Preliminary safety and biological efficacy studies of ethyl pyruvate in normal mature horses

Reasons for performing the study: Endotoxaemia causes substantial morbidity and mortality in horses with colic and sepsis. Ethyl pyruvate is a novel anti‐inflammatory medication that improved survival in preclinical models of severe sepsis endotoxaemia and intestinal ischaemia and reperfusion in rodents, swine, sheep and dogs and may be a useful medication in horses. Hypothesis: Ethyl pyruvate has no adverse effects in normal horses and is biologically active based on suppression of proinflammatory gene expression in endotoxin stimulated whole blood, in vitro. Methods: Physical and neurological examinations, behaviour scores, electrocardiograms and clinicopathological tests were performed on 5 normal healthy horses receiving 4 different doses of ethyl pyruvate. Doses included 0, 50, 100 and 150 mg/kg bwt administered in a randomised crossover design with a 2 week washout period between doses. Biological efficacy was assessed by stimulating whole blood with endotoxin from the horses that received ethyl pyruvate prior to and 1 and 6 h after drug infusion. Gene expression for TNFα, IL‐1β and IL‐6 was assessed. Results: There were no effects of drug or dose (0, 50, 100 or 150 mg/kg bwt) on any of the physical or neurological examination, behaviour factors, electrocardiogram or clinical pathological results collected from any of the horses. All parameters measured remained within the normal reference range. There was a significant reduction in TNFα, IL‐1β and IL‐6 gene expression in endotoxin stimulated whole blood from horses 6 h after receiving 150 mg/kg bwt ethyl pyruvate. There were no detectable effects on gene expression of any of the other doses of ethyl pyruvate tested. Conclusion: We were unable to detect any detrimental effects of ethyl pyruvate administration in normal horses. Ethyl pyruvate significantly decreased proinflammatory gene expression in endotoxin stimulated blood 6 h after drug administration. Clinical relevance: Ethyl pyruvate may be a safe, effective medication in endotoxaemic horses.     

Isolation and molecular characterisation of Neospora caninum in cattle in New Zealand

AIM: To isolate Neospora caninum from the brains of naturally infected cattle and use molecular techniques to characterise the isolates.

METHODS: Neospora caninum tachyzoites were isolated in Vero cell culture from the brains of a cow and two calves. The isolates were characterised using polymerase chain reaction (PCR) methods, DNA sequencing, an immunofluorescent anti-body test (IFAT), transmission electron microscopy (TEM), and immunohistochemistry (IHC). The brains of the three cattle were subjected to histopathological examination. A pathogenicity study was conducted in 120 BALB/c mice.

RESULTS: Neospora caninum tachyzoites were isolated from all three cases and first observed in vitro between 14 and 17 days post-inoculation. Parasites were sub-cultured and maintained in Vero cell culture for more than 6 months. PCR products were generated for all three isolates, using two different primers. Sequencing of the PCR products and a subsequent BLAST search identified the isolates as N. caninum. In addition, the isolates tested positive using IFAT and IHC, and ultrastructure revealed by TEM was characteristic of N. caninum. Histopathological examination revealed lesions characteristic of N. caninum in 1/3 brains. In the pathogenicity study using BALB/c mice, the mortality rate was 3–7%.

CONCLUSION: This was the first successful isolation of N. caninum in New Zealand confirmed using molecular characterisation tests.     

Enzymatic removal of off-flavors from apple juice

Contaminating microorganisms such as Actinomycetes, Alicyclobacillus, and Chlostridium can generate off-flavors in apple juices. Such bacterial metabolites represent, besides phenol types such as guaiacol and 2,6-dibromophenol, a broad range of other chemicals, for example, geosmin, 2-methylisoborneol, or alpha-terpineol. A laccase from Trametes hirsuta was purified, immobilized, and applied for the selective elimination of off-flavor substances in apple juice caused by microbial contamination. The evaluation using GC-MS showed that enzymatic treatment could reduce the amount of guaiacol and 2,6-dibromophenol in apple juice significantly by 99 and 52%, respectively. Upon addition of mediators, the degradation could be increased and the spectrum of substrates extented. Furthermore, commercial apple juices spiked with off-flavors were treated in a continuous-flow reactor and tested by sensory evaluation.     

Atmospheric concentrations and deposition of Hg to A deciduous forest atwalker branch watershed,Tennessee, USA

Aerosol and total vapor-phase Hg concentrations in air have been measured at Walker Branch Watershed, Tennessee for ≈ 2 yr. Airborne Hg at this site is dominated by vapor forms which exhibit a strong seasonal cycle, with summer maxima that correspond to elevated air temperature. Concentrations in this forest are near background levels; however, concentrations at a site within 3 km are significantly elevated due to emissions from Hg-contaminated soils. The concentration data have been combined with a recently modified dry deposition model to estimate dry deposition fluxes to the deciduous forest at Walker Branch. Weekly mean modeled V_d values for Hg° ranged from <0.01 (winter) to > 0.1 (summer) cm s¹. Weekly dry deposition fluxes ranged from <0.1 μg m⁻² during winter to > 1.0 μgg m⁻² in the summer. Our dry deposition estimates plus limited measurements of wet deposition in this area indicate that dry deposition may be the dominant input process in this forest, at least during the summer.     

Efficient inhibition of the Alzheimer's disease beta-secretase by membrane targeting

beta-Secretase plays a critical role in beta-amyloid formation and thus provides a therapeutic target for Alzheimer's disease. Inhibitor design has usually focused on active-site binding, neglecting the subcellular localization of active enzyme. We have addressed this issue by synthesizing a membrane-anchored version of a beta-secretase transition-state inhibitor by linking it to a sterol moiety. Thus, we targeted the inhibitor to active beta-secretase found in endosomes and also reduced the dimensionality of the inhibitor, increasing its local membrane concentration. This inhibitor reduced enzyme activity much more efficiently than did the free inhibitor in cultured cells and in vivo. In addition to effectively targeting beta-secretase, this strategy could also be used in designing potent drugs against other membrane protein targets.     

Anchoring tick salivary anti-complement proteins IRAC I and IRAC II to membrane increases their immunogenicity

Tick salivary proteins are promising targets for the development of anti-tick vaccines. Recently, we described two paralogous anti-complement proteins, called Ixodes ricinus anti-complement (IRAC) proteins I and II, that are co-expressed in tick I. ricinus salivary glands. However, our previous attempts to immunize rabbits against IRAC via infection with recombinant Bovine herpesvirus 4 (BoHV-4) vectors invariably failed although both recombinants expressed high levels of functional IRAC proteins in vitro. As IRAC are soluble monovalent antigens, one of the possible explanations is that monovalent ligation of the B-cell receptor induces receptor activation but fails to promote antigen presentation, a phenomenon that is thought to induce a state of B-cell tolerance. In the present study, we tried to increase IRAC immunogenicity by expressing them as oligovalent antigens. To this end, IRAC were fused to membrane anchors and BoHV-4 vectors expressing these recombinant forms were produced. The immunization potentials of recombinant viruses expressing either secreted or transmembrane IRAC proteins were then compared. While the former did not induce a detectable immune response against IRAC, the latter led to high titres of anti-IRAC antibodies that only marginally affected tick blood feeding. All together, the data presented in this study demonstrate that the immunogenicity of a soluble antigen can be greatly improved by anchoring it in membrane.     

Suspected primary immunodeficiency syndrome in three related Irish wolfhounds

Three related Irish wolfhound dogs less than one year old presented with a history of chronic nasal discharge and signs of lower respiratory tract disease. These responded well to treatment initially but were chronically recurring. Cursory evaluation of the immune system (full blood counts, globulin determination and fractionation, electrophoresis and lymphocyte blastogenesis) seemed to indicate a cell-mediated immunodeficiency which, because of the age of the patients, Is strongly suspected to be primary.     

Pharmacological evidence for a discrete neurotoxic action of dieldrin (HEOD) in the American cockroach,Periplaneta americana (L.)

The excitatory effects of HEOD poisoning on spontaneous activity, and synaptic function in the sixth abdominal ganglion of the American cockroach are described. These effects are shown to be antagonized by Mg²⁺, hemicholinium-3, atropine, and d-turbocurarine. HEOD poisoning did not alter the nicotine sensitivity of the ganglion. On the basis of the findings it is hypothesized that HEOD acts at presynaptic membranes of cholinergic junctions, causing excessive and spontaneous release of presynaptic stores of acetylcholine.