Amino acids in different layers of the matrix of the normal equine hoof. Possible importance of the amino acid pattern for research on laminitis.

A method was developed for separating different layers of the matrix of the equine hoof wall by dissection, and the layers were then analyzed with respect to their amino acid composition. The results were used to compare the biochemistry of hard keratinization (e.g., in the hoof wall matrix) and soft keratinization (e.g., in the epidermis of the skin). Hard keratinization differed from soft keratinization not only by its previously well known high incorporation of cystine, but also by considerable incorporation of tyrosine and threonine into the outer layers of the keratogenous zone and by the absence of increasing histidine incorporation in those layers. The importance of the results for research on the pathogenesis of laminitis is discussed.

Monoclonal antibody analysis of bovine epithelial keratins. Specific pairs as defined by coexpression.

We have characterized the keratin proteins of various bovine epithelial tissues by one- and two-dimensional gel electrophoresis, coupled with the immunoblot technique using AE1, AE2, AE3, AE5, CA20, BE14, and 6.11 monoclonal antikeratin antibodies. The results indicate that all known bovine keratins can be divided into two subfamilies. The "acidic" (Type I) subfamily consists of 41-, 43-, 45-, 46-, 50-, 54-, 56-, and 56.5-kDa keratins, all of which have a pI of less than 5.6, and most of them are recognized by our AE1 antibody, whereas the "neutral-to-basic" (Type II) subfamily consists of 55-, 57-, 58-, 62-65-, 66-, and 67-kDa keratins, all of which have a pI of greater than 6.0 and are recognized by our AE3 antibody. Tissue distribution data and cell culture studies show that, within the two subfamilies, keratins with similar "size ranks" form a "pair" as defined by frequent co-expression. Furthermore, within most "keratin pairs," the basic keratin is larger than the acidic one by 8-10 kDa. These results provide further support for the concepts of "keratin subfamilies" and keratin pairs and are consistent with the possibility that the acidic and basic members of at least some keratin pairs may interact specifically during in vivo tonofilament assembly and/or function. Immunoblotting data derived from the use of several monospecific antibodies show that although the size, charge, and pattern of expression of most bovine keratins are similar to those of the human counterparts, there are important exceptions to this rule.

Mineral and amino acid composition of beef cattle hooves.

Twenty-three Hereford X Shorthorn cattle were used to evaluate the effects of seasonal and dietary changes on the mineral composition (Ca, Mg, Cu, Zn, and S) of hooves. A seasonal pattern was found in the Ca, Mg, and Zn composition of hooves in the 12 cattle evaluated in 1982 and in the 11 cattle evaluated in 1983, with the concentrations of 3 minerals decreasing in winter when dietary change did not occur. Copper concentrations significantly decreased during the 1st year (1982) and had a tendency to decrease during the 2nd year. During the 1983 pasturing season, when effects of seasonal vs dietary change could not be distinguishable, hoof concentrations of Ca and Mg decreased, whereas the inverse trend was observed for Cu and Zn. Seasonal patterns for hoof concentrations of S were not found. Results of mineral analysis of hooves indicated strong correlations between calcium and the other minerals (except S), and between Zn and Cu. Amino acid analyses of hooves of the 11 cattle in 1983 indicated differences in their composition related to dietary changes (winter feeding vs pasture) or to management.

Cholesteryl sulfate: the major polar lipid of horse hoof.

The lipids of horse hoof have been analyzed by quantitative thin-layer chromatography. The major components include cholesterol (37-40%), six groups of ceramides (10-15%), and cholesteryl sulfate (15-20%). Free fatty acids are abundant (15.8%) in the outer fully keratinized hoof, but are present at only low levels (3.1%) in the softer hyponychium. The material identified as cholesteryl sulfate was isolated by preparative thin-layer chromatography and characterized by a combination of chemical, chromatographic, and spectroscopic methods. The infrared spectrum of the isolated material had absorption bands at 800, 1063, 1200, and 1235 cm-1, indicating a sulfate ester. This sulfolipid was nonsaponifiable, but upon acid hydrolysis yielded cholesterol as the only charrable product, which was identified by its chromatographic behavior and by its electron impact mass spectrum. The isolated sulfolipid also had the same mobility on thin-layer chromatography as authentic cholesteryl sulfate in several different solvent systems. Sulfated gangliosides, which were previously reported to be major horse hoof lipids, were not found among the principal lipid components in the present study. It is concluded that cholesteryl sulfate is the major polar lipid of horse hoof. This may be a significant factor determining the high degree of cohesiveness of this fully keratinized tissue.

A comparative study of the immunologic properties of hoof and nail fibrous proteins.

We have studied the localization of epidermal (soft) and nail and hoof (hard) fibrous keratins in the various anatomic regions of bovine hoof and human nail. Indirect immunofluorescence was performed on frozen sections of various parts of hoof and nail with antibodies demonstrated to be specific for hard and soft fibrous keratins by the Ouchterlony technique. The antibody to hard fibrous keratin reacted with the upper region of hoof bed and matrix tissue but not perihoof epidermis. The antibody to soft fibrous keratin reacted with hoof bed, matrix tissue, and perihoof epidermis. Electrophoretic analysis of the fibrous proteins of hoof bed indicated they contained both soft and hard fibrous keratins while matrix tissue contained only hard keratins. Immunoblot analysis of matrix fibrous proteins indicated that most of the polypeptides reacted with both antibodies. These results indicate that the antibody to soft fibrous keratin cross-reacts with hard fibrous keratin but the antibody to hard fibrous keratin appears to be specific. Immunologic studies, therefore, must be correlated with electrophoretic studies in order to define the localization of the various types of fibrous keratins. Similar results were obtained with human nail.

Mechanical properties of equine hoof wall tissue.

The mechanical properties of pigmented equine hoof wall tissue were determined for samples taken from the inner and outer portions of the stratum medium of the toe. Two properties, the modulus of elasticity and proportional limit, which are measures of the rigidity and yield point, respectively, of the tissue, were studied for samples compressed in 3 orthogonal directions. All samples tested were anisotropic. Inner wall samples were less rigid and had a lower yield point than outer wall samples.

Fibrous proteins of bovine hoof.

The matrix region of calf hoof was identified as the living precursor layer of the hardened hoof plate. Fibrous protein was isolated from the matrix with citrate buffer, pH. 2.65, while Tris buffer, pH. 9.5, with 8 M urea and a reducing agent was required to dissolve the cornified hoof. The purified matrix protein had a sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern similar to hoof plate and different from bovine epidermis. When the s-carboxymethyl derivatives of matrix, hoof plate, and hair proteins was compared by urea-polyacrylamide gel electrophoresis, they were identical, and different from that of epidermal protein. The matrix protein reacted with an antibody to hair fibrous protein. Cultured matrix keratinocytes appeared to be identical to cultured epidermal cells, pointing to the importance of the dermis in epidermal cell differentiation.

Effect of methionine hydroxy analog supplementation on dairy cattle hoof growth and composition.

Fifty lactating Holstein cows were assigned randomly to one of two treatments, control and control plus approximately 30 g methionine hydroxy analog, and confined on concrete for 11 mo. The control diet consisted of sorghum silage and concentrate fed as a blended ration. Sulfur contents of dry matter were .12% and .16% for control and methionine hydroxy analog rations. Hoof growth and hardness were measured on front and rear right abaxial claws in the dorsal and lateral regions. Hoof growth rates were measured for four periods; summer-fall, fall-winter, winter-spring, and spring-summer, each 70 to 90 days. Hooves of cows fed methionine hydroxy analog grew faster than those of control cows during spring-summer in all regions. Variations of growth rates of hooves were seasonal and tended to follow variations in daily photoperiod. Wear rates were not affected significantly by treatment. Hooves of cows fed methionine hydroxy analog were softer in the top dorsal region at the end of winter-spring and in the dorsal toe region at the end of spring-summer. All other locations were not affected significantly by treatment. The toe region was harder than the top of the hoof. Cows fed methionine hydroxy analog had less cysteine and proline in hoof than control cows and greater percentages of methionine lysine, tyrosine, and glutamic acid. These results suggest that a decrease of disulfide bonding occurred in the hoof tissue of cows fed methionine hydroxy analog. Cows fed methionine hydroxy analog produced more actual milk, milk fat, and 4% fat-corrected milk during 180 days than did control cows.

[Total protein, sulfur and bound amino acid content in the hoof horn of cows]. / Sudurzhanie na obshtiia beltuk, siarata i svurzanite aminokiselini v kopitniia rog na kravi.

Comparative investigations were carried out for the evaluation of the total protein and sulfur in the sole hoof horn of cows of the breeds Bulgarian Brown and Black and White with normal and diseased hooves as well as of the bound amino acids in the hoof horn of the same hooves of cows of the Black and White breed. It was found that the total protein as indicated above did not show any essential differences in the two breeds. The amount of total sulfur in Bulgarian Brown cows was relatively higher than that in Black and White cows. It was also found that the lower content of sulfur in the diseased hooves indicated lower resistance. The content of bound amino acids in the hoof horn of Black and White cows with both normal and diseased hooves showed certain variations. Generally, it proved lower in diseased hooves of the hind limbs than in affected hooves of the forelimbs.

[Physicochemical properties of the hoof horn in healthy highly productive cows and in those with hoof diseases]. / Fiziko-khimichni kachestva na kopitniia rog na zdravi i sus zaboliavaniia kopita pri visokoproduktivni kravi.

Comparative investigations were carried out to determine the water content, total protein, the macroelements Ca, P, Na, and K and the trace elements Zn, Mn, and S in the wall and sole horn of the front and hind hooves of high-producing cows of the Bulgarian Brown breed and the Black Pied cattle. The hoof water content in both breeds did not show any substantial differences in normal and diseased cows, and varied within the limits of 19 to 34 per cent. In affected hooves the water content was higher than that in normal hooves. The contents of total protein, Ca, P, K, Na, Zn, and Mn did not show, essential differences of either. The low level of total sulfur in diseased hooves was an indication of the lowered resistance of hoof horn.

Proteins of the hard keratins of echidna, hedgehog, rabbit, ox and man.

In the accompanying paper it has been shown that two major groups of proteins (low-sulphur and high-sulphur) of ovine wool, horn, and hoof contain similar components although the overall proportions of the groups of proteins and the relative proportions of components within the groups may show significant differences. In the present paper it has been shown for five other species (echidna, hedgehog, rabbit, ox and man) that the hard keratins produced by one animal contain the same groups of protein components but in different relative proportions. The wide apparent differences in the type and relative proportions of the the low-sulphur components which comprise the major constituent proteins of the microfibrils suggest that microfibrils can tolerate a considerable variation in the constituent proteins and still produce functional structures. The low-sulphur protein components are sufficiently well resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis to make this procedure potentially useful for animal identification and classification.

Bovine keratohyalin: anatomical, histochemical, ultrastructural, immunologic, and biochemical studies.

Salt extraction studies showed that keratohyalin (KH) could be solubilized and extracted from fresh bovine hoof epidermis. The solubility of KH varied in relation to the molarity of the salt solution used for extraction. Using this information, the extracted KH was aggregated in vitro by dialyzing the high salt extract against distilled water. Histochemical, ultrastructural, and immunologic studies of the resultant particles or macroaggregates showed that the latter had the same properties and immunogenicity as the KH granule in situ and produced antibodies against it. Fractionation of the macroaggregates by polyacrylamide gel electrophoresis demonstrated that the macroaggregates were compsed of sets of 20 polymers whose subunits or monomers had a molecular weight of 16,900. Amino acid analyses showed that the macroaggregates and the various fractionated polymers were similar and that the protein ahd 116 amino acid residues. Serine, arginine, glycine, glutamic acid, and histidine constituted 78% of all residues, and serine alone represented 27%. The molecular weight by amino acid analyses was 16,150 after correction for the 8% ribonucleic acid which appears to be complexed to the protein.