Investigation of some amino acid analogues and metabolites as inhibitors of wool and hair growth

Sheep were given intravenous infusions of ethionine together with cycloleucine or reduced glutathione, in attempts to prevent the inhibition of wool growth by ethionine. Other sheep were given cycloleucine alone to measure effects on wool growth. Twenty-two compounds related to cystine, methionine, ethionine, lysine, phenylalanine and tyrosine were given as intravenous infusions to sheep to investigate their potential as depilatory agents. Nineteen of these compounds were also tested in mice during their first cycle of hair growth. The concurrent administration of cycloleucine with ethionine prevented the weakening of wool fibres caused by ethionine, but reduced glutathione was ineffective. Cycloleucine weakened wool fibres, as judged subjectively, and caused a small reduction in fibre diameter. Selenocystine and selenomethionine caused some hair loss in mice but selenocystine was also toxic. Both seleno-amino acids were toxic for sheep; selenocystine was lethal at 0.025 mmol kg-0.75 and selenomethionine at 0.09 mmol kg-0.75. Doses that permitted survival of sheep did not have depilatory effects. However, the presence of autophagic vacuoles in the cytoplasm of follicle bulb cells of sheep indicated that a toxic dose of selenocystine had potential depilatory activity. Other compounds investigated did not induce loss of wool or hair. Some compounds, notably 3-methylthiopropionic acid and S-(2-aminoethyl)-L-cysteine, were toxic to mice but not sheep. The methionine analogue, methoxinine (O-methyl-DL-homoserine), caused a substantial reduction in the strength of wool fibres and a prolonged alteration of the crimp pattern. It is suggested tentatively that cycloleucine inhibits methionine adenosyltransferase and thereby reduces or prevents the formation of S-adenosylethionine. The failure of various compounds related to methionine and ethionine to have any depilatory activity in sheep supports the view that ethionine influences wool growth via the formation of S-adenosylethionine.     

Studies on the inhibition of synthesis of the tyrosine-rich proteins of wool.

Three treatments known to produce weak wool were imposed on sheep, and the effects on the synthesis of high-tyrosine wool proteins were noted. The treatments were: intravenous infusion of the amino acid mimosine (a potential chemical defleecing agent), intravenous injection of the synthetic steroid Opticortenol (dexamethasone-21-trimethylacetate), and the abomasal infusion of methionine into sheep consuming a diet of wheat. All three treatments caused a partial suppression of high-tyrosine protein synthesis. The inhibition caused by mimosine could not be prevented by the simultaneous infusion of tyrosine or phenylalanine, suggesting that in this system mimosine is not acting as a tyrosine antagonist. The role of phenylalanine in controlling the synthesis of the high-tyrosine proteins in wool was also investigated. Although the infusion of an amino acid mixture minus phenylalanine reduces the level of these proteins, supplements of phenylalanine or tyrosine do not stimulate their synthesis, irrespective of the initial level in the fibre. The improtance of aromatic amino acids in the regulation of the high-tyrosine proteins is therefore uncertain. Suppression of the high-tyrosine proteins is usually accompanied by a stimulation in the synthesis of the ultra-high-sulphur proteins, although there does not seem to be a simple stoichiometric relationship between the two protein types.     

A further study on the dietary-regulated biosynthesis of high-sulphur wool proteins

When the diet of sheep is supplemented by the infusion of sulphur-containing amino acids or casein into the abomasum, the newly synthesized wool shows characteristic changes in its amino acid composition, with significant increases in cystine, proline and serine and decreases in aspartic acid and phenylalanine. This modification seems to be due entirely to an alteration in the overall composition of the high-sulphur proteins and to an increase in their proportion in the fibre. These variations are not the result of a change in the composition of individual proteins, but are due to alterations in their relative proportions and to the initiation of the synthesis of `new' proteins, many of which are extremely rich in cystine. It is suggested that the heterogeneity of the high-sulphur proteins may be due, in part, to similar changes in composition caused by natural variations in the nutrition of sheep.     

Wool proteins of New Zealand Romney sheep

Proteins extracted from the wool of 65 Romney ewes were analysed qualitatively by one- and two-dimensional polyacrylamide gel electrophoresis. Romney wool proteins could be classified into the low-sulfur, high-sulfur, and high-tyrosine protein groups described for wool from other breeds. The wool protein pattern of an individual sheep remained constant despite changes in season, age or nutritional status of the sheep, and did not vary between different body positions. There were between-sheep differences in protein pattern, most variation occurring in the high-sulfur protein group. These differences were presumed to reflect genetic differences between the sheep.     

Monoclonal antibodies to a subfraction of merino wool high-tyrosine proteins

Monoclonal antibodies were prepared which react with members of the high-tyrosine type proteins from Merino wool. Specificity was confirmed by the use of Western transfer immunoassays and by enzyme-linked immunosorbent assay on purified fractions. Immunofluorescent staining of sections of wool follicles using the antibodies showed that the proteins were present in the developing wool shaft but that staining was asymmetric, indicating specific location of the proteins in the orthocortex of the fibres. Immunogold-electron microscopy confirmed that one of the antibodies bound to the keratin microfibril bundles.     

Effects of intravenous infusion of mimosine on wool growth of Merino sheep.

Merino sheep were given continuous intravenous infusions of L-mimosine for periods of 1 1/2, 2 or 21 days; efficacy as a defleecing procedure and effects on subsequent wool growth were measured. In addition, the amino acids tyrosine, phenylalanine and cystine were investigates as antagonists to the effects of mimosine. Infusions for 1 1/2 or 2 days at the daily rate of 80-120 mg/kg caused a cessation of wool growth by 1 1/2-2 days from the start of infusion, and all sheep were subsequently defleeced. It was estimated that, on average, fibre growth stopped for 10 1/2-13 days in four sheep after a 2-day infusion, and for 5 1/2 and 9 1/2 days in two sheep after an infusion for 1 1/2 days. There was considerable variation in the time taken for new fibres to recommence growth. During the period 3-5 weeks after infusion of mimosine, length growth rate was consistently greater than the pretreatment rate. Likewise, fibre diameter was greater in three out of the four sheep. As a result, the volume growth rate of fibres was greater post-treatment than it was pretreatment. Infusion for 3 weeks at the daily rate of 21-24 mg/kg did not stop wool growth. However, both length growth rate and fibre diameter were considerably depressed, and after 12 days' infusion, fibre diameter and volume growth rate were reduced to less than half the pretreatment values, and wool fibres were very weak. After the mimosine infusion stopped, fibre diameter increased to above pretreatment values and remained ther for the period of 2-3 weeks studied. The concurrent infusion of tyrosine, phenylalanine or cystine with mimosine failed to prevent any of the effects of mimosine on wool growth.     

Amino acid chalcogen analogues as tools in peptide and protein research

The chalcogen elements oxygen, sulfur, and selenium are essential constituents of side chain functions of natural amino acids. Conversely, no structural and biological function has been discovered so far for the heavier and more metallic tellurium element. In the methionine series, only the sulfur‐containing methionine is a proteinogenic amino acid, while selenomethionine and telluromethionine are natural amino acids that are incorporated into proteins most probably because of the tolerance of the methionyl‐tRNA synthetase; so far, methoxinine the oxygen analogue has not been discovered in natural compounds. Similarly, the chalcogen analogues of tryptophan and phenylalanine in which the benzene ring has been replaced by the largely isosteric thiophene, selenophene, and more recently, even tellurophene are fully synthetic mimics that are incorporated with more or less efficiency into proteins via the related tryptophanyl‐ and phenylalanyl‐tRNA synthetases, respectively. In the serine/cysteine series, also selenocysteine is a proteinogenic amino acid that is inserted into proteins by a special translation mechanism, while the tellurocysteine is again most probably incorporated into proteins by the tolerance of the cysteinyl‐tRNA synthetase. For research purposes, all of these natural and synthetic chalcogen amino acids have been extensively applied in peptide and protein research to exploit their different physicochemical properties for modulating structural and functional properties in synthetic peptides and rDNA expressed proteins as discussed in the following review.     

Evidence of a unique developmental mechanism specifying both wool follicle density and fibre size in sheep selected for single skin and fleece characters

Skin and fleece traits have been characterized in four lines of Merino sheep selected for high- and low-fibre diameter (D +/-) and staple length (L +/-) from a medium-woolled flock. Over a period of 20 years, each line responded in the desired direction, producing fleeces composed of thick or thin fibres and long or short wool staples. However, variations in the amounts of wool grown that might be expected from these procedures were compensated by changes in unselected characters. Thus a predicted difference in fleece weights between high and low staple length lines was reduced by an increase in fibre crimp frequency in L- sheep. Similarly, changes induced in fibre diameter in the D lines resulted in small effects on fleece weight in comparison to the large (and inverse) effects on follicle numbers. Towards the end of the selection regime, mean follicle density in D- sheep was twice that of D+ sheep. This intriguing response within the follicle population was examined further: an analysis of the relationship between follicle density and fibre diameter amongst the four lines revealed a highly significant, negative linear correlation. The implication of this statistical association is that the numbers of follicles initiated in skin during foetal life had a direct bearing on the sizes of wool fibres eventually produced. It was concluded that both features must be under the control of a single developmental mechanism. Since the expression of each of the characters is separated in time, the mechanism must be activated during the earlier event, i.e. at or before the phase of follicle initiation.     

Wool fibre crimp is determined by mitotic asymmetry and position of final keratinisation and not ortho- and para-cortical cell segmentation

Crimp, a distinguishing feature of sheep fibres, significantly affects wool value, processing and final fabric attributes. Several explanations for fibre bending have been proposed. Most concentrate on relative differences in the physicochemical properties of the cortical cells, which comprise the bulk of the fibre. However, the associations between cortical properties and fibre crimp are not consistent and may not reflect the underlying causation of fibre curvature (FC). We have formulated a mechanistic model in which fibre shape is dictated primarily by the degree of asymmetry in cell supply from the follicle bulb, and the point at which keratinisation is completed within the follicle. If this hypothesis is correct, one would anticipate that most variations in fibre crimp would be accounted for by quantitative differences in both the degree of mitotic asymmetry in follicle bulbs and the distance from the bulb to the point at which keratinisation is completed. To test this hypothesis, we took skin biopsies from Merino sheep from sites producing wool differing widely in fibre crimp frequency and FC. Mitotic asymmetry in follicle bulbs was measured using a DNA-labelling technique and the site of final keratinisation was defined by picric acid staining of the fibre. The proportion of para- to ortho-cortical cell area was determined in the cross-sections of fibres within biopsy samples. Mitotic asymmetry in the follicle bulb accounted for 0.64 (P < 0.0001) of the total variance in objectively measured FC, while the point of final keratinisation of the fibre accounted for an additional 0.05 (P < 0.05) of the variance. There was no association between ortho- to para-cortical cell ratio and FC. FC was positively associated with a subjective follicle curvature score (P < 0.01). We conclude that fibre crimp is caused predominantly by asymmetric cell division in follicles that are highly curved. Differential pressures exerted by the subsequent asymmetric cell supply and cell hardening in the lower follicle cause fibre bending. The extent of bending is then modulated by the point at which keratinisation is completed; later hardening means the fibre remains pliable for longer, thereby reducing the pressure differential and reducing fibre bending. This means that even highly asymmetric follicles may produce a straight fibre if keratinisation is sufficiently delayed, as is the case in deficiencies of zinc and copper, or when keratinisation is perturbed by transgenesis. The model presented here can account for the many variations in fibre shape found in mammals.     

Effects of abomasal supplements of methionine on the wool follicles and skin of wheat-fed sheep

In wheat-fed sheep, supplemented abomasally with 1.5-6.0 g methionine per day, poor formation and improper keratinization of the wool fibres were evident 4 days after the start of the methionine supplementation. This led to kinking of the fibres. Subsequently severe distortion of the fibres, accompanied by gross thickening of the outer root sheaths, occurred in the distal (upper) halves of most follicles. Within this thickened region partial degradation of the distorted fibres occurred before emergence from the skin surface, causing a marked reduction in the tensile strength of the wool. It is postulated that kinking of the fibres stimulated the accumulation of outer root sheath cells, which led to hyperactivity of the process that normally degrades the inner root sheath, so that the poorly keratinized fibres were also partly degraded. Thickening of the epidermis and cellular infiltration of the upper dermis sometimes occurred during the infusions of methionine, whereas there were negligible effects on the sebaceous and sweat glands. Disappearance of the excess accumulation of outer root sheath cells after cessation of the methionine supplementation occurred gradually following improvement in keratinization and elimination of kinking of the fibres.     

Effects of p-chlorophenylalanine and alpha-methylphenylalanine on amino acid uptake and protein synthesis in mouse neuroblastoma cells.

The phenylalanine analogues p-chlorophenylalanine and alpha-methylphenylalanine were used to inhibit phenylalanine hydroxylase in animal models for phenylketonuria. The present report examines the affects of these analogues on the metabolism of neuroblastoma cells. p-Chlorophenylalanine inhibited growth and was toxic to neuroblastoma cells. Although in vivo this analogue increased cell monoribosomes by 42%, it did not significantly affect poly(U)-directed protein synthesis in vitro. P-Chlorophenylalanine did not compete with phenylalanine or tyrosine for aminoacylation of tRNA and was therefore not substituted for those amino acids in nascent polypeptides. The initial cellular uptake of various large neutral amino acids was inhibited by this analogue but did not affect the flux of amino acids already in the cell; this suggested that an alteration of the cell's amino acid pools was not responsible for the cytotoxicity of the analogues. In contrast with p-chlorophenylalanine, alpha-methylphenylalanine did not exert these direct toxic effects because the administration of alpha-methylphenylalanine in vivo did not affect brain polyribosomes and a comparable concentration of this analogue was neither growth inhibitory nor cytotoxic to neuroblastoma cells in culture. The suitability of each analogue as an inhibitor of phenylalanine hydroxylase in animal models for phenylketonuria is discussed.     

Syntheses and biological activities of methoxinine-analogues of the C-terminal octapeptide of cholecystokinin-pancreozymin.

The C-terminal octapeptide of cholecystokinin-pancreozymin and its analogues in which the methionine residues were successively replaced by methoxinine have been synthesized. When these analogues were tested for their gallbladder-contraction activity in an in vitro system the following relative potencies were observed: [Mox3]-CCK-PZ 8 (95%), [Mox6]-CCK-PZ 8 (5%) and [Mox3, Mox6]-CCK-PZ 8 (2%).     

Influence of nutrition on the crimping rate of wool and the type and proportion of constituent proteins.

When the nutritional level of sheep is restricted, the staple crimp frequency of the resultant fleece increases substantially whereas the cystine and high-sulphur protein contents decrease. This is in marked contrast to the direct relationship between crimp frequency and cystine content among sheep. These observations can be reconciled by assuming that variations in crimp frequency are attributable solely to a combination of follicle shape and fibre length growth rate without recourse to the more generally accepted theories relating to the proportion and distribution of ortho- and paracortical cells in the firbre cortex. The major portion of the decrease in the cystine content of high-crimp wools is due to the decreased synthesis of a specific protein fraction (ultra-high-sulphur protein) as would be expected from the results of dietary supplementation experiments. Low-crimp wools do not appear to contain this protein fraction and in this respect they may differ from high-crimp wools.     

角蛋白HGTP及其对羊毛发育的影响

羊毛的主要成分是角蛋白,其组分高甘氨酸-酪氨酸蛋白(HGTP)家族成员KAP6、KAP7和KAP8基因表达对羊毛细度和弯曲等特性具有重要影响。本文从羊毛的组成、角蛋白的生物学特征以及HGTP基因定位和表达对细度的影响等方面进行了综述,旨在为羊毛发育调控研究提供理论参考。     

Effect of phenylalanine on protein synthesis in the developing rat brain

1. Inhibition of the rate of incorporation of [(35)S]methionine into protein by phenylalanine was more effective in 18-day-old than in 8-day-old or adult rat brain. 2. Among the subcellular fractions incorporation of [(35)S]methionine into myelin proteins was most inhibited in 18-day-old rat brain. 3. Transport of [(35)S]methionine and [(14)C]leucine into the brain acid-soluble pool was significantly decreased in 18-day-old rats by phenylalanine (2mg/g body wt.). The decrease of the two amino acids in the acid-soluble pool equalled the inhibition of their rate of incorporation into the protein. 4. Under identical conditions, entry of [(14)C]glycine into the brain acid-soluble pool and incorporation into protein and uptake of [(14)C]acetate into lipid was not affected by phenylalanine. 5. It is proposed that decreased myelin synthesis seen in hyperphenylalaninaemia or phenylketonuria may be due to alteration of the free amino acid pool in the brain during the vulnerable period of brain development. Amyelination may be one of many causes of mental retardation seen in phenylketonuria.     

The dietary-regulated biosynthesis of high-sulphur wool proteins

1. When the diet of sheep is supplemented by the abomasal infusion of sulphur-containing amino acids or casein, a special group of proteins, with a very high content of sulphur (about 8.3%), is incorporated into the high-sulphur proteins of wool. These special proteins cannot be detected in control wool from the same sheep. 2. This is a naturally occurring process, as these special proteins are found in wool from sheep on a high level of nutrition under ordinary conditions of feeding, and in wool of an inherently high sulphur content. 3. This represents a control mechanism in protein synthesis that has not previously been observed, and may be further evidence that the high-sulphur proteins of wool are produced by an unusual synthetic route.     

Unravelling the proteome of wool: towards markers of wool quality traits

With ongoing efforts to make wool more competitive alongside other fibres, notably synthetics, there is a need to obtain a better understanding of the relationship between protein composition and characteristic wool properties to assist sheep breeding programmes. Before this can be achieved, the wool proteome needs to be mapped, by gel and non-gel techniques, and methods developed to reliably quantitate protein expression. Nevertheless, in setting out to achieve this, there are numerous challenges to be faced in the application of proteomics to wool, including the relative lack of wool protein sequence information in the publically accessible databases, the wide variety of proteins in the wool fibre, the high homology within the Type I and Type II keratins, the high degree of homology and polymorphism within individual keratin associated protein families, the dominance of the keratin proteins over others in wool and the peculiar chemistries found in keratins and their associated proteins. This review will discuss the various strategies that have been developed to both identify these proteins in the wool protein map and quantify them with the view to their application to the identification of markers for wool quality traits.     

Genome-Wide Association Study for Wool Production Traits in a Chinese Merino Sheep Population

Genome-wide association studies (GWAS) provide a powerful approach for identifying quantitative trait loci without prior knowledge of location or function. To identify loci associated with wool production traits, we performed a genome-wide association study on a total of 765 Chinese Merino sheep (JunKen type) genotyped with 50 K single nucleotide polymorphisms (SNPs). In the present study, five wool production traits were examined: fiber diameter, fiber diameter coefficient of variation, fineness dispersion, staple length and crimp. We detected 28 genome-wide significant SNPs for fiber diameter, fiber diameter coefficient of variation, fineness dispersion, and crimp trait in the Chinese Merino sheep. About 43% of the significant SNP markers were located within known or predicted genes, including YWHAZ, KRTCAP3, TSPEAR, PIK3R4, KIF16B, PTPN3, GPRC5A, DDX47, TCF9, TPTE2, EPHA5 and NBEA genes. Our results not only confirm the results of previous reports, but also provide a suite of novel SNP markers and candidate genes associated with wool traits. Our findings will be useful for exploring the genetic control of wool traits in sheep.     

Screening for mutants temperature sensitive in protein synthesis by using methionine analogues

Summary Two new mutants are described which are temperature sensitive in protein synthesis. The mutants were obtained by a screening procedure using methionine analogues. The method is based on two findings: a) that in E. coli, and in other members of the Enterobacteriaceae, sensitivity to methionine analogues increases with the growth temperature, and b) that cells which are not synthesizing proteins during treatment with methionine analogues have a shorter lag in resuming growth subsequent to removal of the analogue.     

Characterisation of white and black merino wools: a proteomics study

Wool is an important agricultural commodity with merino wool being rated alongside the finest quality fibres, which include the goat fibres Mohair and Cashmere. Although pigmented wool merinos have become extremely rare, the market for this wool is increasing. In Portugal, there are two merino breeds: white and black, descendants of animals originally bred on the Iberian Peninsula. These breeds have the potential to assist in our understanding of how protein expression relates to wool traits of importance to the textile industry. Herein, we study the characteristics and protein expression profiles of wool from ewes of the Portuguese black and white merino (n=15). Both breeds had very similar results for fibre diameter (25 µm) and curvature (105 to 111°/mm). Significant between-breed differences were found in the two types of keratin-associated proteins (KAPs): high-sulphur proteins (HSPs) and high-glycine–tyrosine proteins (HGTPs). The expression of HSPs, KAP2-3 and KAP2-4, decreased expression in the pigmented animals, whereas KAP13-1 was found in higher amounts. Likewise, the expression of the ultra-high-sulphur proteins, KAP4-3 and KAP4-7-like, was reduced in black sheep to half the levels of the white wools, whereas the HGTPs, KAP6, KAP6-1, KAP6-2 and KAP16-2, were more abundant in black sheep. These results suggest structural differences between the black and white merino wool, because of differences among some KAPs. These differences have important implications for the textile industry.