Protein synthesis in tissues cultured from the bovine hoof

Abstract

The mechanical strength of the bovine hoof depends on keratinization of cells in the germinal layers of the epidermis. Histological examination of hoof tissues in calves and young heifers has identified disturbances in this keratinization process which would result in ineffective hoof development and could precipitate lameness. Short-term culture of bovine hoof tissue was used to investigate epidermal keratinization. Cell function remains viable in these cultures. The rate of protein synthesis, measured by [35S]-methionine incorporation, continued for at least 3 h in culture. Radiolabelled proteins in tissue homogenates were separated by SDS-polyacrylamide gel electrophoresis and characterised by fluorography and were representative of the proteins found in hoof tissue. Three prominent radiolabelled bands were identified as keratins and actin by Western blotting. Immunohistochemistry showed that keratin was localised principally in the epidermal layers, and microautoradiography indicated that this was the major site of protein synthesis. Hoof tissues cultured under these conditions provide a useful system for studying the acute regulation of epidermal keratinization.

L-methionine uptake, incorporation and effects on proliferative activity and protein synthesis in bovine claw tissue explants in vitro

“…These differences in response probably reflect differences in the quantities of sulphur-containing amino acids required to support synthesis of sheep wool or mohair fibre (kg quantities per year) compared with requirements for claw horn deposition (c. 700 g/year). Major similarities in the morphology of the dermis and epidermis of the hair follicle and horn-producing structures of the bovine claw are well recognized (Hendry et al 1995;Galbraith 1998).…”

“…Methods for the collection of claws, preparation of explants and culture conditions were as described previously (Hendry et al 1995(Hendry et al , 1999(Hendry et al , 2001. Briefly, right hind lateral claws of crossbred female beef cattle (18-20 months old) and Holstein-type dairy cattle of varying ages, but all in excess of 30 months, were collected immediately post mortem at a commercial abattoir and rapidly transported in ice to the laboratory, typically within 1 h of slaughter.…”

“…Explants were prepared as described above and incubated for 21 h in a range of concentrations and proportions of L-methionine, L-cysteine and L-cystine in DMEM/F12 followed by 3 h in similarly composed medium containing L-[ 35 S]-methionine (6 mCi/ml) and [methyl-3 H]-thymidine (2 . 5 mCi/ml) to provide measures of (a) L-methionine incorporation and protein synthesis and (b) DNA synthesis, respectively (Hendry et al 1995;Goulet et al 1996). These concentrations were guided by information on concentrations in blood of sulphur-containing amino acids from studies on dairy cattle (Galbraith et al unpublished) that, for L-methionine, ranged from approximately 16 to 25 mmol/litre and 50 mmol/ litre (Shennan et al 1997).…”

“…The work reported herein was part of a programme that measured the responses of bovine claw tissue to methionine supply in vivo and in vitro. It utilized a well-established bovine in vitro claw explant system (Hendry et al 1995(Hendry et al , 1999(Hendry et al , 2001) which previously demonstrated synthesis of proteins, under a variety of conditions, by incorporation of 35 S-label from supplied L-[ 35 S]methionine or L-[ 35 S]-methionine and L-[ 35 S]-cysteine mixtures, and incorporation of 3 H-methyl thymidine into DNA as an indicator of cellular proliferation. The present study was designed to extend this work by quantifying the relationships between L-methionine and L-cysteine supply in the culture medium, uptake into intracellular pools and incorporation into protein fractions using L-[ 35 S]-methionine.…”

Synthesis and distribution of cytokeratins in healthy and ulcerated bovine claw epidermis

“…Two dimensional electrophoresis markers pI range 7n6-3n8 were from Sigma. The sources of other histological reagents, culture media and laboratory reagents were as described previously (Hendry et al 1995(Hendry et al , 1999.…”