The release of membrane-associated calcium from rabbit neutrophils by fixatives. Implications for the use of antimonate staining to localize calcium

Summary The addition of oxalate to a suspension of rabbit peritoneal neutrophils before fixation with glutaraldehyde and postfixation with osmium tetroxide-antimonate greatly enhanced the amount of calcium antimonate precipitate subsequently detectable with the electron microscope. Using chlortetracycline as a fluorescent probe for membrane-associated calcium, it was found that both glutaraldehyde and osmium tetroxide release calcium from membrane-associated stores in suspensions of living neutrophils. These findings suggest that some of the calcium released from cellular stores during fixation with glutaraldehyde is trapped within the neutrophil by oxalate which then reacts with potassium antimonate. This produces a more copious precipitate of calcium antimonate than fixation without oxalate. It is suggested, therefore, that the histochemical localization of calcium by antimonate techniques may not always represent thein vivo situation. The use of oxalate during fixation, however, may give a better indication of the amount of calcium stored within a cell.     

Degranulation of rat omental mast cells by A(1) receptor agonists in vitro

The haemodynamic effects of adenosine are thought to result in part from a release of mast cell amines via A3 receptor stimulation. To investigate the nature of the receptors involved in adenosine-induced mast cell degranulation in the rat isolated omentum we have used adenosine analogues with varying specificities as activators of the A(1), A(2) and A(3) receptors, and antagonists with differing specificities for A(1) and A(2) receptors. Analogues which act predominantly as A(1) (e.g. N(6)-cyclopentyladenosine) or as mixed A(1)/A(2) receptor agonists (e.g. adenosine, inosine, 5'-(Nethylcarboxamido) adenosine) caused mast cell degranulation, whereas a predominantly A3 receptor agonist (IB-MECA) was inactive. Pre-treatment of the omentum with the A(1)/A(2) receptor antagonist 8-phenyltheophylline or with the more specific A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine significantly reduced agonist-induced degranulation. Pre-treatment with disodium cromoglycate or with BN52021 also reduced degranulation of mast cells in response to N(6)-cyclopentyladenosine. In the rat isolated omental mast cell we conclude that degranulation is an indirect result of A(1) receptor stimulation. Platelet-activating factor release appears to mediate at least part of the degranulation.     

Identification of a novel species of Eimeria Schneider, 1875 from the woylie,Bettongia penicillata Gray (Diprotodontia: Potoroidae) and the genetic characterisation of three Eimeria spp. from other potoroid marsupials

Faecal samples (n = 1,093) collected from the woylie Bettongia penicillata Gray, in south-western Australia were examined for the presence of coccidian parasites. Eimeria sp. oöcysts were detected in 15.2% of samples. Faecal samples obtained from the eastern bettong Bettongia gaimardi (Desmarest) (n = 4) and long-nosed potoroo Potorous tridactylus (Kerr) (n = 12) in Tasmania, were also screened for the presence of Eimeria spp. (prevalence 50% and 41.7%, respectively). Morphological and genetic comparison with other known species of Eimeria indicates that the material identified in woylies is novel. This study aimed to (i) morphologically describe and genetically characterise Eimeria woyliei n. sp. found in woylies; and (ii) genetically characterise Eimeria gaimardi Barker, O’Callaghan & Beveridge, 1988, Eimeria potoroi Barker, O’Callaghan & Beveridge, 1988, and Eimeria mundayi Barker, O’Callaghan & Beveridge, 1988, from other potoroid marsupials. Molecular phylogenetic analyses conducted at the 18S rDNA and mitochondrial cytochrome c oxidase subunit 1 (cox1) loci revealed that E. woyliei n. sp. was most closely related to Eimeria setonicis Barker, O’Callaghan & Beveridge, 1988, at the 18S rDNA locus, and Eimeria trichosuri O’Callaghan & O’Donoghue, 2001, at the cox1 locus. Eimeria woyliei n. sp. is the sixth species of Eimeria to be formally described from potoroid marsupials.

     

Genetic mapping of hereditary mixed polyposis syndrome to chromosome 6q.

Hereditary mixed polyposis syndrome (HMPS) is characterized by atypical juvenile polyps, colonic adenomas, and colorectal carcinomas. HMPS appears to be inherited in an autosomal dominant manner. Genetic linkage analysis has been performed on a large family with HMPS. Data did not support linkage to the APC locus or to any of the loci for hereditary nonpolyposis colorectal cancer. Evidence that the HMPS locus lies on chromosome 6q was, however, provided by significant two-point LOD scores for linkage between HMPS and the D6S283 locus. Analysis of recombinants and multipoint linkage analysis suggested that the HMPS locus lies in a 4-cM interval containing the D6S283 locus and flanked by markers D6S468 and D6S301.     

Mast cell amines and inosineinduced vasoconstriction in the rat hind limb

Under certain circumstances injected inosine causes a net vasoconstrictive effect on the arterioles, which has been attributed to 5-hydroxytryptamine (5HT) released in response to adenosine type 3 (A(3)) receptor stimulation of mast cells residing in the adventitia. We have sought further evidence for this hypothesis using blood vessels of the rat hind limb perfused in vitro at constant rate with a gelatin-containing physiological salt solution. Injection of inosine (2.7 mg) caused a rise in perfusion pressure, which was only slightly increased by inclusion of N-nitro-L-arginine methyl ester (100 muM) in the perfusate. Inclusion in the perfusate of cyproheptadine (1 muM), compound 48 80 (1 mug ml), 8-phenyltheophylline (1 muM) or 8-cyclopentyl-1,3 dipropylxanthine (0.1 muM) greatly reduced the pressor response to inosine. The pressor effect of injected 5HT (400 mug) was abolished by pre-treatment with cyproheptadine, but not by pre-treatment with compound 48 80. These results suggest that the net pressor response to injected inosine was mainly the result of an A(1) receptor-mediated release of 5HT, most probably from mast cells. No evidence was found for an involvement of A(3) receptor stimulation.     

Evaluating Stress Physiology and Parasite Infection Parameters in the Translocation of Critically Endangered Woylies (Bettongia penicillata)

Translocation can be stressful for wildlife. Stress may be important in fauna translocation because it has been suggested that it can exacerbate the impact of infectious disease on translocated wildlife. However, few studies explore this hypothesis by measuring stress physiology and infection indices in parallel during wildlife translocations. We analysed faecal cortisol metabolite (FCM) concentration and endoparasite parameters (nematodes, coccidians and haemoparasites) in a critically endangered marsupial, the woylie (Bettongia penicillata), 1–3 months prior to translocation, at translocation, and 6 months later. FCM for both translocated and resident woylies was significantly higher after translocation compared to before or at translocation. In addition, body condition decreased with increasing FCM after translocation. These patterns in host condition and physiology may be indicative of translocation stress or stress associated with factors independent of the translocation. Parasite factors also influenced FCM in translocated woylies. When haemoparasites were detected, there was a significant negative relationship between strongyle egg count and FCM. This may reflect the influence of glucocorticoids on the immune response to micro- and macro-parasites. Our results indicate that host physiology and infection patterns can change significantly during translocation, but further investigation is required to determine how these patterns influence translocation success.     

Effect of artificial surveillance cues on reported moral judgment: Experimental failures to replicate and two meta-analyses

Several papers have reported that artificial surveillance cues, such as images of watching eyes, cause anonymous participants to behave as if they are actually under surveillance, thus increasing moral behavior. In a series of four experiments, we found no evidence that artificial surveillance cues impact reported moral judgment, self-rated possession of positive traits, or religiosity. Two small meta-analyses, both comprising six experiments investigating the effect of artificial surveillance cues on moral judgment, provided mixed conclusions. One meta-analysis produced a mean effect size not significantly different from zero and the other produced a mean effect size on the edge of significance. On the whole, artificial surveillance cues have inconsistent effects, or possibly no effect, on moral outcomes.     

Evaluating the Effects of Ivermectin Treatment on Communities of Gastrointestinal Parasites in Translocated Woylies (Bettongia penicillata)

Wildlife species are often treated with anti-parasitic drugs prior to translocation, despite the effects of this treatment being relatively unknown. Disruption of normal host–parasite relationships is inevitable during translocation, and targeted anti-parasitic drug treatment may exacerbate this phenomenon with inadvertent impacts on both target and non-target parasite species. Here, we investigate the effects of ivermectin treatment on communities of gastrointestinal parasites in translocated woylies (Bettongia penicillata). Faecal samples were collected at three time points (at the time of translocation, and 1 and 3 months post-translocation) and examined for nematode eggs and coccidian oocysts. Parasite prevalence and (for nematodes) abundance were estimated in both treated and untreated hosts. In our study, a single subcutaneous injection of ivermectin significantly reduced Strongyloides-like egg counts 1 month post-translocation. Strongyle egg counts and coccidia prevalence were not reduced by ivermectin treatment, but were strongly influenced by site. Likewise, month of sampling rather than ivermectin treatment positively influenced body condition in woylies post-translocation. Our results demonstrate the efficacy of ivermectin in temporarily reducing Strongyloides-like nematode abundance in woylies. We also highlight the possibility that translocation-induced changes to host density may influence coinfecting parasite abundance and host body condition post-translocation.

     

Rate of perfusion modulates colloidal carbon leakage from rat intestinal microvessels in vitro

In order to investigate the effects of varying the rate of flow on endothelial integrity the rat isolated small intestinal vasculature was perfused at 1, 5, 10 or 20 ml/min with a gelatin-containing physiological salt solution (GPSS), followed by an injection of colloidal carbon suspension (CC). Significantly greater microvascular CC leakage occurred at 1 or 5 ml/min than at 10 or 20 ml/ mitt. CC leakage at the two slower rates of flow was reduced by adding red blood cells to the GPSS, suggesting that the microvascular endothelium became hypoxic when perfused with GPSS at 1 or 5 ml/min. After perfusion at 20 ml/min with GPSS containing resiniferatoxin (1 muM) or 5-hydroxytryptamine (100 muM), CC leakage was significantly lower than after similar perfusion at 10 ml/min. Two nitric oxide (NO) synthesis blockers, N-nitro-L-arginine methyl ester (L-NAME, 100 muM) and methylene blue (20 muM), and an NO scavenger CPTIO (100 muM) each increased CC leakage. This suggests that NO was being produced at perfusion rates of 10 or 20 ml/min. Sodium nitroprusside (10 muM), 8-bromo-cGMP (100 muM) and BN52021 (10 muM) each significantly reduced CC leakage in the presence of L-NAME.