Wool Keratin Research Articles

Transgenic sheep and wool growth: possibilities and current status.

Merino wool is the result of generations of selection, yet improvements in wool quality and performance are still being sought. Through gene manipulation, sheep transgenesis offers possibilities of understanding the relationship between wool keratin protein composition and fibre structure and properties and of introducing novel changes to fibre properties and growth rates. We have established an efficient sheep transgenesis programme with an overall transgenic rate of 2.1% of zygotes injected. However, by incorporating in vitro culture and assessment of injected zygotes, this equates to a transgenic rate of 13% from 516 lambs born. With the first keratin gene construct, a wool keratin type II intermediate filament gene, four live F0 transgenic sheep have been produced and all express the transgene. In one of them, the highest expressor, phenotypic and ultrastructural changes were evident in the fleece. To improve wool growth rate by increasing the supply of cysteine to the follicle, transgenic sheep are being produced carrying the two genes necessary for endogenous cysteine synthesis. Three promoters have been tested driving the cysteine synthesis genes: two general promoters, the Rous sarcoma virus long terminal repeat and mouse phosphoglycerate kinase promoter, and a rumen-specific promoter from the sheep small proline-rich protein gene. To date, one transgenic sheep (bearing the small proline-rich protein promoter constructs) has produced cysteine in the rumen, although the amount was low at 3 months of age and not detectable at 6 months.

MspI RFLP in the gene for a type I intermediate filament wool keratin.

MspI RFLP in the gene for a type I intermediate filament wool keratin.

The effect of various chromium species on wool keratin*

The effects of different chromium species and chroming processes on wool keratin have been studied. Infra‐red studies revealed that in dichromate‐treated wool the disulphides oxidise mainly to Bunte salt residues (S‐thiosulphate). As expected, trivalent chromium treatments produce little or no oxidative degradation of wool keratin. These results were supported by fibre strength tests and SEM observations.

Formation of Hydroxyl Radicals from Reaction of Cu(II)/Wool Keratin Complex with Hydrogen Peroxide

In order to determine thatthe degradation of wool keratin molecules in the Cu(II)/ wool keratin complex / hydrogen peroxide system may be brought about by the hy droxyl radical formed in this system, hydroxyl radical formation was measured using the thiobarbituric acid method. The Cu(II)/wool keratin complex can yield hydroxyl radicals by reaction with hydrogen peroxide, but under anaerobic conditions the amount of hydroxyl radical formation is less than that under aerobic conditions. Epi nephrine oxidation, an indicator of superoxide radical generation, occurs in the reaction of this system. These results indicate that the Cu(II)/wool keratin complex appears to catalyze hydroxyl radical formation from the hydrogen peroxide superoxide radical being formed as an intermediate. The sequence of reactions leading to hydroxyl radical formation is essentially the metal catalyzed Haber-Weiss reaction.

Characterizing keratins using high‐pressure differential scanning calorimetry (HPDSC)

AbstractApplying differential scanning calorimetry (DSC) for temperatures up to 180°C, the denaturation transition of the α‐helical material of various keratins in excess water was studied at conditions of equilibrium water vapor pressure (high‐pressure DSC). The results show a generally cystine content and material invariate denaturation range of 20–30°C with peak temperatures around 140°C. Though analysis of variance and multiple comparison tests show a pronounced inhomogeneity of the enthalpy data, the results generally support the material invariance of the denaturation enthalpy and hence of the helix content of the keratins. The denaturation enthalpy for the helical coiled‐coil‐structures in the intermediate filaments is determined as ΔH = 6.5–7.8 kJ/mol. A significant positive correlation was found between the denaturation temperatures and the cystine content. It is concluded, that the helix denaturation temperatures are kinetically controlled by the amount and/or the chemical composition of the surrounding nonhelical matrix, and that the double‐peak endotherms observed for wool and other keratins originate from two cell types that are sufficiently different in sulfur content to allow endotherm separation. In the case of wool the cell types can be identified as “ortho‐” and “paracortical” cells. © 1993 John Wiley & Sons, Inc.

Sites for Cu ( II ) Stabilization in Wool Keratin

Light green copper(II)-wool samples prepared under conditions of pH < 5 have been treated for short periods at about pH 11.5. The color changes and the electron spin resonance spectra of these new samples are described. All observations are con sistent with the metal ion being transferred from the carboxylate binding sites of the light green complex to new ones involving nitrogen donor groups.

Relaxation and quenching of the excited states of tryptophan in keratin

The temperature dependence of the fluorescence and phosphorescence originating from tryptophan residues in the solid protein keratin was studied. The position of the tryptophan fluorescence spectral maximum was measured as a function of temperature and was found to be substantially Stokes shifted even at 77 K. The activation energies for non-radiative relaxation of the singlet and triplet states of tryptophan in wool keratin, derived from Arrhenius plots of the fluorescence and phosphorescence yields, are 580 cm −1 and 995 cm −1 respectively. One possible fluorescence quenching mechanism which is consistent with these observations is a very effective, thermally induced mixing of the 1L a and 1L b excited singlet states which results in enhanced internal conversion apparent as a low fluorescent yield even at low temperatures. The phosphorescence decay from keratin in the absence of oxygen following excitation at 265 nm is non-single exponential. At room temperature it is dominated by a short-lived component with more than 80% of the decay completed within 3 μs of the flash. Longer living components with first-order lifetimes as long as 2–3 s were also observed.

Effects of Added Metal Ions on the Interaction of Wool Keratin Derivatives and an Azo Dye Carrying Hydroxyl Groups

The extent of binding of chrome violet, which is a monoazo dye involving two hydroxyl groups in the o and o' positions to an azo group, by wool keratin derivatives such as S-carboxymethylated, S-cyanoethylated, and regenerated keratin derivatives is markedly enhanced in the presence of Co2+ ion. The amount of binding in the presence of 1 times 10-4 mol/I of Co2+ ion increases by a factor of 12∼20 compared to that in the absence of the metal ion. Cu2+, Ni2+, and Zn2+ ions do not perceptibly influence the binding affinity of the dye. Al3+ ion, in contrast, suppresses the binding. To further investigate the action of added metal ions, we prepared a cobalt-complex dye and compared its binding property for the polymers with that of chrome violet in the presence of metal ions. We also measured the binding of the metal ions by the polymers. Co2+ ion did not exhibit remarkable binding affinity toward the polymers. Some possible mechanisms for enhancing chrome violet binding by the addition of Co2+ ion are described.

Singlet oxygen produced by UV irradiation of wool keratin

Oxygen was found to react rapidly with the triplet state species in wool keratin which is excited by radiation at 350 nm and which is responsible for phosphorescence observed at 500 nm. As a result of this reaction, singlet oxygen is produced which emits luminescence at 1270 nm. Although various photo-oxidation products of tryptophan are known to be capable of producing high yields of singlet oxygen as yet these compounds have not been positively identified in wool keratin. Tyrosine related species are also suggested as possible sources of singlet oxygen in wool. Irradiation of wool keratin at 265 nm produces the triplet state of tryptophan, but no singlet oxygen could be detected as a result of irradiation at this wavelength. It is suggested that this is because of the low yield of the triplet state of tryptophan in wool and/or inefficient generation of singlet oxygen in the reaction of this triplet state with ground state oxygen.

Degradation of Cu(II)-partly alkylated wool keratin complex by hygrogen peroxide.

In order to obtain some information regarding the degradation or morphological components of wool keratin fiber brought about by hydrogen peroxide under conditions in which the Cu (II) ion is bound to wool keratin the degradation of partly alkylated wool keratin fiber prepared by alkylating the generated sulfhydyl groups with 2-vinylpyridine was studied for the Cu (II)-partly alkylated wool keratin complex/hydrogen peroxide system. At the early stage of the degradation, peptide fragments in the treat. ment solution were found to contain a large amount of s-β-(2-pyridylethyl) cysteine residue and the tyrosine content in the wool fiber residue, which belongs in quantity to high-glycine-tyrosine proteins of an amorphous matrix, increased as the initial degradation proceeded. These results suggest that the high-sulfur proteins were easily degraded in the peptide bond cleavage of these proteins, which are contained especially in the cuticle.

Determination of dehydroalanine residues in proteins and peptides: An improved method

Dehydroalanine residues in peptides and proteins react with 4-pyridoethanethiol in alkaline solution to form 4-pyridoethyl cysteine residues, which after acid hydrolysis, give the corresponding amino acid. An experimental protocol has been developed, and with simple dehydroalanine derivatives, conversions of 96-99% are achieved. Tests with peptides and proteins show that serine and cysteine/cystine residues do not interfere with this analytical procedure. A sample of wool keratin contained 57 mumol dehydroalanine/g.

MYC Protooncogenes of Wool and Hair Growtha

The growth of hard keratin fibers such as wool and hair is dependent on the proliferation of cells in the follicle bulb. If the cells leaving the bulb could be induced to undergo an extra division, then fiber growth should increase. The cellular division within the follicle is complex and probably involves one or more growth factors, which act by altering the expression of transcription factors and other nuclear proteins. We propose that the expression of the myc protooncogenes is a central part of this mechanism. In support of this hypothesis we have detected the mRNAs for TGF-beta 1, basic FGF, TGF-alpha, and c-myc in plucked wool follicles using PCR amplification. We have also shown that the TGF-beta 1, TGF-beta 2, TGF-beta 3, EGF, TGF-alpha, basic FGF, N-myc, and c-myc genes are expressed in mouse skin, and we looked for changes during the hair cycle. The PCR data suggest that in whole skin the levels of mRNA for TGF-beta 1, TGF-beta 2, TGF-alpha, and c-myc do not change. In Quackenbush mice the levels for N-myc, TGF-beta 3, and basic FGF mRNA appear to be lower at the end of the hair cycle. We have confirmed in CBA/C57 black mice that lower levels of N-myc mRNA are detected when hair growth ceases in catagen and telogen. To test our hypothesis further and to assess its practical application, we are making transgenic mice in which the N-myc gene is overexpressed in the hair follicle by way of a wool keratin promoter. The transgene consists of 3.3 kb of 5' sequence from an ovine type 1 IF gene, the murine N-myc genomic coding sequence, and an SV40 polyadenylation signal. The native keratin type 1 IF gene is expressed exclusively in the wool follicle, as shown by in situ hybridization. However, in mice the injection of the transgene has resulted in high embryonic mortality and some embryos with large body size and head malformations. Since these mice were not transgenic, this is likely to be an effect of transient expression of the transgene during embryogenesis. The two transgenic mice produced so far have a normal phenotype.

Hair-Specific Keratins: Characterization and Expression of a Mouse Type I Keratin Gene

A genomic clone for a member of the mouse type I hair keratin protein family has been isolated and analyzed in order to study the regulation of this keratin during the hair growth cycle. The coding sequence is divided into seven exons. The gene structure is typical of keratins in particular and intermediate filaments in general in that the intron-exon borders are not located at the domain borders of the protein. Comparison with a sheep wool keratin gene shows that the splice sites in the two hair keratin genes are found at identical locations relative to the amino acid sequence of the proteins. Similarly, comparison of the promoter areas of these genes shows several areas of nucleotide sequence conservation, including the area around the TATA box and an SV40 core enhancer sequence. In addition, a high degree of sequence identity exists in the fourth intron. In situ hybridization shows that transcripts of this gene are first found in the relatively undifferentiated proximal cortex area in the keratogenous zone of mouse vibrissae.

Binding of Acid and Basic Dyes by Wool Keratin Derivatives

Wool keratin derivatives such as S-carboxymethylated, S-cyanoethylated, S-sulfo nated, and regenerated keratin derivatives have been prepared to study the binding of acid and basic dyes (methyl orange, chrome violet, and acridine orange) by the derivatives in aqueous solution. The first binding constants and the thermodynamic parameters accompanying the binding were evaluated. The first binding constants of the proteins for the dyes were on the order of 105-106, which is similar to or higher than that of serum albumin. The favorable free energy of the binding of chrome violet and acridine orange observed for the formation of the dye-protein complex seems to be a result of a favorable enthalpy change rather than any favorable entropy change. The binding of methyl orange by the regenerated keratin derivative is accompanied by an entropy gain and an exothermic enthalpy change. The nature and phenomena of dyes binding with the keratin derivatives are discussed.

Sequences of wool keratin proteins: the CSIRO connection

Wool, a dead tissue of epithelial origin, derives many of its properties as a textile fibre from the structure and arrangement of the proteins from which it is comprised. Much of the progress in the elucidation of wool protein structures, as a step towards understanding this relationship between structure and properties, has been made in the Division of Protein Chemistry of Australia's Commonwealth Scientific and Industrial Research Organization.

Conformational characterization of wool keratin and S-(carboxymethyl)kerateine in the solid state by carbon-13 CP/MAS NMR spectroscopy

Conformational characterization of wool keratin and S-(carboxymethyl)kerateine in the solid state by carbon-13 CP/MAS NMR spectroscopy

Degradation of Fe(III)-wool keratin complex by hydrogen peroxide.

The relation between the decomposition of hydrogen peroxide and the degradation of wool keratin fiber was studied for the Fe(III) -wool keratin complex-hydrogen peroxide system. The catalytic action of the Fe(III)-wool keratin complex in hydrogen peroxide decomposition was also studied. The Fe(III) ion showed a high catalytic activity, while the Fe(III)-wool keratin complex was less active. The decomposition of hydrogen peroxide occurred along with the degradation of wool keratin fiber by the scission of keratin molecules. The wool keratin fiber did not react with hydrogen peroxide in the absence of metal ions, but small quantities of the Fe(III) ion caused degradation. The degradation rate of wool keratin fiber was dependent on the pH of the treatment solution and became progressively higher as the pH of solution was lowered. The activity of Fe(III) ion was lower than that of the Cu(II) ion in both hydrogen peroxide decomposition and degradation of wool keratin fiber.

Investigation of the moisture adsorptivity of polar and hydrophilic groups in wool keratin by its chemical modifications.

For investigating the stoichiometry of water sorption by amino and carboxyl groups in wool keratin, two series of chemically modified specimens were prepared by partial deamination (van Slyke's reaction) and partial esterification (methylation) of a New Zealand Luster 48's wool, for which the moisture sorption isotherm and the heat of wetting were determined over the entire range of relative humidity at 30°C. Because of difficulty in preparing fully modified specimens by the chemical methods, the isotherm data were extrapolated to zero residual concentration of amino and carboxyl groups to estimate the isotherms of fully deaminated and decarboxylated specimens.By analyzing the differences in the isotherms between the untreated/partly modified specimens and the fully modified specimen in terms of the B. E. T.'s multilayer adsorption model, the maximum number of water molecules adsorbed in monolayer per each amino or carboxyl group was estimated as 1.0mole/mole and 1.3moles/mole at low concentration of the polar groups, and 2.5moles/mole and 2.0moles/mole at high concentration of the polar groups, respectively, The values for the carboxyl group is acceptable, while the values for the amino group must be underestimated due to the effects of replaced hydroxyl groups by the deamination.The hydration energies of the amino and carboxyl groups were evaluated as 6.7 and 9.1kcal/mole of the polar group, respectively, from the heat of wetting of the specimens at dryness. The value for the amino group must be underestimated due to the same reason as above. The differential heat of moisture sorption at dryness (hydration enthalpy) was evaluated as 4.91kcal/mole of liquid water for the untreated specimen, which was somewhat smaller than the value of 5.8kcal/mole of the alpha water proposed by Speakman.

Flash photolysis studies of a sulphonated bis-styryl biphenyl fluorescent dye

Long-lived (μ-ms) oxygen-insensitive transient species have been detected during laser flash photolysis of solutions of a sulphonated bis-styryl biphenyl dye. The transient species are readily quenched by the reducing agents iodide and sulphite ions but their yield is enhanced in the presence of oxidising ions. It is proposed that the transients are dye radical cations formed following monophotonic photoionization. Transient quenching is also observed in the presence of indole, tryptophan and tryptophyl peptides and the relevance of these results to the sensitized photodegradation of the protein fibre, wool keratin, in the presence of this fluorescent whitening dye is discussed.

Physical properties of wool keratin yarn modified with dibasic acid anhydrides

AbstractThe structure and physical properties of wool keratin yarns modified with dibasic acid anhydrides, namely, succinic and glutaric anhydride are analysed by thermomechanical analysis (TMA), differential scanning calorimetry (DSC), thermogravimetry (TG), and on the basis of measurements of the tensile properties. The optimum temperature for the reaction with dibasic acid anhydride can be achieved at 65°C. The weight gain of wool yarn treated with glutaric anhydride was 3% larger compared with that of the sample treated with succinic anhydride for the reaction time of 4 h. However, the acyl contents of the wool yarn treated with succinic and glutaric anhydride was the same. The moisture regain values of the wool yarn treated with glutaric anhydride were much lower by about 2% compared with those for the wool yarn treated with succinic anhydride. The original properties in mechanic of the wool yarn are not damaged even after the modification with dibasic acid anhydride. Taking into account the dyeability and thermal properties of the chemically modified wool keratin yarns, it would seem that the ester crosslinks are easily formed in keratin much more by the glutarylation than by succinylation.