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  • Author or Editor: Tonatiuh Melgarejo x
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Abstract

Objective

To improve a previously described purification process by producing a higher yield and purity of α1-protease inhibitor (α1-PI) from canine plasma.

Animals

Plasma pool from 10 clinically normal male dogs.

Procedure

Canine α1-PI was purified by use of ammonium sulfate precipitation, ion-exchange chromatography, and 3 affinity chromatographic procedures: concanavalin A-Sepharose, thiol, and hemoglobin-Sepharose. Characterization was performed by gel electrophoresis, isoelectric focusing, and immunoblot analysis. The N-terminal amino acid sequence was obtained by use of the Edman degradation method and a gas amino acid sequencer.

Results

Canine α1-PI was purified with a yield of approximately 7% and a 54-fold increase in specific inhibitory activity. The inhibitor had a molecular weight of 59,000 and had 2 major patterns after isoelectric focusing: fast and intermediate in homozygous and/or heterozygous forms. Edman degradation revealed glutamic acid as the starting amino acid from the N-terminal sequence. Homologies of the N-terminal sequence of canine α1-PI with those of sheep, horse, and human α1-protease inhibitors were 54, 46, and 41 %, respectively.

Conclusions

Canine protease inhibitor is analogous to the α1-protease inhibitors of sheep, human beings, and mice in terms of molecular weight, amino acid composition, and inhibitory activity against trypsin. Although the method described had a yield of 7%, the final product retained inhibitory activity and was pure.

Clinical Relevance

The availability of pure canine α1-PI, as well as the specific antibodies, will facilitate studies on the fecal excretion and structural heterogeneity of this protein in dogs with naturally acquired protein-losing enteropathy.(Am J Vet Res 1996; 57:258-263)

Free access
in American Journal of Veterinary Research

Abstract

Objective—To analyze and compare contents of the preocular tear films of llamas and cattle.

Animals—40 llamas and 35 cattle.

Procedure—Tear pH was determined by use of a pH meter. Total protein concentration was determined by use of 2 microtiter methods. Tear proteins were separated by use of electrophoresis and molecular weights of bands were calculated. Western blot immunoassay was used to detect IgA, lactoferrin, transferrin, ceruloplasmin, α1-antitrypsin, α1-amylase, and α2-macroglobulin. Enzyme electrophoresis was used to detect proteases.

Results—The pH of llama and cattle tears were 8.05 ± 0.01 and 8.10 ± 0.01, respectively. For results of both methods, total protein concentration of llama tears was significantly greater than that of cattle tears. Molecular weights of tear protein bands were similar within and between the 2 species, although llama tears had a distinct 13.6-kd band that was not detected in cattle. Lactoferrin, IgA, transferrin, ceruloplasmin, α1-antitrypsin, α1-amylase, α2–macroglobulin, and proteases were detected in both species.

Conclusions and Clinical Relevance—Llama tears have significantly greater total protein concentration than cattle tears, whereas pH is similar between species. Because little variation was detected within species for the number and molecular weight of protein bands, pooling of tears for analysis is justified. Results suggest that lactoferrin, ceruloplasmin, transferrin, α1-antitrypsin, α2-macroglobulin, α1-amylase, and IgA are present in the tears of llamas and cattle. (Am J Vet Res 2000;61:1289–1293)

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in American Journal of Veterinary Research

Abstract

Objective—To develop and validate an ELISA for quantitative analysis of feline trypsin-like immunoreactivity (fTLI).

Sample Population—Purified feline cationic trypsin (fCT) and rabbit anti-fCT antiserum; blood samples from 63 healthy cats.

Procedures—A sandwich capture ELISA was developed, using anti-fCT antiserum purified by affinity chromatography that underwent biotinylation. Purified fCT was used for standards. The assay was validated by determination of sensitivity, working range, linearity, accuracy, precision, and reproducibility. A reference range was established by assaying serum samples from the 63 healthy cats.

Results—Sensitivity was 1.23 µg/L; working range was 2 to 567 µg/L. Ratios of observed versus expected results for 4 samples tested at various dilutions ranged from 90.0 to 120.7%. Ratios of observed versus expected results for 5 samples spiked with various concentrations of fCT ranged from 82.0 to 101.8%. Intra- and inter-assay coefficients of variability ranged from 9.9 to 11.1% and from 10.2 to 21.7%, respectively. The reference range for serum fTLI measured with this ELISA was 12 to 82 µg/L.

Conclusions and Clinical Relevance—Results suggest that an ELISA can be used to measure serum fTLI in cats. The ELISA was sufficiently sensitive, linear, accurate, precise, and reproducible for clinical use. (Am J Vet Res 2000;61:620–623)

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in American Journal of Veterinary Research

Abstract

Objective—To evaluate intestinal permeability and gluten sensitivity in a family of Soft-Coated Wheaten Terriers (SCWT) affected with protein-losing enteropathy (PLE), protein-losing nephropathy (PLN), or both.

Animals—6 affected adult dogs.

Procedure—Intestinal biopsy specimens, urine protein- to-creatinine ratio, serum concentrations of albumin and globulin, and concentration of α1-protease inhibitor in feces were evaluated before, during, and 13 weeks after daily administration of 10 g of gluten for 7 weeks. Eosinophils and lymphocytes-plasmacytes were enumerated in intestinal biopsy specimens. Intestinal permeability was evaluated before and during the sixth week of gluten administration via cellobiose-mannitol and chromium-EDTA absorption tests.

Results—Serum globulin concentration decreased significantly after prolonged administration of gluten. Although not significant, there was an increase in lymphocytes- plasmacytes and a decrease in eosinophils in intestinal biopsy specimens. Furthermore, these counts were greater than those reported for clinically normal dogs. Gluten administration did not increase intestinal permeability.

Conclusions and Clinical Relevance—Daily administration of gluten was associated with a significant decrease in serum globulin concentration in SCWT affected with PLE or PLN, but other variables remained unchanged. Although enhanced wheatgluten sensitivity may be one factor involved in the pathogenesis of PLE or PLN in SCWT, this syndrome does not appear to be the result of a specific sensitivity to gluten. (Am J Vet Res 2000;61:518–524)

Full access
in American Journal of Veterinary Research