Characterisation of a 34 kD protein from potato cyst nematodes, using monoclonal antibodies with potential for species diagnosis

Two monoclonal antibodies, which differentially recognise the two species of potato cyst nematodes (PCN), Globodera pallida and G. rostochiensis, are described. They have been shown to have potential for quantification of these two species, recognising proteins of the same molecular weight (34 kD) in both species. Further investigation showed these proteins to have isoelectric points at pH values of 5.7 in G. pallida and 5.9 in G. rostochiensis, in common with the proteins used by Fleming & Marks (1983) to differentiate the species of PCN. They are likely to be structurally very similar, with the same physiological function (and therefore similar concentrations) in the two species. In cross-reactivity tests with a wide range of soil nematode species, the antibodies reacted strongly only with species of the genus Globodera, and thereby confirmed their potential as the basis of a quantitative immunoassay likely to be useful in management of PCN populations.     

A search for associations between major histocompatibility complex restriction fragment length polymorphism bands and resistance to Haemonchus contortus infection in sheep

Polymorphic bands were detected within the DQB and DRB regions of the ovine major histocompatibility complex by probing TaqI digested DNA from three large sheep half-sib families derived from a highly resistant ram. All animals were phenotypically assessed for Haemonchus contortus resistance by faecal egg counts and associations with RFLP bands and haplotypes were estimated using mixed model, best linear unbiased prediction statistical methods. Although the highly resistant sire was homozygous at the MHC, no significant associations were found between any band or haplotype and faecal egg count.     

Newly Imported Rieske Iron-Sulfur Protein Associates with Both Cpn60 and Hsp70 in the Chloroplast Stroma

The precursor of the Rieske FeS protein, a thylakoid membrane protein, was imported by isolated pea chloroplasts, and the mature protein was shown to be integrated into the cytochrome bf complex of the thylakoid membranes. Insertion into the thylakoid membrane was sensitive to the ionophores nigericin and valinomycin, suggesting a requirement for a proton motive force. A considerable proportion of the imported Rieske protein was detected in the stromal fraction of the chloroplasts, and this increased when membrane insertion was blocked with ionophores. Electrophoresis of the stromal fraction under nondenaturing conditions resolved two distinct complexes containing the Rieske protein. One of these complexes was identified as an association of the Rieske protein with the chaperonin Cpn60 complex by its electrophoretic mobility, Mg-ATP-dependent dissociation, and immunoprecipitation with anti-Cpn60 antibodies. Coimmunoprecipitation of imported Rieske protein with anti-heat shock protein 70 (Hsp70) antibodies indicated that the Rieske protein was also associated, in an ATP-dissociable form, with a chloroplast Hsp70 homolog. Immunoprecipitation analysis of an import time course detected the highest amounts of the Cpn60-Rieske protein complex early in the time course, whereas highest amounts of the Hsp70-Rieske protein complex were formed much later. The disappearance of the Cpn60-Rieske protein complex correlated with increased amounts of the Rieske protein in the thylakoid fraction.     

Echistatin disulfide bridges: selective reduction and linkage assignment.

Echistatin is the smallest member of the disintegrin family of snake venom proteins, containing four disulfides in a peptide chain of 49 residues. Partial assignment of disulfides has been made previously by NMR and chemical approaches. A full assignment was made by a newly developed chemical approach, using partial reduction with tris-(2-carboxyethyl)-phosphine at acid pH. Reduction proceeded in a stepwise manner at pH 3, and the intermediates were isolated by high performance liquid chromatography. Alkylation of free thiols, followed by sequencer analysis, enabled all four bridges to be identified: (1) at 20 degrees C a single bridge linking Cys 2-Cys 11 was broken, giving a relatively stable intermediate; (2) with further treatment at 41 degrees C the bridges Cys 7-Cys 32 and Cys 8-Cys 37 became accessible to the reagent and were reduced at approx. equal rates; (3) the two bicyclic peptides produced in this manner were less stable and could be reduced at 20 degrees C to a peptide that retains a single bridge linking Cys 20-Cys 39; and (4) the monocyclic peptide can be reduced to the linear molecule at 20 degrees C. Some disulfide exchange occurred during alkylation of the bicyclic intermediates, but results unambiguously show the pattern to be [2-11; 7-32; 8-37; 20-39]. A comparison is made with kistrin, a longer disintegrin whose disulfide structure has been proposed from NMR analysis.     

Biosynthesis and function of membrane bound and secreted forms of recombinant CD11b/CD18 (Mac-1)

Full-length (membrane bound) and truncated (secreted) forms of the beta 2 integrin heterodimer, CD11b/CD18 (Mac-1), were expressed in a human kidney cell line (293) that normally does not express leukocyte adhesion molecules (Leu-CAMs). The biosynthesis of recombinant Mac-1 in 293 cells differed from that reported for leukocytes in that heterodimer formation was not required for CD11b to be exported to the cell surface. A stable cell line was constructed that constitutively secreted the recombinant, truncated Mac-1 heterodimer into growth conditioned cell culture medium. A novel monoclonal antibody that enabled an immunoaffinity method for the selective purification of recombinant Mac-1 heterodimers was identified. Sufficient protein was purified to allow the first measurement of the 50% inhibitory concentration (IC₅₀) for CD11b/CD18 and for the direct comparison of the inhibitory activity of recombinant soluble Mac-1 with that of various CD18 and CD11b specific monoclonal antibodies. Purified recombinant soluble Mac-1 inhibited the binding of neutrophils, activated by opsonized zymosan or fMet-Leu-Phe peptide, to human umbilical vein endothelial cells. Similarly, the recombinant integrin was effective in inhibiting the binding of unactivated neutrophils to tumor necrosis factor (TNF-alpha) activated endothelial cells. The availability of an abundant source of purified, biologically active Mac-1 will enable direct physical and chemical investigations into the relationship between the structure and function of this leukocyte adhesion molecule.     

Viability of Teschen Disease Virus

A portion of spinal cord taken from a pig infected with the Konratice strain of Teschen Disease virus was found to be infectious for swine after an eleven year period of storage at dry ice temperature. The virus was recovered in tissue culture from the brains of two experimentally infected pigs, titrated, and a serum-virus neutralization test performed.