Investigation of the structure of equine articular cartilage link protein (LP) from individuals ranging in age from 1 to 15 years identified 3 distinct isoforms having molecular weights of 46,000, 43,000, and 41,000. The relative amounts of each of the 3 isoforms altered with age. The largest form did not change with age; however, amounts of the Mr 43,000 and 41,000 forms increased with increasing age. The results suggested that an accumulation, in the extracellular matrix of cartilage, of these 2 smaller products may have arisen from proteolytic cleavage. The complete amino acid sequence of the protein core was determined from complementary DNA products prepared by polymerase chain reaction amplification of cartilage LP mRNA. The sequence had 96% similarity with human LP and with that of other species for which the primary structure has been determined. This high degree of sequence conservation and the isoform data indicate that extracellular processing of LP occurs by similar mechanisms in various species. At the transcription level, equine chondrocytes were found to express LP as 2 abundant mRNA of 5.0 and 3.0 kb, and a smaller mRNA of 1.5 kb. Processing of the LP mRNA in horses, thus, appears to be similar to that found in other species investigated, and although multiple transcripts are present, the coding region remains unaltered and only 1 protein product is made.

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).

The major difficulty in analysis of nitrofurans in feed, feed water, and food of animal origin is that nitrofurans have low molecular weights and fast metabolism. The principal goal of this study was to prepare a procedure for the determination of nitrofurans and their metabolites by a single method in different types of feed, feed water, and food of animal origin. Two-gram samples were subjected to hydrolysis and derivatisation processes by addition of hydrochloric acid and 2-nitrobenzaldehyde. After incubation the sample was purified by solid phase extraction technique. Nitrofurans were analysed using ultra-high-pressure liquid chromatography-MS/MS (UHPLC-MS/MS). The results of validation fulfil the requirement of the confirmatory criteria according to the European Commission Decision 2002/657/EC regarding apparent recoveries (88.9%–107.3%), repeatability (2.9%–9.4%) and within-laboratory reproducibility (4.4%–10.7%). The method can be successfully applied to monitor nitrofurans and their metabolites in different matrices.

Objective-To use transient and stable transfection of Chinese hamster ovary cells to clone the gene encoding feline erythropoietin (feEPO) protein, characterize the expressed protein, and assess its biological activity. Sample Population-Cultures of Chinese hamster ovary or TF-1 cells. Procedure-The gene encoding feEPO was cloned into a eukaryotic expression plasmid. Chinese hamster ovary cells were transiently or stably transfected with the plasmid. Expressed recombinant feEPO (rfeEPO) protein was purified from transiently transfected cells. The protein was characterized by use of SDS gel electrophoresis and western blot analysis. Biological activity was assessed by measuring thymidine incorporation by TF-1 erythroleukemic cells. Results-Purified rfeEPO from supernatants of transiently transfected cells was determined to be 34 to 40 kilodaltons (kd) by use of SDS gel electrophoresis, whereas the molecular weight predicted from the amino acid sequence was 21.5 kd. The banding pattern and high molecular weight suggested the protein was glycosylated. The rfeEPO proteins derived from transient or stable transfections subsequently were determined to be biologically active in vitro. Conclusions and Clinical Relevance-The gene encoding feEPO can be transfected into eukaryotic cells, and the expressed rfeEPO protein is biologically active in vitro. Cats with chronic renal failure often are anemic as a result of reduced expression of erythropoietin (EPO). Treatment with human-derived EPO stimulates RBCs in anemic cats; however, treatment is often limited by the development of antibodies directed against the recombinant human protein, which can then cross-react with endogenous feEPO. Recombinant feEPO may prove beneficial for use in cats with chronic renal failure.

Thirty healthy male horses were allotted to 3 groups and treated blindly during 4 days. Group-1 horses received unfractioned calcium heparin (100 IU/kg of body weight, SC, q 12 h). Group-2 horses received a single dose of a low-molecular-weight heparin (50 anti-Xa IU/kg, SC) every morning, and a similar volume of saline solution every evening. Group-3 horses received the vehicle (saline solution), SC, every 12 hours. Citrated and EDTA-anticoagulated blood samples were collected before starting the medication (T-0) and once daily 3 hours after each morning injection (T-3, T-27, T-51, and T-75). The PCV, hemoglobin concentration, RBC and platelet counts, and clotting times (activated partial thromboplastin time and thrombin time) were determined, and a microscopic examination to detect hemagglutination was performed. Plasma concentration of heparin was measured by use of the antifactor Xa activity assay. Bleeding time was determined on the first and fourth days, using a double-template method. The horses given unfractioned heparin had marked agglutination of erythrocytes after the first injection that became more pronounced as treatment progressed. Also, significant decrease in PCV, hemoglobin concentration, and RBC count was observed during treatment. Platelet count was significantly decreased after the first day, and clotting times were significantly prolonged. In contrast to the horses given unfractioned heparin, those given low-molecular-weight heparin did not have any agglutination of erythrocytes during the 4 days of treatment, and there were no significant changes in PCV, hemoglobin concentration, or RBC and platelet counts. Activated partial thromboplastin time increased slightly in the horses given low-molecular-weight heparin, although the values remained within reference range. Both groups of horses achieved adequate concentrations of heparin in plasma for prophylactic purposes, but those given low-molecular-weight heparin achieved those values after the first injection. Bleeding times were not significantly different between heparin-treated horses and horses given saline solution during treatment. We conclude that low-molecular-weight heparin may be used more safely and conveniently in horses, because it does not affect equine erythrocytes, platelets, or clotting and bleeding times.

Biological and biochemical characteristics of the leukotoxin of Fusobacterium necrophorum were determined. Culture supernatant of F necrophorum was toxic to polymorphonuclear neutrophilic leukocytes from cattle and sheep, but not to those from pigs and rabbits. Culture supernatant and sonicated bacterial cell fractions had low hemolytic activity and did not cause dermonecrosis in a guinea pig. Supernatant-derived leukotoxin was inactivated at 56 C for 5 minutes and became unstable at pH > 7.8 or < 6.6. Chemical treatment with 0.1% sodium dodecyl sulfate, 0.25% sodium deoxycholate, 5.2% sodium sulfide, or 0.25 mM titanium (III) citrate markedly decreased leukotoxicity. Enzymatic treatment with protease, trypsin, and chymotrypsin inactivated the toxin completely, whereas amylase had no effect. Use of protease inhibitors failed to prevent loss of leukotoxin activity. Using membrane partition chromatography and gel filtration, the estimated molecular weight of the toxin was > 300,000. On reduction and denaturation, the toxin dissociated into several components by use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The neutrophil plasma membrane has a major role in migration, phagocytosis, and destruction of microorganisms. Neutrophils isolated from blood and mammary secretions were homogenized, and the plasma membrane fraction was isolated on discontinuous sucrose gradient (20, 32, and 50%). Purity of plasma membrane preparation was determined by use of marker enzyme analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the membrane proteins was performed under reducing conditions for polypeptide characterization. The membrane proteins were also labeled with 125I externally, using 1,3,4,6-tetrachloro-3 alpha-6 alpha-diphenylglycouril, and proteins were separated by SDS-PAGE and autoradiographed. Compared with whole cell homogenate, the plasma membrane fraction obtained at the 20/32% interface was enriched for the marker enzymes, 5'-nucleotidase (16-fold), alkaline phosphatase (5.5-fold), and total phosphatase (26-fold). The membrane fraction had minimal specific activity for beta-glucuronidase (0.4-fold), compared with whole cell homogenate. Plasma membrane protein yield was about 500 micrograms/ 10(9) bovine blood neutrophils. The SDS-PAGE of plasma membrane proteins revealed 25 protein bands, of which 13 were major bands. There were 3 distinct bands (18, 36, and 65 kd) in the plasma membrane-enriched fraction (20/32 interface) that were not seen in other fractions (30/50% and pellet). Further, 125I-labeling identified 5 distinct protein bands (205, 140, 65, 35, and 30 kd). Blood and mammary neutrophils had similar polypeptide patterns, except that 36- and 65-kd bands were more prominent for blood neutrophils than for mammary neutrophils.

We purified porcine whey lactoferrin by affinity chromatography on a heparin-sepharose column, followed by high-performance liquid chromatography. Molecular mass of purified lactoferrin (PLF) is 78,000 daltons. The iron-binding activity of PLF had a UV/ visible-light absorption spectrum indistinguishable from that of human and bovine lactoferrins (absorbance ratio [465 nm/280 nm] approx 0.046). The growth ratio of WIL-2 cells in PLF-supplemented medium is 70% of that in serum-containing medium. The aforementioned characteristics are similar to those of human and bovine lactoferrins. Immunoblot analysis, using polyclonal antibody raised in rabbits against porcine whey lactoferrin, revealed high specificity for PLF, and low cross-reactivity with commercial human and bovine lactoferrins.

Monoclonal antibodies were prepared against 40- and 56- to 64-kd antigens of Chlamydia pecorum strain Maeda, which was isolated from a cow with pneumonia. Using the monoclonal antibodies, 5 strains of C pecorum, 25 strains of mammalian and 19 strains of avian C psittaci, 1 strain of C pneumoniae, and 3 strains of C trachomatis were analyzed for immunologic reactivity by use of the indirect immunofluorescent test. Monoclonal antibody analysis revealed immunologic relatedness between C pecorum and mammalian strains of C psittaci, which were completely differentiated from the other avian strains. Bovine strains were distinguished from ovine strains. Antigenic diversity mm observed for bovine and ovine strains. Feline- and guinea pig-derived strains were shown to be immunologically different from bovine and ovine strains. Results provide the basis for typing and epidemiologic study of bovine and ovine strains of C pecorum and C psittaci.

Protein concentration was determined, using the Bradford technique, in tears from cats with normal corneas and from cats with corneal sequestrum. Tears from the former group contained 5.81 +/- 2.29 mg of protein/ml; those from cornmeal sequestrum-affected cats contained 6.21 +/- 2.21 mg/ml. Difference between the 2 values was not significant. Molecular weight determination was made, using 4 to 20% sodium dodecyl sulfate-polyacrylamide gels. Molecular mass of proteins ranged from 263 to 14 kDa. There was no detectable difference in the band patterns for the 2 groups.