. The cycling conditions were 95°C for 30 s, followed by 40 cycles of 95°C for 30 s, and 55°C for 40 s.To identify the hUII expression levels in transgenic rabbits, protein samples extracted from alveolar macrophages of the transgenic rabbits and their nontransgenic littermates were prepared, as described previously, and analyzed by electrophoresis on 10% SDS-polyacrylamide gels, followed by Western blotting and probing with a polyclonal antibody against hUII  [n = 5, male) and their nontransgenic littermates  were fed a high cholesterol diet  for 16 weeks to induce atherosclerosis.The hUII transgenic rabbits  to obtain the plasma. The plasma triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and glucose levels were measured using commercial assay kits .At the end of the experiment, rabbits were anesthetized with sodium pentobarbitone. The blood pressure was examined directly via a carotid artery catheter, with a pressure transducer and amplifier attached to a digital PowerLab data acquisition system . The data were collected 10 minutes after the rabbits became calm and there were no blood pressure fluctuations. The blood pressure measurements were calculated using Chart 5 Pro v5.5 software .φ)  and smooth muscle cells (SMCs) . The sections for microscopic quantification were examined and photographed under a microscope equipped with a digital camera  and measured with image analysis software .At the end of the experiment, the rabbits were sacrificed by an overdose of sodium pentobarbitone for the analysis of the atherosclerotic arterial lesions. The aortas were en face stained with Sudan IV  for evaluation of the gross atherosclerotic lesions, as described previously , 14. Briµm thick) were stained routinely by hematoxylin and eosin (H&E).At the end of the experiment, the heart, liver, lung, kidney, and spleen were carefully removed, weighed, and fixed in 10% buffered formalin for subsequent histological examination. Adipose tissue from the subcutaneous  and visceral regions  were collected and weighed-wet . The tist-test for the data with an equal F value or Welch's t-test when the F value was not equal. A p value of less than 0.05 was considered statistically significant.The results were expressed as the means ± SEM. The statistical analysis was performed using Student's In this study, we successfully generated transgenic rabbit macrophage-specific expressing hUII. A total of 769 zygotes of rabbits were microinjected with fragmented hUII cDNA, and 40 pups were born. Two pups were found to be integrated with the transgene by PCR genotyping . The hUIThe plasma TC and TG levels were measured biweekly, and the LDL-C and HDL-C were measured every 4 weeks during the entire period of the experiment. As shown in As shown in The aortas of the rabbits were collected, and the lesions were characterized by the following: (1) Sudan IV staining of the positive area of the aortic arch and thoracic and abdominal aortas; (2) microscopic measurement of the intimal lesion size; and (3) assessment of the lesional cellular components (macrophages and SMCs). The expression of the autocrine exogenetic hUII did not significantly change the gross lesion area in the cholesterol fed rabbits . The hisAs a vasoactive cyclic peptide, circulating UII is involved in the progression of atherosclerosis and identified to be a proatherogenic factor, based on the current information from several laboratories and our previous study –10. The κB pathway and to reduce the cholesterol efflux to promote macrophage-foam cell formation [β [One of the important roles that circulating UII plays in the development of atherosclerosis is to promote macrophage-derived foam cell formation by regulation of the cholesterol metabolism and inflammation molecules , 10, 15.ormation . Circulaation [β –19. UII ation [β . The colThe effect of autocrine hUII was shown more specifically in the promotion of the macrophage-derived foam cell formation than in the circulating UII, without any changes in the plasma lipid levels and blood pressure. The circulating UII was reported to have significant effects on the circulating plasma lipids, blood pressure, weight, visceral fat, proatherogenic cytokines, and glucose tolerance –26. HoweThe results of this study showed that the autocrine hUII changes the cellular components in atherosclerotic lesions by enhancement of the macrophage-derived foam cell formation in transgenic rabbits. To the best of our knowledge, this study was the first to elucidate the role of autocrine UII in the development of atherosclerosis.


The complex spatial and temporal organization of the ensuing intracellular Ca2+ signals allows selective regulation of diverse physiological responses. Interactions of IP3Rs with other proteins contribute to the specificity and speed of Ca2+ signalling pathways, and to their capacity to integrate information from other signalling pathways. In this review, we provide a comprehensive survey of the proteins proposed to interact with IP3Rs and the functional effects that these interactions produce. Interacting proteins can determine the activity of IP3Rs, facilitate their regulation by multiple signalling pathways and direct the Ca2+ that they release to specific targets. We suggest that IP3Rs function as signalling hubs through which diverse inputs are processed and then emerge as cytosolic Ca2+ signals.Inositol 1,4,5‐trisphosphate receptors (IP ACadenylyl cyclase2RBtype 2 bradykinin receptorcAMPcyclic adenosine monophosphateCREBcAMP response element‐binding proteinEB3end‐binding protein 3ERendoplasmic reticulumGPCRG protein‐coupled receptorIBC3‐binding coreIP3IPinositol 1,4,5‐trisphosphate3RIP3 receptorIPIRBIT3R‐binding protein released with IP3IP1RMtype 1 muscarinic acetylcholine receptorPKAprotein kinase APLCphospholipase CSDsuppressor domainTMDtransmembrane domain2+ signals within cells are spatially and temporally intricate, allowing them to elicit a multitude of specific downstream effects , the most widely expressed class of intracellular Ca2+ channel, release Ca2+ from intracellular stores in response to binding of IP3 and Ca2+  . These changes are proposed to disrupt interactions between the N‐terminal regions of the four subunits of the IP3R, leading to opening of the channel. The latter is formed by transmembrane domains (TMDs) towards the C‐terminus of each IP3R subunit , which can form homo‐tetrameric or hetero‐tetrameric channels  , but continued progress and the advent of high‐throughput proteomics methods , which binds directly to both proteins , the cell type and the expression levels of the interacting proteins and IP3Rs. Thirdly, interactions that occur in cellular lysates may be precluded within intact cells. For example, the interaction of two proteins may be prevented by their physical separation within the cell or by mutually exclusive binding of other proteins or ligands. IRBIT, for example, binds to IP3R subunits only when they have no IP3 bound. Lastly, some forms of experimental evidence are more discriminating than others, and it will be necessary to verify the putative interactions indicated by methods such as yeast two‐hybrid screening and mass spectrometry.Searches of proteomic databases and published literature reveal a large number of proteins that form complexes with IPs Tables , 2, 3, 4ped Fig. . At the 3Rs to release Ca2+ from intracellular stores, IP3Rs have additional roles. For example, binding of IP3 is proposed to release IRBIT from the IP3‐binding site, freeing IRBIT to regulate additional targets that include ion channels, transporters and the enzyme ribonucleotide reductase  channels is proposed to stimulate opening of the latter , the epidermal growth factor receptor (EGFR), the erythropoietin receptor, the Gβγ complexes of G proteins, and some forms of PLC. IP3Rs also associate with other signalling proteins linked to PLC signalling, including protein kinase C (PKC), RACK1 (receptor of activated PKC) and the phosphoinositide phosphatase PTEN. The interactions extend also to proteins from other signalling pathways, including adenylyl cyclase (AC), the small G protein K‐Ras, and the protein kinases AKT1 (RAC‐α serine/threonine protein kinase), mTOR , c‐Src and MAPK1/MAPK3 (mitogen‐activated protein kinase 1/3)  for association of messengers with their targets. Rapid de‐activation requires rapid destruction or dissipation of the messenger and a fast dissociation rate (k−1). By facilitating delivery of messengers at high local concentrations to their targets (e.g. IP3 to IP3Rs), signalling complexes contribute to both rapid activation and de‐activation, the latter because diffusion of messengers away from the site of delivery may be sufficient to allow their concentration to fall below that required for activation as soon as synthesis of the messenger ceases. Secondly, targets can have fast off‐rates (k−1) with a corresponding loss of affinity  that does not compromise their capacity to respond to high local concentrations of messenger. We suggest, then, that assembly of proteins around IP3Rs contributes to fast and specific signalling, while providing opportunities for signal integration and plasticity.The advantages of these signalling complexes are clear. They allow information to be directed selectively from specific extracellular stimuli to specific intracellular targets through conserved signalling pathways. Furthermore, associated proteins can integrate signals from different signalling pathways and so modulate traffic through the complex. Hence, protein complexes confer both specificity and plasticity. A third advantage is speed. Signalling pathways must be able to turn on and off quickly. Fast activation benefits from high concentrations of reactants and fast on‐rates (3Rs under four somewhat arbitrary (and overlapping) headings: proteins that enhance or inhibit the activity of IP3Rs  are a well‐defined example. In sympathetic neurons, both muscarinic M1 receptors (M1Rs) and B2Rs activate PLC, but only activation of B2Rs evokes Ca2+ release through IP3Rs  that may mediate the universal inhibition of IP3Rs by Ca2+, are prime candidates for mediating this negative feedback. Proteins that inhibit IP3Rs fall into two broad categories: those that bind reversibly to interfere with binding of IP3 and/or Ca2+ or their links to gating; and those that cause post‐translational modifications of the IP3R . Residue S68 is the ‘master’ phosphorylation site. When it is phosphorylated by a Ca2+‐dependent kinase, perhaps a Ca2+/calmodulin‐dependent protein kinase (CaMK), it allows casein kinase I‐mediated phosphorylation of the two residues  that are critical for binding of IRBIT to IP3Rs (and its other targets) , allows Ca2+ release at lower concentrations of IP3  .IRBIT inhibits all three IP. 3 Fig. A. Resid3Rs by associated proteins may be reversible (e.g. phosphorylation) . By covalently modifying a glutamine residue within the C‐terminal tail of IP3R1, TGM2 causes irreversible cross‐linking of adjacent IP3R subunits via a lysine residue and the modified glutamine. This prevents the conformational changes required for activation of IP3Rs, and so inhibits IP3‐evoked Ca2+ release , PKA and AKT1 coordinates gluconeogenesis  and thereby AKT1. The latter phosphorylates IP3Rs and attenuates their activity. Hence insulin, by inhibiting IP3Rs, opposes the actions of glucagon by restraining the activation of calcineurin and so maintains CRTC2 in its inactive phosphorylated state  can ensure targeted delivery of IP3, but Ca2+ is most often provided by neighbouring IP3Rs. An important interaction, therefore, is that between IP3Rs themselves, because their proximity to neighbours dictates whether Ca2+ released by an active IP3R can ignite the activity of other IP3Rs. Considerable evidence suggests that clustering of IP3Rs within the plane of the ER membrane is dynamically regulated by IP3 and/or Ca2+  evoked by low stimulus intensities, by both bringing IP3Rs together and retuning their Ca2+ sensitivity  and end‐binding protein 3 (EB3) are disrupted. EB3 belongs to a family of proteins that bind to the plus‐end of growing microtubules and recruit other proteins, often via an S/TxIP motif (where x denotes any residue)  and the Wellcome Trust (101844).


We have previously identified that the first intracellular loop that contains a high density of acidic residues mediates voltage- and calcium-dependent gating of Ano1. Mutation of the four consecutive glutamates (444EEEE447) inhibits the voltage-dependent activation of Ano1, whereas deletion of these residues decreases apparent Ca2+ sensitivity. In the present study, we further found that deletion of 444EEEEEAVKD452 produced a more than 40-fold decrease in the apparent Ca2+ sensitivity with altered activation kinetics. We then systematically mutated each acidic residue into alanine, and analyzed the voltage- and calcium dependent activation of each mutation. Activation kinetics of wild type Ano1 consisted of a fast component (τfast) that represented voltage-dependent mode, and a slow component (τslow) that reflected the Ca2+-dependent modal gating. E444A, E445A, E446A, E447A, E448A, and E457A mutations showed a decrease in the τfast, significantly inhibited voltage-dependent activation of Ano1 in the absence of Ca2+, and greatly shifted the G-V curve to the right, suggesting that these glutamates are involved in voltage-gating of Ano1. Furthermore, D452A, E464A, E470A, and E475A mutations that did not alter voltage-dependent activation of the channel, significantly decreased Ca2+ dependence of G-V curve, exhibited an increase in the τslow, and produced a 2–3 fold decrease in the apparent Ca2+ sensitivity, suggesting that these acidic residues are involved in Ca2+-dependent gating of the channel. Our data show that acidic residues in the first intracellular loop are the important structural determinant that couples the voltage and calcium dependent gating of Ano1.Anoctamin1  is a Ca It has been reported that calmodulin (CaM) regulates Ano1 via at least two binding motifs in the channel 2+/CaM mediated increase in HCO3− permeability of Ano1 is lost 2+2+-binding site on the channel may be responsible for activation of Ano1 by Ca2+. The explicit Ca2+-binding site in Ano1 has not been identified. We have previously found that the first intracellular loop that contains five consecutive acidic residues are critical for both calcium- and voltage-dependent gating of the channel 448EAVK451) in the first intracellular loop decreases the apparent Ca2+ sensitivity 2+ gating of the channel Although recent studies have investigated the mechanisms underlying Ca2+ in several Ca2+-binding proteins 448EAVK451 in the first intracellular loop dramatically decreases apparent Ca2+ sensitivity of Ano1, and mutating the adjacent 444EEEE447 alters voltage-dependent activation of Ano1 without significant changes in the apparent Ca2+ sensitivity 2+-dependent gating of Ano1. Here, we mutated each acidic residue to alanine in an attempt to identify the role of each acidic residue in voltage- and calcium-dependent gating of Ano1. We found that the five consecutive glutamates 444EEEEE448 and E457 were critical for voltage-dependent gating of Ano1, and acidic residues D452, E464, E470, and E475 following 448EAVK451 contribute to regulation of Ano1 by Ca2+.Acidic amino acids are known to contribute to coordinate Caac) tagged with enhanced green fluorescent protein (EGFP) was obtained from Dr. U. Oh . Site-specific mutations were generated using PCR-based mutagenesis . All constructs were confirmed by sequencing.Ano1 were cultured in DMEM supplemented with 10% fetal bovine serum and 0.5% penicillin-streptomycin at 37°C. Low-passage HEK cells were transiently transfected with Fugene-6 (Roche) with 1 µg Ano1 per 35-mm dish. Cells were also transfected with 1 µg of pEGFP for fluorescence detection. Transfected cells were plated at low density and investigated between 24 and 72 h after transfection.2+ pipette solution contained (in mM): 146 CsCl, 2 MgCl2, 5 EGTA, 10 sucrose, and 8 HEPES, pH 7.3 adjusted with NMDG. The “high” Ca2+ pipette solution contained 5 mM Ca2+-EGTA instead of EGTA, which supplied free Ca2+ of about 25 µM. Solutions contained different concentrations of free Ca2+ were made by mixing the “0’ Ca2+ and “high” Ca2+ solutions. Free Ca2+ concentrations were verified using Fure-2 and Fura 6F (Invitrogen). The standard external solution contained (in mM): 140 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES (pH 7.3). For voltage-dependent activation, cells were voltage clamped with 50-ms voltage steps from −100 to +200 mV in 20-mV increments. For calcium-dependent activation, cells were voltage clamped with 750-ms voltage steps from −100 to +100 mV. For excised patches, the standard external solution was used as pipette solution. For better buffering the Ca2+, 3.5 mM Dibromo-BAPTA ethane-N,N,N′,N′-tetraacetic acid, Kd = 1.6 µM, Molecular Probe, Inc) was used in the excised patch to make 1 µM and 2 µM free Ca2+ concentrations. Patches were voltage clamped with 300-ms voltage steps from −160 to +200 mV in 20-mV increments at 10-s intervals. Osmolarity was adjusted with sucrose to 305 mOsm for all solutions.Transfected cells were identified by EGFP fluoresce. The electrophysiological recordings were performed in whole-cell and inside-out patch clamp configurations. Patch pipettes had resistances of 2–4 MΩ. Data were acquired by an Axopatch 200B amplifier controlled by Clampex 9 via a Digidata 1322 A data acquisition system . For whole-cell recordings, the “0” Ca2+ concentrations. The values of EC50 and Hill coefficients were calculated by fitting the current densities with the Hill equation, I/Imax = 1/(1+ (EC50/[Ca2+])n). G-V curves were generated from amplitudes of tail currents measured 200 µs after repolarization to −100 mV. G-V relations were fitted with a Boltzmann function, G/Gmax = 1/1+exp(- (V-V0.5) zF/RT), where z is the equivalent gating charge, V0.5 is the voltage at half-maximal activation, F is Faraday’s constant, R is the gas constant, and T is temperature. Z values were calculated from the slope of the G-V curve. Results were represented as mean±SEM. One-way analysis of variance (ANOVA) were performed to compare the differences between the mutations and wild type Ano1, followed by post-hoc Bonferroni test. A value of p<0.05 was considered statistically significant.We used Origin7 software for the calculations and graphical presentations. The activation kinetics of currents traces were fitted by two exponentials with Clampfit9, and visually inspected for proper fit. Dose-response curve of wild type Ano1 and mutations were generated by plotting the steady state current densities at +100 versus Ca444EEEEE448). We previously found that substituting the first four glutamates with alanines (444EEEE/AAAA447) inhibited the voltage-dependent activation of Ano1 without changes in the apparent Ca2+ sensitivity, whereas deleting 448EAVK451 (ΔEAVK mutation) decreased the apparent Ca2+ sensitivity with enhanced voltage-dependent activation of Ano1 444EEEEEAVKD452 (Δ444EEEEEAVKD452) 2+ sensitivity of the Δ444EEEEEAVKD452 mutation, compared with wild type (WT) (2+ concentration ([Ca2+]i) <1 µM, WT Ano1 was strongly outwardly rectifying. At 25 µM Ca2+, outward rectification was greatly reduced, and the currents were much less time-dependent   . At intrependent  [24]. Fo]i ≤1 µM  D,E. At ge as WT . In contge as WT . The Δ44(0.4 µM) . These f2+ concentrations for WT, 444EEEE/AAAA447, ΔEAVK, and Δ444EEEEEAVKD452 Ano1. For WT, the activation of Ano1 current was usually poorly fit to a single exponential, but was well-fit by two exponentials, suggesting that the activation of Ano1 in the presence of Ca2+ requires at least two conformational changes . For wild type, the z values at 1 µm Cantration .50 for Ca2+ was 1.11 µM for D452A, 0.81 µM for E464A, 0.75 µM for E470A, and 0.93 µM for E475A, which were approximately 2–3 fold higher than that of the WT (0.4 µM). These findings further confirmed that acidic residues D452, E464, E470, and E475 were important in Ca2+ -dependent gating of Ano1.We further examined the calcium sensitivity of the D452A, E464A, E470A, and E475A mutations  and S1. 2+-dependent activation 444EEEEE448) and 448EAVK451, which is lacking in the splice variant c. We have previously found that the 444EEEE/AAAA447 mutation inhibits the voltage-dependent activation of Ano1 without changes in the apparent Ca2+ sensitivity, whereas the ΔEAVK mutation decreased the apparent Ca2+ sensitivity with enhanced voltage-dependent activation of Ano1 2+-dependent gating of Ano1. In agreement with our previous finding, the present study further showed that deletion of 444EEEEEAVKD452 produced a more than 40-fold decrease in the apparent Ca2+ sensitivity, accompanied with slow kinetics of channel activation. By analyzing the kinetics of current activation at different Ca2+ concentrations for the WT and 444EEEE/AAAA447, ΔEAVK, and Δ444EEEEEAVKD452 mutations, we found that τfast and τslow of the channel were associated with voltage- and Ca2+-dependent activation of Ano1, respectively. Among the single alanine substituted mutations, E444A, E445A, E446A, E447A, E448A, and E457A mutations showed a decreased in the τfast, suggesting that these glutamates are involved in voltage gating of the channel. In agreement with this idea, these mutations significantly inhibited voltage-dependent activation of Ano1 in the absence of Ca2+, and greatly shifted the G-V curve to the right. Because we have previously found that the 444EEEE/AAAA447 mutation does not alter Ca2+ sensitivity, we believe that these glutamates  are involved in voltage-gating of Ano1. Furthermore, we found that D452A, E464A, E470A, and E475A mutations exhibited an increase in the τslow. Because these mutations did not alter voltage-dependent activation of the channel, but significantly decreased Ca2+ dependence of G-V curve and produced a 2–3 fold decrease in the calcium sensitivity, suggesting that they may be involved in Ca2+-gating of Ano1. Our findings demonstrate that the acidic amino acids in the first intracellular loop likely contribute to both voltage- and Ca2+-gating of Ano1.Ano1 has been identified as a novel CaCC that plays key roles in many physiological processes 0.5 shift in the G-V curve is different for each alanine substituted mutation, with V0.5 shifts most for E446A and least for E444A. Furthermore, mutations in the voltage sensor alter equivalent gating charge as measured as slope in G-V curve. However, all these mutations tested in this study do not alter the slope in G-V curve, further suggesting that they indirectly regulate a voltage sensor. The equivalent gating charge is about 0.33, about 4–6 fold less than that of voltage gated ion channel such as BK channel, suggesting that structure of the voltage sensor is different from that of voltage gated ion channels. Because the voltage sensitivity of Ano1 is very shallow, it seems unlikely that the voltage sensor is comprised of charged amino acids within a transmembrane segment like voltage-gated cation channels. The sequence of Ano1 provides no clear clues where the voltage sensor might be located. There are 3 basic amino acids at the interface between transmembrane domain 2 and the first intracellular loop, but mutation of these does not significantly affect Ca2+-independent gating (data not shown).Though we identify E444, E445, E446, E447, E448 and E457 as the critical residuals for voltage sensing, a lack of structural information precludes insight into how these residues are related to the voltage sensor. This region has been demonstrated to be cytoplasmic in both Ano1 and Ano7 − vs SCN− or NO3−) in the pore abc) It remains unknown how the first intracellular loop may couple to channel gating, because the location of the channel gate and selectivity filter remains to be established. The pore of Ano1 has been proposed to be located in a re-entrant loop between transmembrane domains 5 and 6, because mutations in this region alter the relative anion/cation selectivity of the channel 444EEEEE448) significantly reduced voltage-dependent activation fast without significant change in τslow  is also present in Ano2. Similar to Ano1, deletion of the five glutamates in Ano2 resulted in a right shift of the G-V curve, and modified voltage-dependent gating of the channel The cluster of five consecutive glutamates  exhibits an approximately 50-fold decrease in the Ca µM Ca2+ . These r2+ sensitivity of Ano1 2+ sensitivity 2+ sensitivity has been confirmed in Ano2 and Ano6 2+ is commonly coordinated by 6–8 oxygen atoms 2+ in the Ca2+ binding site in Ano1. However, we can not exclude the possibility that the acidic residues in the first and third intracellular loops contribute to separate Ca2+ binding sites. Although it is possible that Ano1 has more than one type of Ca2+ binding site, there is no direct evidence to support this.Recently, in a revise model of Ano1, the two acidic residues E702 and E705 in the third intracellular loop have been identified to be critical for the Caabc) channel activation by Ca2+abc), and activation of Ano1 by Ca2+ is not mediated by CaM abc) mediates channel activation by Ca2+/CaM. Jung et al. found that CaM modulated HCO3− permeability of Ano1(ac) that did not contain the b segment, but did not alter the ability of Ca2+ to activate the channel ac) by Ca2+ac) by Ca2+ is not mediated by CaM, but via the calcium binding sites on the channel. In the present study, our findings that mutations in acidic residues D452, E464, E470, and E475 altered the apparent calcium sensitivity of Ano1(ac) further suggest that these acidic residues may contribute to Ca2+-binding site on the channel. Since Ano1(ac) can interact with many Ca2+-binding proteins 2+ -dependent gating of Ano1.Recent studies have shown that calmodulin (CaM) can directly bind and regulate Ano1 2+ of Ano2 is lower compared with Ano1 2+ sensitivity greatly, it is possible that the backbone carbonyl groups of the EAVK contribute to the Ca2+-coordinating oxygen atoms in the Ca2+ binding site. If this is true, it is reasonable to hypothesize that the difference in the sequence of the corresponding residues (ERSQ) in Ano2 may not contribute to the difference in the Ca2+ sensitivity between Ano1 and Ano2. Consistent with our idea, Scudieri et al. found that replacement of the first intracellular loop of Ano1 with the corresponding domains of Ano2 did not alter the apparent Ca2+ sensitivity 2+ sensitivity between Ano1 and Ano2 2+ sensitivity of Ano2 2+ sensitivity between Ano1 and Ano2.Ano2 has been identified as a CaCC in the photoreceptors 2+ coordination. Among the acidic residues adjacent to EAVK, the cluster of five consecutive glutamates (444EEEEE448) and E457 are important for voltage-dependent gating of the channel. Other acidic residues D452, E464, E470, and E475 are involved in Ca2+ -dependent gating of Ano1. The present study identifies that acidic residues in the first intracellular loop is the important structural determinant that couples the voltage- and calcium-dependent gating of Ano1.In summary, the present study supports the conclusion that the first intracellular loop is critical for voltage- and calcium-dependent gating of Ano1. The backbone carbonyl oxygen atoms of the EAVK are likely to play a key role in CaFigure S12+ concentrations ranging from 74 nM to 25 µM.Representative traces of D452A (A), E464A (B), E470A(C) and E475A (D) activated by Ca Cells were voltage clamped from a holding potential of 0 mV to various potentials between −100 mV to +100 mV in 20 mV increments for 700 ms, followed by a 100-ms step to −100 mV.(TIF)Click here for additional data file.


Arabidopsis thaliana were shown to restore the specific growth phenotype of APC yeast loss-of-function mutants and to interact with calcium via their N-terminal EF-hand motifs in vitro. In this study, we performed biochemical characterization of all three APC isoforms from A. thaliana to gain further insights into their functional properties.Adenine nucleotide/phosphate carriers (APCs) from mammals and yeast are commonly known to adapt the mitochondrial adenine nucleotide pool in accordance to cellular demands. They catalyze adenine nucleotide - particularly ATP-Mg - and phosphate exchange and their activity is regulated by calcium. Our current knowledge about corresponding proteins from plants is comparably limited. Recently, the three putative APCs from 2+-binding to the EF-hand motifs in the N-terminal domain of the carrier.Recombinant plant APCs were functionally reconstituted into liposomes and their biochemical characteristics were determined by transport measurements using radiolabeled substrates. All three plant APCs were capable of ATP, ADP and phosphate exchange, however, high preference for ATP-Mg, as shown for orthologous carriers, was not detectable. By contrast, the obtained data suggest that in the liposomal system the plant APCs rather favor ATP-Ca as substrate. Moreover, investigation of a representative mutant APC protein revealed that the observed calcium effects on ATP transport did not primarily/essentially involve Cain vitro it is arguable whether ATP-Ca formation and thus the corresponding transport can take place in vivo.Biochemical characteristics suggest that plant APCs can mediate net transport of adenine nucleotides and hence, like their pendants from animals and yeast, might be involved in the alteration of the mitochondrial adenine nucleotide pool. Although, ATP-Ca was identified as an apparent import substrate of plant APCs The online version of this article (doi:10.1186/s12870-015-0616-0) contains supplementary material, which is available to authorized users. The mitochondrial carrier family (MCF) comprises structurally related but functionally diverse proteins that are characteristic for and generally restricted to eukaryotes –5. MCF paac2 lethality) as well as of various mammalian homologs became more and more clarified -ATP, [α32P]-ADP, [45Ca], MgCl2 and CaCl2 and incubated at 30 °C. At the given time points import was terminated by removal of external import medium via vacuum filtration as described in [45Ca] uptake measurements import was terminated and non-imported Ca2+ was removed by EGTA addition (2 mM) and incubation for 15 s prior to vacuum filtration and washing.For preparation of proteoliposomes 100 μg of the solubilized proteins were mixed with 20 mM Hepes, pH 7.0 and 1 mM PMSF. To obtain vesicles with internal counter exchange substrates 5 mM of phosphate or adenine nucleotides were added to the protein mixture. Preparation of mixed detergent-lipid micelles  loaded liposomes  into the Mg2+-complexed form (ATP-Mg2−)  increase in adenine nucleotide/Pi exchange compared to ATP without Mg2+ . ATP-Mg2+ on ATP uptake is due to general preference for ATP-Mg as substrate or rather due to the electroneutrality of the corresponding transport process we investigated Mg2+-effects on ATP homo-exchange. Homo-exchange of ATP is electroneutral but becomes electrogenic when ATP-Mg2− is exchanged with ATP4−. Comparison of the transport rates indicates that AtAPC1 highly, AtAPC3 markedly and AtAPC2 slightly prefer ATP homo-exchanges over the corresponding ATP/Pi hetero-exchanges  largely resemble the rates of the corresponding Mg2+-stimulated ATP transport (in exchange with Pi or ATP)  revealed that in fact traces of both, Ca2+ and Mg2+, are present in the media .Transport studies with reconstituted APCs might provide a suitable tool to overcome interfering metabolic and physiological effects and to study the impact of Caaddition  and also2+ is essential for carrier activation and under the assumption that the proteoliposomes still contain a certain amount of inactive (Ca2+-free) APC proteins, an addition of extra Ca2+ should result in transport stimulation. To investigate a possible Ca2+-induced increase in transport activation we performed uptake studies with and without 200 μM Ca2+. Elevated Ca2+ availability generally stimulated nucleotide uptake of all three plant APCs (Table 2+-induced activation of previously inactive (Ca2+-free) carrier proteins. Studies with the two separately expressed sub-domains  of the human SCaMC1 led to the conclusion that the N-terminal domain acts as a lid that either opens or closes the translocation pathway in response to Ca2+ availability  from the liposomal surface was achieved by additional EGTA treatment of the vesicles subsequent to the uptake measurements (prior to vacuum filtration and washing). The correspondingly modified transport assay allowed determination of small but significant time dependent Ca2+ uptake by full-length and truncated AtAPC2.The observed Ca2+ uptake into Pi loaded vesicles  are required to cause approximately half maximal transport inhibition whereas 25-fold excess of Mg2+ completely blocks Ca2+ uptake. Because of the generally low [45Ca] transport rates of the truncated AtAPC2 reliable interpretation of the corresponding results obtained with this protein is complicated. Nevertheless, the tendency of Mg2+ impact on Ca2+ uptake generally resembles that of the full-length protein atractyloside insensitive (AAC independent) uptake of adenine nucleotides –28. In ti . Later oP uptake . These iArabidopsis thaliana encodes three MCF proteins (AtAPC1-3) that represent promising candidates for net adenine nucleotide transport. First of all, AtAPC1-3 exhibit significant amino acid similarities to APCs from animals or yeast and contain the characteristic N-terminal domain with EF-hand motifs . Thisoforms . Moreoveisoforms , 51) is isoforms , 52–54. AtAPC3 and AtAPC1 apparently prefer homo-exchanges of ATP and ADP over the corresponding hetero-exchanges with Pi  hetero-exchange or that hetero-exchange with Pi is not electrogenic at all. Interestingly, ATP transport of AtAPC2 was totally inhibited by EGTA or EDTA and could be restored by Mg2+ or Ca2+  Ca2+-dependent transport stimulation, (ii) Vmax increase , and (iii) transport reduction by Ca2+-depletion (with EGTA) independent of the exchanged substrates. Moreover, truncation of the N-terminal domain should cause constantly active carriers that are no longer influenced by Ca2+. However, the data obtained in this work suggest that this is not the case. We therefore hypothesize that in the in vitro system ATP-Ca acts as substrate of the plant APCs and is even favored over ATP-Mg or free ATP. By contrast, ADP-Ca seems to be rather discriminated against when compared to free ADP. Ca2+-induced alterations of the apparent transport affinities most likely reflect these specific substrate preferences of the respective APC isoforms e.g. higher preference for ATP-Ca (when compared with the Mg-complexed or free ATP) and lower preference of ADP-Ca (when compared to free ADP)  enhances the imbalance caused by the ADP/ATP hetero-exchange. In addition, effects of EGTA, EDTA, Mg2+ and Ca2+ on ATP transport inhibition, stimulation or reactivation suggest a competition between these cations during complex formation and provide further evidences for ATP-Ca as a potential in vitro substrate of recombinant plant APCs  Table . AccordiCs Table , 5. We cAtAPC2 and the N-terminally truncated version support the assumption that ATP-transport stimulation by Ca2+ is not (or not exclusively) caused by activation of previously inactive (Ca2+-free) carriers. ATP transport of both, the full-length carrier and the truncated version, can be stimulated by Ca2+ and inhibited by EGTA whereas ADP transport was not significantly affected . Moreover, this observation also demonstrates that the chelator at the liposomal interior is apparently physically separated from Ca2+ at the exterior  which indicates that both, EGTA and Ca2+, do not pass the lipid barrier freely.The fact that external but not internal EGTA caused inhibition of 2+-dependent regulation, we were able to identify the in vitro function of Ca2+ as co-substrate with the applied system.Notwithstanding or even because of the missing Ca32P]-ATP provided evidence for a possible ATP-Ca transport it would still have been imaginable that Ca2+ stimulates transport of unchelated ATP and impedes ATP-Mg transport in a different way. However, the specifically adapted uptake assay using [45Ca] provided a direct proof that ATP-Ca is de facto transported via reconstituted  [2+ concentrations within plant mitochondria ATP is nearly completely complexed with Mg2+, which argues against any potential ATP-Ca formation in the matrix [2+ levels in the cytosol increase the accessibility of free ATP, it is unclear whether conditions or microdomains of high Ca2+ availability at the mitochondrial surface might allow ATP-Ca formation [2+ uptake via AtAPC2 was low and completely blocked by 25-fold excess of Mg2+. If these characteristics  also represent the in vivo situation, ATP-Ca transport via plant APCs is highly unlikely to occur.Although plant mitochondria contribute to Ca 100 nM) –57. Moree matrix . Althougormation , 58–63. 2+ translocation. SCaMC3 was shown to physically interact with the (low affinity) Mitochondrial Calcium Uniporter (MCU) and lack of SCaMC3 apparently decreases ATP and Ca2+ import into mitochondria [2+ uptake system, a supercomplex formed by channels and carriers in microdomains for enhanced Ca2+-sensitivity [2+ release might be advantageous to guarantee fast Ca2+-dependent activation and response of plant APCs.Although, a direct role of plant APCs in ATP-Ca transport is therefore arguable, recent data suggest an indirect function of a mammalian isoform in Cachondria , 64. AccA. thaliana in the liposomal system revealed that the recombinant carriers mediate ATP, ADP and phosphate exchange. Accordingly, plant mitochondria harbor a subset of carriers capable of net adenine nucleotide translocation, however in contrast to yeast and mammalian orthologs they show no high preference for ATP-Mg as import substrate. Surprisingly, we instead obtained evidence for a possible ATP-Ca transport by the reconstituted plant APCs in the liposomal context but it is arguable that physiological Mg2+ and Ca2+ concentrations most likely prevent ATP-Ca formation and its subsequent transport in vivo. Although we were not able to detect EF-hand based Ca2+-dependent carrier regulation, this was shown recently to exist in plant APCs [2+ concentrations regulate activity of plant APCs via EF-hands of the N-terminal domain whereas high Ca2+ concentrations can induce its own transport as co-substrate of ATP in vitro. While this study deepens our knowledge about mitochondrial net nucleotide transport of plants it also gives rise to new intriguing questions. In the future, it is important to investigate the in vivo function of plant APCs and the impact of divalent cations on the corresponding transport.Determination of the biochemical characteristics of three putative APC isoforms from ant APCs . Summari


This study empirically investigates the mediating role of psychosocial mentoring support on emotional stability personality disposition and career resilience relationship. In addition, this research also focuses on estimating the interrelationship between emotional stability, psychosocial mentoring support and career resilience. The results show substantive direct relations between emotional stability and psychosocial mentoring as well as between emotional stability and career resilience. Psychosocial mentoring is also seen as a significant predictor of career resilience. Further, it mediates partially the relationship between emotional stability personality and career resilience. Future and practical implications of research have also been provided. Of thesIn this study, we propose to examine the interrelationships between emotional stability, psychosocial mentoring and career resilience with a specific focus on investigating the mediating role of psychosocial mentoring on the relationship between emotional stability and career resilience. The proposed model of the study has been represented in Emotional stability dimension is recognized as a significant predictor of job performance , and is In the past, various perspectives have been represented to guide the concept of resilience that includes both psychological and environmental perspectives . Bryant One such form of resilience is career resilience that highlights “the ability to adapt to change even when the circumstances are discouraging . Accordirole-model; conveying unconditional positive regard toward protégé through unconditional acceptance and confirmation; encouraging protégé to discuss his anxieties and fears without any hesitation and counseling him by informally interacting with him by becoming his friend  is characterized by instability, depression and also display a lack of personal insecurity. Such people have a tendency to remain hostile and impulsive because of which they often show lack of psychological adjustment . MoreoveH1: Emotional stability personality disposition will be strongly related to career resilience.Past research has depicted that people, who are low on emotional stability are anxious, carry negative emotions because of which they do not show greater interest in initiating mentoring relationships . FurtherH2: Emotional stability personality disposition will be strongly related to psychosocial mentoring.Numerous studies have suggested that managerial support plays a key role in the employee’s career development  and otheWe have, thus, hypothesized that:H3: Psychosocial mentoring support will be strongly related to career resiliencefriendly nature and admiration of the mentor in psychosocial mentoring also contributes to the establishment of interpersonal comfort in the mentor-protégé relationship. This helps in reducing the stress and tension prevalent during the times of turbulence  and public sector (54.9%) organizations in North India. Of these 233 participants, about 85.4% were males and 14.6% were females with about 39.1% aged within 26–30 years. Participants were from all the three hierarchical levels, junior level management (24.5%), middle level management (60.5%) and senior level management (15%). Overall participants had a good educational background with 6% as diploma holders, 43.8% as graduates, 46.4% as post graduates and 3.8% held degrees higher than post-graduation.The organizations were targeted by contacting HR managers. These organizations were located in the same geographical region and had similar kind of organizational structure. None of these organizations had a formal mentoring scheme in place at the time of the study. Survey questionnaires were distributed among the participants during the office hours with the support of human resource department of the organization. The aim of the study was then explained to the participants and those managers, who provided their consent, were then administered with a paper and pencil survey. The survey questionnaires, which were distributed, were prefaced with a cover letter that explained the importance of participation. Participants were also assured regarding the anonymity and confidentiality of their responses. Survey questionnaires were initially administered among 240 respondents, seven of whom responded to be not involved in any current mentoring relationship, which ultimately resulted in overall relatively complete data of 233 participants.For the measurement of emotional stability, ten items were adopted from We have measured psychosocial mentoring support using the 14-item scale from the study of This was assessed using a single item. An established definition of mentor was also provided to the participants to help them understand meaning of mentor as given below:A mentor is generally defined as a higher ranking, influential individual in your work environment who has advanced experience and knowledge and who is committed to provide upward mobility and support to your career. Your mentor may or may not be in your organization and may or may not be your immediate supervisor , p. 2.Following this, they were also asked to respond whether they currently had a mentor (coded No: 1 and coded Yes: 2) in line with previous studies .To measure the career resilience, four items were adopted from the scale of Career Commitment Measure (CCM) . CCM rep2 goodness-of-fit statistic; (b) the comparative fit indices  = 413.059; CFI = .958; GFI = .890, RMSEA = .036  and IFIFollowing variables were set as controls in order to control their spurious effects on dependent variable and were coded as given below:Age (years): 21–25 (1), 26–30 (2), 31–35 (3), 36–40 (4), 41–45 (5), above 45 (6)Gender: Males (1), females (2)Marital status: Married (1), unmarried (2)Educational level: Diploma holder (1), graduates (2), post-graduates (3), higher than post-graduate (4)Hierarchical level: Junior level (1), middle level (2), senior level (3)Organization sector: Private sector (1), public sector (2).Multiple hierarchical regression analysis was used for the testing of both direct and indirect effects. Specifically, for the testing of mediation hypothesis, guidelines of Baron and Kenny‘s mediation approach was adopted in alignment with earlier studies . This teBesides these four conditions, another unique distinction was illustrated in context to condition supporting ‘full mediation’ and ‘partial mediation’. According to them, the condition of full mediation is well established when relationship between IV and DV completely disappears while controlling the effect of MV. On the other hand, the condition of partial mediation is considered to be established when the relationship between IV and DV is significantly reduced while controlling MV, but does not completely disappear .r = .211, p < .01); and psychosocial mentoring support also relates positively but has weak correlation with career resilience .The descriptive statistics and inter-correlations among the study variables have been detailed in p < .01), which supported H1. Second, for the evaluation of H2, direct impact of emotional stability personality dimension on the psychosocial mentoring was measured. p < .05) as a significant predictor of psychosocial mentoring that provided support to H2. Similarly, psychosocial mentoring support  was also seen as a significant predictor of career resilience , but still remained significant while controlling the effects of MV. This indicates partial mediating effect of psychosocial mentoring support on emotional stability and career resilience relationship. Hence, H4 is partially supported.This study makes useful contribution to the available literature on mentoring and occupational psychology from the perspective of understanding the role of socio-emotional support as a mediator of the relationship between emotional stability personality factor and career resilience. To the best of our knowledge, the present study appears to be the first research to explore mediating role of psychosocial mentoring between the two variables. In addition, this study also focuses on understanding the interrelationships between emotional stability and career resilience, emotional stability and psychosocial mentoring, psychosocial mentoring and career resilience, as not many studies have investigated the influence of personality and mentoring on career resilience. In support of Hypothesis H1, emotional stability personality disposition was found to be a positive significant predictor of career resilience. This implies that those protégés, who exhibit greater self-control, remain calm and composed and regulate their emotions, are higher on career resilience. On the other hand, people with low emotional stability (i.e. high neuroticism) are more vulnerable to emotional distress and exhibit poor coping mechanisms, which further makes them a low scorers on the resilience dimension .As proposed, for Hypothesis H2, emotional stability personality was found to be significant predictor of psychosocial mentoring support. This means that protégés with higher scores on emotional stability show greater interest and willingness to receive psychosocial mentoring support and vice-versa. Besides this, such individuals may also perceive greater psychosocial mentoring support in comparison to protégés, who are low on emotional stability. This is because protégés with low emotional stability lack confidence for establishing relationships with upper-level managers , and areFurther evaluation of Hypothesis H3, showed that among Indian managers’ psychosocial mentoring acts as a significant predictor of career resilience, which is similar to the proposed hypothesis. This highlights the subjective nature of psychosocial mentoring in influencing employees’ affective reactions for individual level and organizational level outcomes . SimilarThe results of the study provide meaningful implications from both practical and theoretical perspectives. These are discussed as below:As, emotional stability personality disposition is closely related to “successful functioning” in all areas of life including situations of adversity, both in and out of the workplace ; this alGiven the crucial role of protégé’s positive perceptions of psychosocial mentoring in the development of positive attitudes, such as organizational commitment, organizational attractiveness and intentions to pursue employment in the mentor’s organizations ; we alsoSpecifically, the findings of this research have implications for incorporating counseling practice in the mentorship model of the organizations so as to guide the workforce during the times of turbulence. For example, if a protégé is low on emotional stability, his anxiousness and negative affectivity can be reduced to some extent with mentor’s counseling skills. As counseling helps in assessing the degree of anxiety and depression that may influence the client’s decision-making ability; additionally, it may also help protégés in overcoming challenges of their multiple roles by enhancing their self-efficacy and self-confidence .The findings of the study might be useful for preparing guidelines for designing mentoring programs in the organization. One of the noteworthy, implications of this research is that it informs both mentors and protégés about how important it is to maintain friendly and unconditional support in the mentor-protégé relationship in order to achieve outcomes in the long run.There are some limitations associated with the present research. First limitation is the use of a cross-sectional survey based research design, which is based on protégé’s self-reported data that limits the scope for making any inferences about causality. For instance, our study revealed psychosocial mentoring as the significant predictor of career resilience. Yet, one might think of a career-resilient protégé to perceive higher levels of psychosocial mentoring support. Perhaps, such kind of causal inferences could be determined through the adoption of longitudinal research designs.Second concern is related to the common-method bias (CMB) due to the use of self-reported measures in the study. However, this does not represent serious threat to our study, as checked by using the procedural guideline of Harman’s single factor , which sFurther, as scope of the study was limited to study the linkage between psychosocial mentoring, emotional stability and career resilience; sample inclusion took into consideration only the mentored individuals. Further study is recommended using the experimental and quasi-experimental research designs while considering both mentored as well as non-mentored individuals.As consent prescreening is considered as one of the promising techniques for increasing response rates ; the datAnother concern is the sample intake from different industries; hence, findings cannot be generalized to any specific sector. Future studies are recommended for testing the proposed interrelationships in the specific business setting. In this regard, scholars and academicians may seek to extend the work by exploring various probable subsets of psychosocial mentoring functions in a specific organizational context. Also, given the shortcomings in the development of a psychosocial mentoring relationship in organizations with formal mentorship scheme, a qualitative research investigation may be of interest for preparing comparative account of the formal mentoring versus informal mentoring.


Homoacetogenic bacteria have received attention as a hydrogenotrophic population that offers a significant energetic advantage to the host animal. Reductive acetogenesis is likely an important hydrogen disposal mechanism in the cecum of rabbits. However, molecular ecology information about cecal acetogen candidates has rarely been reported. To better understand the effect of host selection in the rabbit cecal acetogen community with respect to growth, rabbits at four different age stages  with the same diet were studied. Although the abundance of potential acetogens and methanogens was high in the cecum of rabbits undergoing growth, many novel potential acetogen populations were observed in the cecum of rabbits across all age groups. Young and adult rabbits had their own distinct acetogen community although they received the same diet, which suggests that as the rabbit ages, acetogens in the cecum undergo developmental changes because of host selection that are independent of diet, and perhaps the different acetogen communities result in different hydrogenotrophic characteristics. The within-group similarity increased with age, indicating that the acetogen community converges to a more homogeneous and stable arrangement with aging. Acetogehabitats . Acetogehabitats , but onloacetica , 6, Closngdahlii , 8, Closagsdalei  and theiagsdalei . A betteMethanogens have a greater thermodynamic advantage than acetogens when competing for hydrogen in anaerobic habitats –13. RabbThe structure and composition of the gut microbiota is driven by lifestyle strategies, such as the growth rate, substrate utilization patterns, and host selection for specific bacteria with emergent collective behavior that is beneficial to the host . StudiesThe cellulolytic capability of the gut microbiota is enhanced with an increase in the growth stage of the host , 26, andThe Animal Care Committee of Zhejiang University  approved all experiments, and the experimental procedures used in this study were in accordance with the university’s guidelines for animal research. Rabbits at four different age stages were fed the same diets and were used to study the change in the acetogen population in the cecum. Six male New Zealand White rabbits at ages 30, 60, 120 and 180 days were purchased simultaneously from the Zhejiang Academy of Agricultural Science. They were housed in indoor three-layer cages (60×50×35 cm) with natural lighting and raised with the same commercial pellet diets consisting of 12% corn, 18% bran, 8% soya bean cake, 31% meal, 10% malt root, 16% chaff, 0.8% powder, 0.5% salt and 4% commercial additives. Only one rabbit was housed in each individual cage. All rabbits were sacrificed on the same day. The performance of euthanasia and removal of cecum were undertaken by trained animal technician . Body weight was measured for each rabbit to ensure intravenous injection of 100 mg/kg (body weight) phenobarbital sodium  . Once thThe samples for the measurement of volatile fatty acids (VFA) and ammonia nitrate were prepared as described by Yang et al. . Approxifhs) [mcrA) [2 and 109 target gene copies calculated from the concentration of plasmids. The copy numbers of the standards and the copy numbers of the target genes per gram of cecal sample were calculated according to the method of Ritalahti et al. [Total DNA was extracted from 0.1 gram of each of the stored samples using the cetyltrimethylammonium bromide (CTAB) method as described by Gagen et al. . Primersfhs)  and the ) [mcrA)  were use) [mcrA) . Real-tii et al. .acsB) from each extracted DNA sample was performed using the TaKaRa PCR Kit  with 1.0 ng template DNA per reaction. The specific primers for acsB [Amplification of subunit B of the acetyl-CoA synthasegene  and chimera removal of the raw data were performed using the pipeline in QIIME . The seqn ExPASy . The obtn ExPASy . Addition ExPASy –45. RAxMn ExPASy  and ARB n ExPASy  were usen ExPASy . The seqfhs and mcrA genes, and the concentration or molar percentage of the total and individual VFA were analyzed by one-way analysis of variance in R 3.2 with individual rabbits as the experimental unit and age as the main effect. Multiple comparisons of means among different age groups were conducted using Turkey multiple range tests. Significance was declared at P<0.05.The abundance of the 16S rRNA and The pH of the cecum increased with age. However, the total VFA and ammonia nitrate decreased with age . The ratacsB from the cecum of rabbit clustered into 6101 OTUs, suggesting that many acetogen species exist in the cecum of rabbits. However, most of the detected sequences lack representatives from previously described sequences, which is in consistent with previous studies [acsB sequences amplified in our study expand the database of ACS amino acid sequences for acetogens. Alpha diversity, which is described in terms of richness or the number of different species present, and evenness were initially investigated to observe changes in acetogen diversity in the cecum of rabbits during growth and development  . Clear aThe between-group similarity was further analyzed using a heat map , and theacsB gene database, most of the detected novel ACS OTUs in the cecum of rabbits could not be classified at the species or genus level. Therefore, a maximum likelihood tree  increased in adult rabbits aged 180 days compared to the young rabbits aged 30 days. The presence of OTU 43, which was phylogenetically affiliated with Ruminococcaceae, significantly (P<0.05) increased in older rabbits aged 180 days. In addition, another 13 ACS OTUs (group 10) and OTUs 41 and 456 were phylogenetically affiliated with Clostridiaceae, showing a significant increase (P<0.05) in rabbits aged 180 days. Another 8 clusters  of ACS sequences containing the dominant OTU 9 were phylogenetically affiliated with the Lachnospiraceae family and were more highly present (P<0.05) in rabbits aged 180 days but were far away from any known cultured acetogens. The presence of OTUs 7, 23, and 541 and group 5 in the trees was affiliated with Lachnospiraceae but were far away from any known cultured acetogens and was higher (P<0.05) in young rabbits aged 30 days. A previous study indicated that over 90% of the bacterial sequences in the cecum of rabbit belong to the Firmicutes phylum [Ruminococcaceae and Lachnospiraceae were the dominant families, accounting for 45% and 35% of all sequences, respectively [Lachnospiraceae, Ruminococcaceae, Blautia, and Clostridiaceae, which were shown from the maximum-likelihood trees of the 16S rRNA gene and the ACS amino acid sequences for comparison to the tree topology reported by Gagen et al. [Blautia coccoides, was associated with carbon dioxide and hydrogen metabolism [Due to the limited ood tree  was consood tree . The mosia group . The preia group . These sia group . Comparis phylum . Ruminocectively . In our tabolism .fhs and mcrA to determine the potential abundance of acetogens, which is currently the best target for quantifying acetogens [mcrA gene to determine the methane producing hydrogenotrophy, which is likely the dominant hydrogen disposal method in most anaerobic environments. The abundance of the mcrA gene was at 106 to 108 gene copy numbers per gram of cecal content in most of the samples, which is consistent with a previous study  [fhs gene was at 105 to 106 gene copy numbers per gram of cecal content in most of the samples and showed no significant differences (P>0.05) with growth stage  , which icetogens , 58, and day 35)  and was th stage .Blautia family, in which the known strains Blautia coccoides, Blautia hydrogenotrophica and Blautia schinkii can use carbon dioxide and hydrogen to synthesize acetate as a main product [Blautia producta with carbon monoxide or carbon dioxide and hydrogen as a substrate is only 1.5 or 5 h [Compared to the published acetogen strains used in microbial production platforms, many new acetogen strains were found in the cecum of rabbits in our study, with the dominant acetogen affiliated with the  product , 60. In 5 or 5 h , 62, sugBlautia group, Ruminococcaceae and Clostridiaceae. Blautia schinkii and Blautia hydrogenotrophica are known to use carbon dioxide and hydrogen as substrates for growth, and Blautia spp. may play a key role in the kangaroo foregut and are associated with carbon dioxide and hydrogen metabolism [Clostridiaceae family contains the Clostridium genus, which is now an important candidate in the biofuel industry. Therefore, because of the growth effect from host selection, the cecum of adult rabbits may be a good source for enriching acetogen candidates for biofuel production.The true extent of acetogen biodiversity in the cecum of rabbits is not known because most organisms cannot be cultured ex vivo. However, there was a clear rabbit-specific effect on the cecal acetogen community in individuals used in this study. Only rabbits of the same species  and with the same type of diet were used, which minimized both the genetic variability and dietary variation in the study to examine the effect of growth. However, because of the limited database of known acetogens, the acetogen strain changes with growth could not be classified accurately. Some of the acetogen strains were more present in adult rabbits (180 days) and were affiliated with the tabolism . The CloThe inter-individual variation of the cecal bacteria in rabbits was previously observed, although acetogen was not specifically tested . ComparamcrA and fhs were observed in our study, a previous study indicated that the cecum of rabbits harbors a methanogenic community with low complexity of the genus Methanobrevibacter, and it is possibly one-species dominated because all sequences detected presented similarity values of 99% or higher [As a hindgut fermenter, the greatest numbers of bacteria reside in the cecum of rabbits. Relationships between the gut bacterial communities and their mammalian hosts contribute to the host’s well-being and proper function , 64. In r higher . HoweverTo better understand the function of acetogens in the cecum of rabbit, further studies, such as tracking of the biological fate of carbon dioxide in the cecum of rabbits using RNA stable isotope probing (RNA-SIP) techniques, should be performed to identify the dominant organisms and pathways involved in hydrogenotrophy of rabbit cecum. This was previously performed in the kangaroo forestomach to explain the dominance of reductive acetogenesis as a hydrogen removal pathway .The cecum of the rabbit is a good resource to identify and develop new acetogen products. Hosts select preferable microbiota for their growth, and the microbiota in the cecum are more homogeneous and stable with growth and development. To better understand the contribution of acetogen populations to the host, stable isotope tracing can be performed to identify the dominant organisms and pathways involved in hydrogenotrophy in the cecum of rabbit.


The nature and duration of the effects of pindone poisoning in rabbits were evaluated through observational monitoring of affected animals and necropsy. Using the resulting data in a formal assessment framework, the welfare impacts of pindone poisoning were ranked as relatively higher than other vertebrate toxic agents currently used for rabbit control.Control methods used to manage unwanted impacts of the European rabbit in Australia and New Zealand include the use of toxic bait containing the anticoagulant pindone. Towards increased certainty in evaluating the animal welfare impacts of pindone poisoning in rabbits, we recorded behavioral and post-mortem data from rabbits which ingested lethal quantities of pindone bait in a laboratory trial. Pindone poisoning in rabbits resulted in welfare compromise, primarily through functional impairments related to internal haemorrhage over a maximum duration of 7 days. Applying this data to a formal assessment framework for ranking animal welfare impacts indicated that pindone had relatively high severity and also duration of welfare impacts in comparison to other rabbit control methods. Those tasked with managing impacts or populations of unwanted (“pest”) wild animals often have a variety of lethal and non-lethal control methods available to them. While cost-efficacy and human safety have been traditional drivers of the development, selection and application of control methods, animal welfare outcomes of pest animal management is an increasingly important criterion e.g., ,2). In t. In t2])Oryctolagus cuniculus), a species introduced to New Zealand and Australia following British colonisation. In both countries, establishment and expansion of rabbit populations into favourable habitats was accompanied by unwanted effects on biological conservation and agricultural production values. Rabbits can compete with grazing livestock and some native species for food, cause soil erosion, modify native vegetation and influence dynamics of predator-prey interactions [One such example is the management of European rabbits , which are considered a significant pest in New Zealand [vs. duration) for each method/animal species combination to produces scores, which can then be ranked in terms of relative welfare impacts. This framework [In the context of using VTAs to control unwanted animals, early approaches to evaluating the humaneness of poisoning ,8 identi Zealand ,11. More Zealand  was desi Zealand . The fraramework , with soramework . In compraison d'être of the study [One such information gap concerned the use of the VTA pindone  for rabbit control. Pindone is a first-generation indandione anticoagulant and its main toxic activity is to inhibit Vitamin K metabolism in the liver to the point where depletion of circulating active blood clotting factors results in failure of coagulation and fatal haemorrhage . Paparelhe study  within the study ) to recoet al. [The study was conducted in 2011 under approval from the Landcare Research Animal Ethics Committee (Project No. 10/11/01). Laboratory housing and acclimation conditions for rabbits are described fully by Fisher et al. . In brieThese treatments were intended to simulate low and high lethal intakes that could occur in operational application of bait for rabbit control. In Australia and New Zealand, carrot, oat or cereal pellet baits are used in bait station or broadcast applications, where rabbits typically have access to bait over a number of days. As for other ‘first generation’ anticoagulants, pindone is most effective when ingested in multiple, consecutive exposure over a few days, but a relatively much higher single exposure can also be lethal .2 floor enclosure for a minimum of one hour per day. Voluntary ingestion of potentially lethal quantities of pindone bait by rabbits was required to meet the main objective of the trials, which was to quantify residual pindone concentrations in rabbit tissue [During these trials we sought to simulate movement and conspecific interactions typical of free-ranging rabbits, as potentially important influences on the effects of anticoagulant ingestion. Rabbits were acclimated for a minimum of two weeks to free exercise in same-sex groups in a 3 mt tissue . We sougThese endpoints were based on described effects of anticoagulant poisoning in rats and possums . They wei.e., advanced progression of pindone poisoning. This enabled relatively precise estimates of times to first visible signs of poisoning, and description of the progression of poisoning to mortality. Statistical comparison of times to first signs of poisoning, and times to death was through one-way ANOVA and Tukey multiple comparisons of means in the program R .Behavioural observations of each rabbit were recorded against time from the first potential intake of pindone bait, and recorded during the free exercise periods and at least twice-daily observations (morning and afternoon) of rabbits in their individual cages. Frequency of observations was increased to three-hour intervals where individual rabbits appeared to be approaching any endpoint After pindone bait was offered monitoring of rabbits continued for 21 days. Any rabbits alive after that period were assumed to have been sublethally exposed or not exposed to pindone at all (as measured by bait intake) and were killed by lethal intracardiac injection. Post-mortem, all rabbits were stored chilled (−4 °C) for 3–24 h prior to necropsy and sampling of liver, muscle and fat tissue to test for residual concentrations of pindone (as described in ). At necDuring July–August 2011, intact rabbit carcasses were collected after two pindone-pellet baiting operations in the South Island of New Zealand, one in a pine plantation at the northern end of Lake Tekapo and the other at various locations around Wanaka . Whole carcasses were stored frozen for up to two weeks, then defrosted and necropsied as for the laboratory rabbits.3. 2,24 = 1.1854, p = 0.32). “Illness” behaviours during the progression of poisoning included a general reluctance to move and reduced responsiveness to the presence of other rabbits during the exercise periods. Inappetance and inactivity became generally more pronounced in the one or two days preceding death. Evidence of physical injury associated with anticoagulation was observed in 10 of the 27 rabbits that ingested lethal doses, including lameness favouring one limb, uncoagulated bleeding from minor wounds or visible subcutaneous bruising. In particular, severe haematoma in or around one eye triggered alternative endpoint euthanasia in six rabbits  or rabbit survival  between the three trials. rabbits , and oneLaboratory rabbits that had died of poisoning or were euthanased at an alternative endpoint had (at least) one large internal haemorrhage which was ascribed as causing death. The haemorrhage sites from the most to least common were; in cardiac and/or thoracic cavity, in hind or forelimbs extending down into the paws, haemorrhage around the gut including the outer stomach wall, intestines and mesenteric membranes, and in the muscle of the hind quarters, shoulder or neck. Most bleeding in limbs occurred in the outer muscle layers and was usually extensive throughout an affected limb, but bleeding into bone joints was not observed. Necropsy also showed no consistent patterns of haemorrhage in rabbits related to the magnitude of the lethal dose ingested, and death in poisoned rabbits was considered due primarily to hypovolaemic shock with contributing effects of anaemia.i.e., the magnitude of a lethal exposure does not significantly influence the extent or duration of the effects of poisoning.Of the nine rabbits that ate sublethal amounts of bait , signs oOf twelve rabbit carcasses collected following a pindone baiting operation at Lake Tekapo, three could not be fully necropsied because they had been partially scavenged. In the nine Tekapo rabbits necropsied there was no visible external bleeding, their stomachs were at least half full and there was no haemorrhaging in the cardiac area or limbs. In seven of these rabbits extensive haemorrhage was concentrated around the gut and mesentery. One rabbit also had a pale mottled liver, suggesting disease may have contributed to mortality. Necropsy of nine rabbits collected after a pindone baiting operation near Wanaka revealed some visible external bruising and more generalised haemorrhage in the abdominal cavity and the musculature of limbs compared to the rabbits from Tekapo. The most likely cause of death for four of the rabbits from Wanaka was abdominal haemorrhage. Two other individuals had haemorrhaged in the hind quarters, and no major identifiable haemorrhage was observed in the remaining three rabbits . Compared to the necropsy observations of laboratory rabbits, field-collected rabbits showed much more diffuse haemorrhage that was less easily defined as originating from particular tissues or locations and a greater incidence of free, uncoagulated blood pooled in the abdominal cavity. This was considered at least partially due to the difference in the time between death and necropsy in laboratory versus field-collected rabbits, including effects of frozen storage e.g., haemolysis, in the field-collected rabbit carcasses and subsequent defrosting for necropsy.i.e., from first signs of poisoning to death) following lethal pindone exposure ranged from 1.43–4.33 days  are considered rare  that produce negative affective states (Domain 5), over a maximum duration of 7 days . ‘Time te .RelativWhen selecting control methods for unwanted animals there is an expectation that the degree and extent of animal welfare compromise will be minimised where possible . HoweverWithin an ethically defensible approach to pest animal management, improved information about the relative welfare impacts of different control methods is important to enable both on-ground managers and regulatory agencies to optimise choices in everyday practice and policy making. Here we have added to the information base regarding the animal welfare impacts of one method for rabbit management (pindone baiting), which potentially affects many thousands of rabbits annually. The relatively high animal welfare impacts of pindone poisoning, in comparison to at least other vertebrate toxic agents, suggest that the use of this control method should be limited to situations where there are no other acceptable or appropriate alternatives for mitigating the unwanted impacts of rabbits on natural environments and production values.