peer reviewed | Increases of plasma concentrations of neutrophil myeloperoxidase (MPO) can be used as markers of polymorphonuclear leucocytes (PMN) activation in pathological situations (sepsis, acute lung injury, acute inflammation). To develop an assay for measurement of plasma MPO in horses during the above-mentioned infectious and inflammatory conditions, MPO was purified from equine PMN isolated from blood anticoagulated with citrate. PMN were extracted in a saline milieu (0.2 M Na acetate, 1 M NaCl, pH 4.7) to eliminate most of cellular proteins. Pellets were then extracted in the same buffer containing cationic detergent (1% cetyltrimethyl ammonium bromide). The supernatant was further purified by ion exchange chromatography (Hiload S Sepharose HP column 0.5 x 26 cm, equilibrated with 25 mM Na acetate, 0.2 M NaCl, pH 4.7) with a NaCl gradient (until 1 M). Most of the peroxidase activity of MPO (spectrophotometrically measured by the oxidation of orthodianisidine by hydrogen peroxide) was eluted at 0.65 M NaCl. MPO was further purified by gel filtration chromatography (Sephacryl S 200 column 2.6 x 42 cm with 25 mM Na acetate, 0.2 M NaCl, pH 4.7). MPO (specific activity: 74.3 U/mg) was obtained with a yield of 30% from the detergent extraction supernatant. Electrophoresis (non-reducing conditions) showed 3 bands identified, by comparison with human MPO, (i) the mature tetrameric enzyme (150 kDa) with 2 light and 2 heavy subunits, (ii) the precursor form (88 kDa) and (iii) a form of the heavy subunit without the prosthetic heme group (40 kDa). The mature enzyme and its precursor were glycosylated and possessed peroxidase activity. Equine MPO showed strong similarities with human and bovine MPO, with an absorption peak at 430 nm (Soret peak) characteristic of ferrimyeloperoxidase. Enzymatic activity was pH dependent (optimal value at pH 5.5).

Restriction endonuclease patterns of genomic fragments separated by use of pulsed-field gel electrophoresis were used to differentiate Brucella abortus strain RB51, a rifampin-resistant mutant of the standard virulent strain 2308, from other brucellae. Results were compared with results obtained by use of standard methods for characterizing brucellae. Electrophoretic patterns of the ATCC type strains allowed identification of the strains to the level of species. Genomic profiles of B abortus biovars 1, 2, and 4 were similar, as were those of biovars 5, 6, and 9. The profile of biovar 3 was similar to that of biovars 5, 6, and 9, except for a missing band at 93 kb and additional bands at 65 and 67 kb. A different fingerprint was detected in B abortus strain RB51, using the pulsed-field gel electrophoresis patterns of genomic DNA digested with restrictive endonuclease Xba I. The profile of B abortus strain RB51 contained a band at 104 kb, as opposed to a 109-kb fragment within profiles of B abortus isolates from naturally infected cattle, bison, and elk. Despite known biochemical and biological differences between RB51 and its parent strain (2308), restriction endonuclease analysis results were similar.

Adhesion of equine spermatozoa to homologous oviduct epithelial cells (OEC) in vitro results in specific changes in spermatozoa and OEC function. To test the hypothesis that adhesion of spermatozoa affects protein synthesis and secretion by OEC, the following treatment groups were established in culture: OEC with culture medium only; control spermatozoa in culture medium only; OEC in coculture with spermatozoa; and OEC and spermatozoa in coculture, but physically separated by a microporous membrane. The experiment was replicated within each of 4 ejaculates from 3 stallions. De novo protein secretion by OEC was measured and compared by incorporation of [35S]methionine, and evaluated, using two-dimensional polyacrylamide gel electrophoresis and fluorography. Monolayers of OEC secreted a large number of proteins of molecular mass ranging from 14 to 205 kd. Adhesion of spermatozoa consistently caused reduced synthesis of 2 OEC secretory proteins and new or increased synthesis of 6 proteins. When spermatozoa and OEC were separated by a microporous membrane, some but not all of these changes were duplicated. Synthesis of 3 OEC secretory proteins, unaffected by binding of spermatozoa, was reduced when spermatozoa were prevented from contact with OEC by a microporous membrane. Adhesion of equine spermatozoa to homologous OEC monolayers and presence of equine spermatozoa resulted in qualitative and quantitative changes in synthesis and secretion of proteins by OEC. These changes have implications for storage, longevity, and maturation of spermatozoa.

The 43 monoclonal antibody raised against feline T cells was found to react with a single-chain glycoprotein of Mr 72,000 that is present on most thymocytes, 60% of lymph node cells, 20% of splenocytes, and 45% of blood mononuclear cells. All CD4+ and CD8+ T cells were found to express the 43-reactive determinant, as did a small subpopulation of CD4-/CD8-/IgM- lymphocytes in the periphery. The 43-reactive determinant was not detected on B cells, macrophages, or other types of blood cells. The 43 antigen was phosphorylated in resting and activated T cells. Its expression was upregulated by stimulation with phorbol myristate acetate and with phytohemagglutinin. When added to concanavalin A-stimulated T-cell cultures in low concentrations, the 43 antibody was found to augment mitogenesis. The data indicate that this antibody may identify a CD5 homologue on feline T cells.

In mammalian species studied previously, pepsinogen consisted of biochemically different groups of isozymogens. By use of gel filtration chromatography and electrophoresis, we isolated a predominant pepsinogen from the gastric mucosa of a horse. Peptide mapping with V8 protease revealed differences with its porcine homologue. However, porcine and equine pepsinogens, when activated to pepsin, had a similar pattern of activity when hemoglobin was used as substrate. Those results suggest that differences must exist in the primary structure of the pepsinogens of the 2 species.

Articular cartilage explants from 3 horses were maintained in tissue culture to test the effects of a polysulfated glycosaminoglycan on proteoglycan biosynthesis. Cultures were exposed to concentrations of 0, 50, or 200 microgram of the drug/ml for either 2 days or 6 days, and labeled with 35S, before measuring the content of sulfated proteoglycan in the culture media and in extracts of cartilage. In a second experiment, the explants were incubated with the isotope and subsequently exposed to the same concentrations of the polysulfated glycosaminoglycan for 4 days. Subsequently, the amount of remaining labeled proteoglycan was determined. Gel filtration chromatography was used to compare the hydrodynamic size of proteoglycans from the cartilage explants in each experiment. Polysulfated glycosaminoglycan caused a dose-dependent depression of sulfated proteoglycan synthesis, which was statistically significant after 6 days of exposure. Radioactive proteoglycan content in explants was similar in the experiment involving isotopic labeling prior to exposure to the drug. Proteoglycan monomer size was similar in all treatment groups. It was concluded that polysulfated glycosaminoglycan caused a modest depression in proteoglycan synthesis, had little effect on endogenous proteoglycan degradation, and did not influence the size of sulfated proteoglycans synthesized by normal equine chondrocytes in explant culture.

Conditions for purification of the ninth component of bovine complement (C9) were established. The conditions for binding and elution from diethylaminoethyl cellulose and hydroxylapatite were different than for human C9. Serum albumin, a frequent contaminant of bovine C9 preparations, was removed by chromatography on reactive-red agarose. The calculated molecular weight of bovine C9 was 66,000, and reduction with 2-mercaptoethanol affected its migration on polyacrylamide gel electrophoresis. Some preparations of bovine C9 migrated as 2 bands when partially reduced, but extensively reduced preparations had a single band.

Viral RNA oligonucleotide fingerprinting was used to discriminate 3 cytopathic vaccine bovine viral diarrhea viruses (BVDV) grown in medium supplemented with serum contaminated with noncytopathic BVDV from the same 3 viruses grown in cell culture free of BVDV. Oligonucleotide tide fingerprinting also effectively discriminated between reference Singer BVDV, NADL BVDV, and New York-1 BVDV grown in BVDV-free noncontaminated or BVDV-contaminated cell cultures. Oligonucleotide fingerprint mapping of viral RNA maybe used to determine the purity of virus stocks, as well as that of BVDV vaccines.

C-reactive protein (CRP) was isolated from equine serum by use of calcium-dependent affinity chromatography conjugated pneumococcal C-polysaccharide, anion exchange chromatography, and gel filtration. It was identified as genuine CRP by its immunochemical cross-reactivity with anti-human CRP, its homology with human CRP in amino acid composition, and its pentameric structure as revealed by electron microscopy. Purified equine CRP had a molecular weight of approximately 118,000 and was composed of 5 identical, nonglycosylated and noncovalently associated subunits with molecular weight of approximately 23,000 each. Equine CRP migrated in the region between beta- and gamma-globulin by results of immunoelectrophoresis, and its isoelectric point was about 7.0. In horses, increased CRP concentration was associated with clinical pneumonitis, enteritis, and arthritis, compared with values obtained in clinically normal horses by use of single radial immunodiffusion method. After IM administration of turpentine oil or castration, serum CRP concentration increased to 6 times higher than baseline values. Results indicate that CRP may be an acute-phase reactant protein in horses.

Cytoplasmic proteins from unsporulated and sporulated Eimeria maxima oocysts were analyzed by gel-filtration column chromatography. Unsporulated oocysts were characterized as having 3 major cytoplasmic proteins and sporulated oocysts as having 5 major cytoplasmic proteins. Molecular weights ranged from 5 X 10(3) to 1.4 X 10(6). Larger molecular weight proteins were detected in sporulated and unsporulated oocysts, but were associated more with sporocysts of sporulated oocysts.