Wool Keratin Research Articles

The photodegradation of wool keratin II. Proposed mechanisms involving cystine

A possible mechanism for the formation of cysteine, cysteic acid and partially oxidized cystine species in keratin following exposure to UVC radiation at 254 nm is proposed. The primary photolysis products are the radical ions RSSR'' and RSSR'-, and it is suggested that partially oxidized cystine species are formed from reactions of the radical cation only. The mechanisms m is supported by Fourier transform IR (FT-IR) and optical reflectance spectroscopy of UVC-treated wool exposed to air, oxygen and nitrogen atmospheres, and by previous electron spin resonance (ESR) studies on disulphides exposed to both UV and ionizing radiation. The mechanism supports the Symons assignment for the sulphur radical “species X” as [RS(SR)SR]', rather than RSS'. Exposure of wool to less energetic UV radiation produces different products via a quenching mechanism involving the formation of the radical anion RSSR'- only, which precludes the formation of oxidized cystine intermediates. The formation of S-sulphonate residues at longer wavelengths suggests that a competing singlet oxygen process may also occur.

Novel proteinous microcapsules from wool keratins

Sonication of an aqueous solution of wool keratins in the presence of an organic solvent such as toluene and xylene furnished the spherical microcapsules with about 2–10 mm in diameter and 0.05–0.1 mm in membrane thickness. When oil, fat and dyes were dissolved in the solvent, they were trapped within the vesicles in a yield of more than 90%. Although the other proteins such as a-lactalbumin gave the capsules of the similar dimensions, the keratin capsules were the most tight container. The barrier property of the keratin membrane was discussed in conjunction with high content of a cysteine residue in the keratins.

FTIR Analysis to Study Chemical Changes in Wool Following a Sulfitolysis Treatment

Sulfitolysis of wool keratin is important to many industrial processes, including shrink resistance, setting, and bleaching; its extent therefore requires rapid estimation. Currently, time consuming chemical analysis is used to convert the cysteine-S-sulfonate to cysteine and then to measure total cysteine colorimetricaily. Using ftir and the attenuated total reflectance (atr) method, our experiments quantify the relative amount of cysteine-S-sulfonate on the wool fiber surface. The cysteine-S-sulfonate (Bunte salt) shows a strong symmetrical stretching vibration at 1022 cm−1; working in the absorbance mode and taking second derivatives to enhance resolution allows changes in the amount of Bunte salt to be followed. The keratin Bunte salt is stable to acid but labile in alkali; a treatment with ammonium thioglycolate at pH 7.6 completely removes the Bunte salt. Results show the same trend as those obtained using the classical chemical method.

Sequences and differential expression of three novel human type-II hair keratins

As part of a program designed to characterize human hair keratin genes and their expression, we present the cDNA sequences and deduced amino acid sequences of three type-II hair keratins hHb3, hHb5, and hHb6, which by virtue of their amino acid homologies are the orthologs of the previously described sheep wool keratins, K2.10, K2.12, and K.211 [29]. Amino acid sequences comparisons of these keratins, including the previously characterized human K2.9 ortholog hHb1, show extreme conservation not only in the α-helices but also in the aminoterminal and proximal carboxyterminal domains. They also demonstrate higher sequence relationships between hHb1, hHb3, and hHb6 as compared to hHb5, which exhibits chain-specific sequences in both the head and tail domains. In situ hybridization studies using specific 3′-probes for the four type-II hair keratins reveal sequential patterns of gene expression in human anagen follicles. Remarkably the onset of hHb5 mRNA synthesis occurs immediately above a small population of matrix cells at the base of the hair bulb and the trichocytes lining the dermal papilla. hHb5 mRNA synthesis extends upward through the matrix and ends in the lower part of the cortex of the hair shaft. In contrast, both hHb1 and hHb3 mRNA synthesis begins simultaneously in the cortex 10–15 cell layers above the apex of the dermal papilla, thus partially overlapping that of hHb5 but continuing to a point well beyond hHb5 in the upper cortex. Synthesis of hHb6 mRNA starts slightly higher than either hHb1 or hHb3 mRNA and proceeds much farther up into the keratogenous zone of the hair shaft. Our study demonstrates that the differentiation of human hair in terms of hair keratin expression begins much earlier than previously assumed, i.e. in lower matrix cells of the hair bulb. This early phase of hair differentiation is followed by a late cortical phase of terminal differentiation which comprises at least three type-II hair keratins in the zone of elongation and the keratogenous zone of the hair shaft.

Origin of the bimodal ?melting? endotherm of ?-form crystallites in wool keratin

The melting endotherm of α-form crystallites in wool keratin appears often to be bimodal. The origin of the bimodal endotherm has been considered to correspond to the differential melting of the α-form crystallites in the domains of ortho- and para-cortical cells. An alternative interpretation is presented, in which the bimodal peak arises from the overlapping of the melting endotherm of α-form crystallites and the thermal degradation of other wool histological components. © 1997 John Wiley & Sons, Inc. J Appl Polym Sci 63: 411–415, 1997

Change in Amino Acid Composition of Cu(II)-Wool Complex by Degradation with Hydrogen Peroxide.

In order to obtain some information regarding to the change in amino acid compositions of wool keratin brought about by hydrogen peroxide under conditions in which the Cu (II) ion is bound to wool, the amino acid analyses were made on the residues and the soluble pepetide fragments produced by this oxidation.Cystine was converted into cysteic acid by this oxidation and not detected on the soluble pepetide fragments in filtrates. A relatively large amount of aspartic and glutamic acids was present in the residues and the filtrates. However phenylalanine and tyrosin were not detected in the filtrates, and these amino acids were less in the residues.

96/06362 Fire preventation during use of alternative fuels

Normal hair growth and differentiation requires co-ordinate expression of many hair specific structural protein genes. It has been established that one of the 4 major groups of hair structural proteins, low-sulphur hair keratins, belongs to the intermediate filament (IF) multigene family. Hair keratin IF proteins differ from those of other epithelia as they contain cysteine-rich terminal domains allowing more extensive disulphide bonding to the high-sulphur hair matrix proteins. Until recently, little information concerning the primary sequence of hair keratins was available but cloning of some mouse hair and sheep wool keratins has now been reported. Using these sequences, we have polymerase chain reaction (PCR) amplified genomic fragments of human hair-specific keratin IF genes and isolated cosmid clones containing full length genes. We have sequenced part of these genes and studied their expression in human hair follicles. Hair specific keratin fragments were amplified from placental gDNA by PCR primed with synthetic oligonucleotides. Fragments were cloned and sequenced after ligation into pGEM-3Z and labelled riboprobes were generated for in situ hybridization on human skin sections. A human cosmid library was screened with PCR fragments and clones encoding human hair keratin genes were characterised by southern hybridization and sequencing. The type I human hair-specific keratin clones obtained (HaKA1-b2, 386 bp; hHaKA1-XH1, 1202 bp) encoded 2B helix, C-terminal and 3′nc regions and were 65% homologous to mouse sequences. The type II hair keratin clone (hHaKB2-1, 829 bp) also encoded 2B helix and C-terminal regions and was 95% homologous to mouse. In situ hybridization on human skin sections showed a specific reaction with precortical cells of the hair follicle. One human cosmid clone, isolated with the hHaKB2-1 probe, contained two type II hair keratin genes about 7 kb apart, each of which had 9 exons spanning ∼ 6 kb. The coding sequences were homologous to mouse cDNA (77–88%). These human hair-specific keratin clones are useful molecular tools for studies of hair differentiation.

Genetic variation in Australian Merino sheep.

Variation at 22 gene loci was investigated in a flock of Australian Merino sheep using restriction fragment length polymorphism (RFLP) analysis. Polymorphism was observed at 20 loci, including loci for wool keratin, hormone and immunoglobulin light chain genes. Eleven loci yielded unambiguous genotypes suitable for population data analysis. Average heterozygosity, determined from these and two monomorphic loci, was estimated as 0.107 (SE = 0.024). Average heterozygosity excluding all monomorphic data as estimated at 0.377 (SE = 0.031), which is comparable with human RFLP heterozygosities for loci chosen in the same way that we selected sheep loci.

Effects of epidermal growth factor and transforming growth factor alpha on the function of wool follicles in culture.

The development of a procedure to culture wool follicles from Merino sheep in serum-free conditions has enabled us to investigate the actions of epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) on follicle function, including fibre growth. Follicles grown in the absence of growth factors maintained their anagen morphology for 6 days as determined by light microscopy. During this time they incorporated [3H]thymidine into the DNA of the bulb matrix and outer root sheath (ORS) cells and produced fibre keratins as detected by immunohistochemistry. In the presence of EGF and TGF alpha, fibre production ceased after 4 days, as it does following the administration of EGF in vivo. Cessation of fibre growth was not accompanied by regression of the follicle bulb which occurs in vivo. Follicle length growth did not differ significantly from controls and cells in the bulb continued to proliferate. Usually, the structure of the dermal papillae resembled that in control follicles, which was also in marked contrast to changes reported in vivo. In EGF- and TGF alpha-treated follicles, [3H]thymidine continued to be incorporated into DNA of the ORS and bulb after fibre growth ceased. Although wool keratin synthesis ceased, cytokeratins of the epidermis and ORS continued to be produced in the bulb as detected by immunochemistry. These bulb cells were also positive for the periodic acid-Schiff (PAS) reaction indicating the presence of glycogen, a normal component of ORS cells. The observations that cell proliferation continued in the bulb, that glycogen was present and that soft keratins were expressed in these cells suggest that the bulb cell population was induced to differentiate into an ORS phenotype by EGF and TGF alpha.

Photodegradation of wool keratin: Part I. Vibrational spectroscopic studies

The photodegradation of wool keratin is a very complex process that is not well understood. In this work the techniques of Fourier transform Raman and attenuated total reflectance Fourier transform-infrared spectroscopies have been used to study wool samples irradiated in air using a variety of different sources. The spectra obtained from these samples have been compared with those obtained from wool treated with sodium bisulphite, a reagent well known to produce thiol and S-sulfonate groups. As an aid for spectral interpretation the Raman and infrared spectra of cystine and those of the sodium and potassium cysteine-S-sulfonates have also been obtained. The data suggest that there are two different photolytic reaction pathways involving the cystine residues which are dependent on the wavelength of the applied radiation. © 1996 John Wiley & Sons, Inc.

The photolytic production of chromophores and fluorescence in wool keratin

The differential reflectance and three‐dimensional fluorescence spectra of wet and dry wool following UVB irradiation at 303 nm in the presence and absence of oxygen were measured. Fluorescence originating from tryptophan residues was reduced under all irradiation conditions and a nonfluorescent chromophore with a maximum at 320 nm was produced. These findings suggest that neither singlet oxygen nor triplet states are involved in the formation of the chromophore. Following irradiation of wool at 303 nm in the absence of oxygen, fluorescence with an excitation maximum between 360 and 380 nm and an emission maximum at 440 nm appeared. Fluorescence with an excitation maximum between 410 and 420 nm and an emission maximum at 470 nm from a different species was produced by photolysis in the presence of oxygen. The presence of water accelerates the formation of the latter fluorophore and this observation suggests that singlet oxygen is not involved in its formation.

Sequence Data and Chromosomal Localization of Human Type I and Type II Hair Keratin Genes

A cDNA library constructed with poly(A)+ RNA from human scalp was screened with selected fragments of both murine type I and type II hair keratin cDNAs. Two keratin clones, one type I, phKI-2, and one type II, phKII-1, were isolated and sequenced. In Northern blots, cDNA probes containing the 3′-noncoding sequences of the clones specifically hybridized to scalp mRNA species. Based on sequence homology comparisons with the four known murine type I hair keratins mHa1-4, the phKI-2 encoded keratin could be identified as human hair keratin hHa2. Similarly, sequence comparison with the four type II sheep wool keratins K2.9-12 revealed an orthologous relationship between the largest member of the type II wool keratin subfamily, K2.9 (i.e., sHb1) and the phKII-1 encoded human hair keratin (hHb1). The specific 3′-noncoding sequences of hHa2 and hHb1 were also used to isolate genomic fragments for both keratins from human genomic libraries which were than used for fluorescence in situ hybridization to human rectaphase chromosomes. The hHa2 gene could be mapped to the long arm of chromosome 17, whereas the hHb1 gene was found on the long arm of chromosome 12. DAPI banding of the chromosomes allowed sublocalization of the hHa2 gene to 17q12-q21 and the hHb1 gene to 12q13, i.e., gene loci that have also been previously determined for human type I and type II epithelial keratins.

New trends in photobiology (invited review) photodegradation of keratin and other structural proteins

Keratin and many other proteins are damaged when they are exposed to ultraviolet and visible radiation. In wool keratin, this is apparent as a colour change and a weakening or embrittlement of the fibre. Photodegradation of other structural proteins such as collagen, elastin and eye lens proteins (crystallins) have been linked with problems such as premature skin ageing and cataract formation; a possible involvement in skin cancer has even discussed. Despite several decades of research, the photochemistries of these processes are still imperfectly understood. This review deals with the photophysical and early photochemical events occurring in keratin upon exposure to ultraviolet and longer wavelength radiation. General aspects of the photochemistry of other proteins and model amino acid systems are also reviewed.

The reaction of wool keratin with tetrazonium cations

A novel dyeing method is described in which wool can be treated with salts formed by tetrazotising benzidine, o‐dianisidine and 4,4′‐diaminostilbene. Evidence is provided that the salts react with aromatic nuclei present in certain amino acids that form part of the keratinous molecular structure of wool, to form in situ azo dyes. Details of the process and a number of potential practical advantages are discussed.

Solid-State 31P Nuclear Magnetic Resonance Spectroscopic Study of Phosphorylated Peptide-Resins and Phosphorylated Wool Keratin

Solid-state 31P n.m.r. spectroscopy was found to be a useful method for the analysis of phosphorylated peptide-polystyrene resins and permitted the monitoring of chemical modifications of the phosphate moiety whilst the phosphorylated peptide was bound to the polystyrene support. Solid-state 31P n.m.r. spectroscopy was also applied to the analysis of phosphorylated wool keratin samples and showed that (A) different phosphate functionalities could be identified from the use of particular phosphorylation treatments, and (B) the majority of the phosphorylative modifications was located on the surface of wool keratin. This technique was found to be useful for monitoring the effectiveness of secondary chemical modification treatments on phosphorus trichloride-treated wool fabric samples.

Sequence and expression of human hair keratin genes

Normal hair growth and differentiation requires co-ordinate expression of many hair specific structural protein genes. It has been established that one of the 4 major groups of hair structural proteins, low-sulphur hair keratins, belongs to the intermediate filament (IF) multigene family. Hair keratin IF proteins differ from those of other epithelia as they contain cysteine-rich terminal domains allowing more extensive disulphide bonding to the high-sulphur hair matrix proteins. Until recently, little information concerning the primary sequence of hair keratins was available but cloning of some mouse hair and sheep wool keratins has now been reported. Using these sequences, we have polymerase chain reaction (PCR) amplified genomic fragments of human hair-specific keratin IF genes and isolated cosmid clones containing full length genes. We have sequenced part of these genes and studied their expression in human hair follicles. Hair specific keratin fragments were amplified from placental gDNA by PCR primed with synthetic oligonucleotides. Fragments were cloned and sequenced after ligation into pGEM-3Z and labelled riboprobes were generated for in situ hybridization on human skin sections. A human cosmid library was screened with PCR fragments and clones encoding human hair keratin genes were characterised by southern hybridization and sequencing. The type I human hair-specific keratin clones obtained (HaKA1-b 2, 386 bp; hHaKA1-XH1, 1202 bp) encoded 2B helix, C-terminal and 3′nc regions and were 65% homologous to mouse sequences. The type II hair keratin clone (hHaKB2-1, 829 bp) also encoded 2B helix and C-terminal regions and was 95% homologous to mouse. In situ hybridization on human skin sections showed a specific reaction with precortical cells of the hair follicle. One human cosmid clone, isolated with the hHaKB2-1 probe, contained two type II hair keratin genes about 7 kb apart, each of which had 9 exons spanning ∼ 6 kb. The coding sequences were homologous to mouse cDNA (77–88%). These human hair-specific keratin clones are useful molecular tools for studies of hair differentiation.

Characterization of a Hair (Wool) Keratin Intermediate Filament Gene Domain

In epithelial differentiation keratin intermediate filament genes are expressed in multifarious tissue-specific and stage-specific patterns. Pairs of type I and type II intermediate filament genes, belonging to multigene families, are coordinately regulated, and 4-5 genes of each type are expressed in the hair follicle. Accumulating chromosomal mapping data points to a major locus for each intermediate filament multigene family on separate chromosomes. In this report we describe the isolation of a sheep hair keratin cosmid by chromosome walking that overlaps two previously described cosmids and establishes a continuous 100-kb segment of cloned DNA containing three hair and three hair-like type II intermediate filament keratin genes. A new hair keratin type II intermediate filament gene, KRT2.11, is located in the middle of the cluster, and partial sequence data reveal a striking conservation of its predicted N-terminal region with other sheep hair keratin type II intermediate filament proteins. Expression analyses demonstrate the presence of a 2.4-kb KRT2.11 transcript in wool follicle RNA and show that expression occurs in the follicle cortical keratinocytes above the dermal papilla. The three hair genes are clustered within about 40 kb and flanked by hair-like genes that are not expressed in the hair follicle, thereby demarcating a hair keratin gene domain.

Dietary Cysteine Regulates the Levels of mRNAs Encoding a Family of Cysteine-Rich Proteins of Wool

The abomasal or intravenous infusion of sulphur-containing amino acids such as cysteine or methionine into sheep on low-quality diets increases the sulphur content of the wool by increasing the synthesis of proteins containing a cysteine content of approximately 30 mol %. To investigate the molecular and cellular basis of this nutritional effect, quantitative analyses of wool keratin mRNA and protein levels, and follicle cortical cell type, were undertaken in sheep intravenously infused with cysteine. Northern blot analyses revealed that the mRNA levels of one gene family encoding cysteine-rich keratin-associated proteins (KAP4 family) expressed in the wool follicle cortex, increased approximately 5-6 times. Furthermore, the response was rapid as the mRNA levels increased approximately 3.5 times after 1 d of the cysteine infusion and, by 1 d post-infusion, they had fallen, approaching their basal level. No changes in the mRNA levels encoding the intermediate filament or the other keratin-associated protein families of lower cysteine content were observed. Concomitantly, two-dimensional polyacrylamide gel electrophoresis analysis of wool proteins showed a striking increase in the abundance of a group of cysteine-rich keratin-associated proteins in the wool by the end of the infusion period, returning to basal levels by 3 weeks later. At the cellular level, KAP4 expression was localized to the follicle paracortical cells, and the proportion of paracortical cells and the extent of KAP4 expression paralleled the changes in the cysteine infusion status of the sheep.

Growth of wool follicles in culture.

A procedure for the culture of isolated wool follicles from Merino sheep is described. Follicles were microdissected from midside skin samples of 2-yr-old wethers and transferred, individually, to 24-well tissue culture plates. When maintained in supplemented Williams' E medium containing 5 to 10% fetal bovine serum (FBS), insulin, hydrocortisone, and a trace element mixture, fibre growth rates of 40 to 80 microns/day were observed. Follicles maintained their morphologic integrity for up to 7 days, incorporated [methyl-3H]thymidine into DNA and [35S]methionine into intermediate-filament keratins of the growing fiber. Insulin and hydrocortisone stimulated fiber growth at concentrations of 10 micrograms/ml and 50 ng/ml, respectively, but higher doses were inhibitory. The growth of fibers in response to hydrocortisone and the changes in follicle morphology was similar to those induced in skin after systemic administration of cortisol in vivo. A positive interaction between hydrocortisone and trace elements for follicle survival and hydrocortisone, insulin, and FBS for fiber growth was also found. The successful culture of Merino sheep follicles provides a model with which to study the direct influence of endocrine, nutritional and local factors on wool keratin synthesis independently of systemic shifts in the animals' metabolism.

Comparative study of metal ion interactions with wool keratin using chemometrics

Uptakes of chromium(III), copper(II), iron(II), nickel(II) and zinc(II) by wool were determined by inductively coupled plasma atomic emission and flame atomic absorption spectrometry. The order of uptakes at 100 °C for these metal ions was found to be CuII≫ ZnII > CrIII > FeII > NiII. Further, the metal ion-treated wool samples were submitted to Fourier transform infrared spectrometric analysis, which was facilitated by chemometric methods (principal components analysis, soft independent modelling of class analogies and fuzzy clustering). This revealed the order of structure modification at the carboxyl group as a result of metal ion–wool interactions to be CrIII > CuII≫ ZnII > FeII≈ NiII. The difference between the above two rank orders suggests that relatively large uptakes do not necessarily lead to the most significant structurally interactive effects in wool keratin.