Quantitation of adenine nucleotides in equine colonic mucosal tissue using high performance liquid chromatography.

The objectives were to use high performance liquid chromatography (HPLC) to validate an established method for adenine nucleotide separation in equine colonic mucosal tissue, to determine the inherent variability in the tissue and extraction method, and to determine the stability of ATP, ADP, and AMP in the tissue with time. Equine colonic mucosal tissue obtained from a single horse was immediately submersed in liquid nitrogen, and stored at -70 degrees C. Samples were lyophilized, extracted, and separated by HPLC. The limit of quantitation was 0.05 microg/mL. The coefficient of variation for the instrument was less than 10% for all nucleotides measured. When the tissue was not homogenized prior to sampling, there were significant differences in adenine nucleotide content between samples. However, when the tissue was homogenized prior to analysis, these differences were no longer significant. There was no significant decrease in ATP, ADP, or AMP content over a 54-day analysis period.     

Lupus-Type "Anticoagulant" in a Dog With Hemolysis and Thrombosis

A circulating anticoagulant was detected in a 2-year-old Chesapeake Bay Retriever with hemolytic anemia, nephrotic syndrome, thrombocytopenia, polyarthropathy, and pulmonary thromboembolism. A persistent prolongation of the activated partial thromboplastin time (aPTT) was detected, and it did not correct with repeated administration of fresh frozen plasma. The aPTT was still prolonged, with a 1:1 mixture of patient's plasma and normal dog plasma in vitro, suggesting the presence of a circulating inhibitor. Results of assays to characterize the inhibitor were compatible with those described for the lupus anticoagulant in human patients with systemic lupus erythematosus. Paradoxically, patients having the lupus anticoagulant are at increased risk for thrombosis. Pulmonary thromboembolism has been described as a frequent complication of immune-mediated hemolytic anemia in the dog, and the presence of a circulating anticoagulant should be considered as a potential mechanism.     

A streak disease of pearl millet caused by a leafhopper-transmitted geminivirus

The cause of a streak disease of pearl millet (Pennisetum glaucum), originating from Nigeria, has been attributed to a geminivirus belonging to the African streak virus cluster. A full-length, infectious clone of the virus was obtained which was transmissible by the vectorCicadulina mbila (Naudé). Analysis of the complete nucleotide sequence of the coat protein gene of this virus shows it to be most closely related to sugarcane streak virus. The possible evolutionary implications of this finding are discussed.     

‘Plantibodies’: a flexible approach to design resistance against pathogens

Engineering resistance against various diseases and pests is hampered by the lack of suitable genes. To overcome this problem we started a research program aimed at obtaining resistance by transfecting plants with genes encoding monoclonal antibodies against pathogen specific proteins. The idea is that monoclonal antibodies will inhibit the biological activity of molecules that are essential for the pathogenesis. Potato cyst nematodes are chosen as a model and it is thought that monoclonal antibodies are able to block the function of the saliva proteins of this parasite. These proteins are, among others, responsible for the induction of multinucleate transfer cells upon which the nematode feeds. It is well documented that the ability of antibodies to bind molecules is sufficient to inactivate the function of an antigen and in view of the potential of animals to synthesize antibodies to almost any molecular structure, this strategy should be feasible for a wide range of diseases and pests.Antibodies have several desirable features with regard to protein engineering. The antibody (IgG) is a Y-shaped molecule, in which the domains forming the tips of the arms bind to antigen and those forming the stem are responsible for triggering effector functions (Fc fragments) that eliminate the antigen from the animal. Domains carrying the antigen-binding loops (Fv and Fab fragments) can be used separately from the Fc fragments without loss of affinity. The antigen-binding domains can also be endowed with new properties by fusing them to toxins or enzymes. Antibody engineering is also facilitated by the Polymerase Chain Reaction (PCR). A systematic comparison of the nucleotide sequence of more than 100 antibodies revealed that not only the 3′-ends, but also the 5′-ends of the antibody genes are relatively conserved. We were able to design a small set of primers with restriction sites for forced cloning, which allowed the amplification of genes encoding antibodies specific for the saliva proteins ofGlobodera rostochiensis. Complete heavy and light chain genes as well as single chain Fv fragments (scFv), in which the variable parts of the light (V_L) and heavy chain (V_H) are linked by a peptide, will be transferred to potato plants. A major challenge will be to establish a correct expression of the antibody genes with regard to three dimensional folding, assembly and intracellular location.     

THE EFFECTS OF PATIENT POSITIONING AND SLICE SELECTION ON CANINE ACETABULAR ANGLE ASSESSMENT WITH COMPUTED TOMOGRAPHY

Acetabular angles (AAs) and dorsal acetabular rim angles acquired by computed tomographic (CT) imaging have been used to assess patient response to juvenile pubic symphysiodesis surgery. The purpose of this study was to evaluate the effects of patient positioning and slice selection on these angles, and an attempt was made to devise a repeatable method of measuring these angles that would eliminate positioning effects. We found significant variation in AAs with small differences in pelvic tilt and slice selection. Dorsal acetabular rim angles were not affected. As a result of positioning differences from one CT study to the next, every attempt should be made to standardize pelvic tilt, or eliminate its effect on AAs by standardizing gantry angle in relation to an anatomic landmark that will not change over time. The floor of the sacral vertebral canal may be a reasonable landmark for this purpose and deserves further study.     

Effects of Latent Infection of Stock Plants and Abundance of Thrips on the Occurrence of <Emphasis Type="Italic">Tomato spotted wilt virus </Emphasis>in Chrysanthemum Fields

DAS-ELISA proved to be reliable enough to detect a latent infection by Tomato spotted wilt virus (TSWV) in asymptomatic stock plants of chrysanthemum. A high density of Frankliniella occidentalis, the predominant vector, in the presence of latently infected stock plants resulted in a high incidence of disease in the chrysanthemum production field. The incidence of disease was low when the vector thrips were not abundant in spite of the presence of latently infected stock plants. These results suggest that an infestation of the vector thrips causes severe secondary spread of TSWV originating from latently infected stock plants in chrysanthemum production fields. Received 27 July 2001/ Accepted in revised form 27 November 2001