Keratin Molecules Research Articles

Changes in Cell Shapes and Cytokeratins of Epithelial Cells during the Infiltration of Lymphocytes in the Human Palatine Tonsils

By the immunohistochemical method using anti-keratin antibodies (KL1-antigen, 56 KD keratin; PKK1-antigen, 40-52.5 KD keratin) and electron microscopy, the changes in cell shapes and the cytokeratin components of the epithelial cells during the infiltration of lymphocytes were studied. In the surface epithelium, PKK1 reacts with only the keratinocytes in the basal layer, while KL1 stains in the spinous layer. In the neck portion of the crypt, transformed keratinocytes scattered in the spinous layer react intensely with PKK1. These cells issue prolonged cytoplasmic processes which surround the cavities filled with infiltrating lymphocytes. In the deep portion of the crypt, PKK1-positive cells interconnect with one another constituting the network of the star-shaped reticular cells. The enlarged intercellular spaces of the reticular cells are filled with many lymphocytes. Our observations suggest that the infiltration of lymphocytes into the crypt epithelium induces the change in keratin expression of the epithelial cells in the human palatine tonsils, and the change of keratin molecules makes the stratified flattened epithelium to transform into the network of star-shaped reticular cells.

The Two Dimensional Peptide Mappings of the Nail Low Sulfur S‐carboxymethyl Keratins

AbstractThe peptide map patterns of the nail low sulfur (LS) S‐carboxymethyl keratins were investigated using Staphylococcus aureus V8 protease (V8 protease) and subtilisin. The nail LS‐keratins which were analyzed by two dimensional (2D) polyacrylamide gel electrophoresis (PAGE) comprised eight major polypeptides. Since these nail LS‐keratins were well separated from each other by 2D‐PAGE, we could compare peptide fragments of each LS‐keratin by a 2D peptide mapping. Among the eight major LS‐keratins of the nail, the LS‐keratins of similar size and pI values yielded many same sized peptide fragments with either proteinase. From these results, we could divide the nail LS‐keratins into three groups by their molecular weights, pI values and the PAGE patterns of their peptide fragments by the proteinase digestion.Examinations of smaller sized peptide fragments produced by partial enzymatic hydrolysis with either proteinase showed strong similarities for all the nail LS‐keratins. Several dense bands of same sized peptide fragments were also observed by 2D peptide mappings. On the other hand, considerable differences were observed when we compared the larger sized peptide fragments of the different group of the nail LS‐keratins. These observations suggested that related structural units (domain structures) were present for all the nail LS‐keratins, although they could be divided into three different groups.The LS‐keratins of the epidermal cornified layer analyzed by 2D‐PAGE were also composed of heterogeneous protein groups. However, when they were partially hydrolyzed by V8 protease, there appeared many same sized peptide fragments for all the epidermal LS‐keratins and three dense bands of same sized peptide fragments were observed. Consequently, the 2D peptide map pattern of the epidermal LS‐keratins by V8 protease was very simple as compared to that of the nail LS‐keratins. Thus the molecular species composing the nail keratin filaments were more heterogeneous than those of the epidermis with respect to the distribution of glutamic acid residues in the molecules. The distribution of glutamic acid in the keratin molecules might contribute the morphological and physicochemical differences between the nail and the epidermis.

Intermediates in the conversion of prekeratin into keratin molecules in psoriatic epidermis

Intermediates in the conversion of prekeratin into keratin molecules in psoriatic epidermis

Monoclonal Antibody Studies of Mammalian Epithelial Keratins: A Review a

Significant progress has been made during the past decade with regard to the biochemistry and immunology of keratin filaments. The results indicate that keratin represents a family of more than 17 water-insoluble, cytoskeletal proteins,2 that keratins are restricted to epithelia and their derivatives;d that different subsets of 210 keratins are expressed in different epithelia,1~2~7-12 that at least two keratin species are required for filament a~sembly,'.'~ and that keratin molecules consist of a central helical domain with two nonhelical end^.^^-^^ Despite these advances, however, it remains a puzzle as to why such a large number of keratin species have evolved to form tonofilaments, while other morphologically similar, intermediate-sized filaments are characterized by a much simpler protein composition with only one to three subunits (for reviews, see Lazaridesl' and other chapters in this volume). To address this fundamental question, we have prepared several monoclonal antikeratin antibodies2' and used them as a tool for studying keratin expression. In this

Ichthyosis linearis circumflexa: morphological and biochemical studies

Extensive morphological and biochemical studies were carried out on two sibling cases of ichthyosis linearis circumflexa. The condition was found to be similar to psoriasis in the following ways: effectiveness of PUVA therapy, psoriatic changes on light and electron microscopy, high urinary polyamine levels, elevation of several enzyme activities in the scales, and a remarkable change in keratin molecules. These results may reflect an increased epidermal proliferation with a reduced epidermal transit time, as occurs in psoriasis.

Intermediates in the conversion of prekeratin into keratin molecules in human epidermis

In order to elucidate the relationship between prekeratin and keratin, we performed pulse-chase experiments using [35S]methionine (35S-Met) in vitro. Of 6 prekeratin molecules (49, 52, 55, 62, 69 and 71 kDa) that incorporated 35S-Met, the 55-kDa prekeratin incorporated the most 35S-Met. In 3 molecules (52, 55 and 62 kDa) incorporation was decreased at 30 min after being chased; however, incorporation of only two molecules (55 and 62 kDa) of the 6 prekeratins was increased at 60 min. From these results and our previous data, we conclude that the initial stage of processing is as follows: 3 prekeratin molecules (52, 55 and 62 kDa) are first cleaved in the N-terminal region, then two prekeratin molecules (55 and 69 kDa) are processed to intermediates (52 and 62 kDa) by some proteolytic enzyme(s).

Expression of specific keratin markers by rabbit corneal, conjunctival, and esophageal epithelia during vitamin A deficiency.

Using an in vivo rabbit model system, we have studied the morphological and biochemical changes in corneal, conjunctival, and esophageal epithelia during vitamin A deficiency. Light and electron microscopy showed that the three epithelia undergo different degrees of morphological keratinization. Corneal and conjunctival epithelia became heavily keratinized, forming multiple layers of superficial, anucleated cornified cells. In contrast, esophageal epithelium underwent only minor morphological changes. To correlate morphological alterations with the expression of specific keratin molecules, we have analyzed the keratins from these epithelia by the immunoblot technique using the subfamily-specific AE1 and AE3 monoclonal antikeratin antibodies. The results indicate that during vitamin A deficiency, all three epithelia express an AE1-reactive, acidic 56.5-kd keratin and an AE3-reactive, basic 65-67-kd keratin. Furthermore, the expression of these two keratins correlated roughly with the degree of morphological keratinization. AE2 antibody (specific for the 56.5- and 65-67-kd keratins) stained keratinized corneal epithelial sections suprabasally, as in the epidermis, suggesting that these two keratins are expressed mainly during advanced stages of keratinization. These two keratins have previously been suggested to represent markers for epidermal keratinization. Our present data indicate that they can also be expressed by other stratified epithelia during vitamin A deficiency-induced keratinization, and suggest the possibility that they may play a role in the formation of the densely packed tonofilament bundles in cornified cells of keratinized tissues.

The use of aIF, AE1, and AE3 monoclonal antibodies for the identification and classification of mammalian epithelial keratins

Recent data have indicated that specific keratin molecules can provide useful markers for studying different types and stages of epithelial differentiation. To utilize these protein markers, however, it is important to establish the keratin nature of the molecules and identify unambiguously the individual keratin species. In this paper, we show that this can be done relatively easily by one- and two-dimensional gel electrophoresis combined with immunoblotting using three monoclonal antibodies (aIF, AE1, and AE3). The aIF antibody has previously been shown to crossreact with all classes of intermediate-filament proteins. Using one- and two-dimensional immunoblotting, we establish that this antibody recognizes all known epithelial keratins of human and rabbit, although the reaction is relatively strong for the larger, basic keratins and is relatively weak for some of the smaller, acidic keratins. In contrast, AE1 and AE3 monoclonal antibodies have previously been shown to be highly specific for the acidic and basic subfamilies of the keratins, respectively. The combined use of the broadly reacting aIF antibody and the subfamily-specific AE1 and AE3 monoclonal antikeratin antibodies should facilitate the immunological definition, identification, and classification of mammalian epithelial keratins.

Initial stage of processing from prekeratin into keratin molecules in human epidermis in vitro.

ABSTRACTIn order to clarify the initial formation of keratin from prekeratin molecules, we undertook a pulse‐chase experiment using [U‐14C]‐glycine labelled prekeratin subunit mixture in vitro.The experiment was carried out at 37°C for 3 hours using a specimen (3 mm2×0.1 mm) in Dulbecco minimum essential medium (MEM) containing 14C‐glycine. Incorporation of 14C‐glycine into the prekeratin molecules was exhibited in six prekeratins (M.W. about 71, 69, 62, 55, 52 and 49 K, respectively), and the highest incorporated molecule among them was the 55K‐prekeratin molecule. After 30 minutes at 37°C in the above medium, the intact specimens were transferred into a comparable medium free of 14C‐glycine, and further incubated for 4 hours. The 52K‐ and 62K‐prekeratin molecules had increased at 60 minutes after being chased, as compared with the starting radioactive value. From this result, it was suggested that the two prekeratin molecules might be intermediates in the initial stage of keratinization. Moreover, the human epidermal homogenate was more effective in converting the prekeratin into keratin molecules. Some factor(s) may be present in the fraction which convert the prekeratin into keratin molecules.

Effects of glutaraldehyde and osmium tetroxide on newborn rat epidermal keratin.

The effects of glutaraldehyde and osmium tetroxide (OsO4) fixation on ultrastructural and biochemical changes of epidermal keratin were investigated. Morphometrically, the apparent size of keratohyalin granules was smallest when granular cells were fixed with OsO4, whereas those fixed with glutaraldehyde were larger, and those double-fixed with glutaraldehyde and OsO4 were largest. The ultrastructure of tonofilaments appeared different in each type of fixation. Biochemically, cross-linking glutaraldehyde with purified keratin from cornified cells formed a high molecular weight product with a blue shift of the absorption maximum and an increase in absorbance. Detection of the absorption spectra of OsO4- keratin reactions revealed that a new absorption maximum around 410 nm developed in glutaraldehyde-treated keratin, while one around 315 nm appeared in nontreated keratin. In addition, glutaraldehyde pretreatment increased by 40% the initial reaction rate and by 23% the total amount of OsO4 reduction. Sodium dodecyl sulfate gel electrophoresis demonstrated that degradation of keratin molecules occurred in both glutaraldehyde-treated and untreated keratin after 2 hr of exposure to OsO4. These findings indicate that keratin molecules are chemically modified by fixatives routinely used for electron microscopy. We consider that this chemical information plays an essential role in the appropriate interpretation of the ultrastructure of epidermal cells.

Organisation of the polypeptide chains in mammalian keratin.

THE keratins are insoluble intracellular α-fibrous proteins which represent the major structural proteins of epidermis. X-ray diffraction studies of keratins in the early 1950s1,2 yielded a pattern since shown to be typical of intact epidermal tissue and epidermal α keratins. This pattern, which consists of a meridional arc at 5.15 A and an equatorial reflection at 9.8 A, was suggested to be indicative of polypeptide chains packed in a partially α-helical coiled coil configuration. Crick3 suggested that a three-stranded model was appropriate for α keratins, and although two-stranded and seven-stranded models have also been proposed, the triple α-helical coiled coil configuration has been a dominant concept in keratin structure4. Rudall made the initial contribution of epidermal keratin chemistry by using urea buffers to extract and characterise the α-fibrous proteins from cow snout epidermis. Matoltsy subsequently showed that an α-fibrous protein, which he designated prekeratin, could also be isolated by extraction with citrate buffer, pH 2.65, and purified by isoelectric precipitation. His data indicated that the prekeratin was an α-helical, rod-like molecule with a molecular weight of 640,000 (ref. 5). Detailed studies6–8 have shown that epidermal keratins consist of several different polypeptide chains, and we describe here experiments which suggest how these chains may be organised in the native keratin molecule.

Wettability of some low energy surfaces. II. Oils on solids submerged in water

Experimental values of contact angles in various hydrocarbon liquid/water/solid systems are compared with calculated values based on the Fowkes-Young and Wu-Young equations. Satisfactory agreement is obtained with the Wu equation for Teflon and poly (methylmethacrylate) provided the receding angle in the hydrocarbon liquid is used. Both equations predict complete wetting of paraffin wax by the hydrocarbon liquids, as was observed. On the other hand, the contact angles observed on nylon 11 and bovine hoof keratin (> 170° in oil) are much higher than predicted. These results are attributed to the ability of nylon and keratin to hydrate under water so promoting aqueous phase/solid interaction at the expense of hydrocarbon/solid interaction. These surfaces possess a dual functionality—hydrophobic in air, hydrophilic in water. In addition, keratin has the property that water will not readily displace oil from its surface. Apparently, surface keratin molecules adopt and retain a conformation most compatible with their immediate environment. The agreement between observed and calculated (Fowkes, Wu) angles for n-hexanol/water/ solid systems was poor. This lack of agreement is attributed to adsorption of the alcohol on the solid. Good agreement was, however, obtained with values based solely on the Young equation, utilizing contact angles for the liquids in air, in the case of Teflon.

Vertebral epidermal transamidases

The α-helical fibrous proteins (keratin) of the stratum corneum undergo changes in their physicochemical properties which are not solely explained on the basis of disulfide bond formation. The precursor of the stratum corneum protein, prekeratin, and the final product keratin were studied in the hair-follicle-free skin of calf snout epidermis. All of the lysines of prekeratin can be cyanoethylated while keratin molecules has 7–8 nmoles lysine per mg protein not available to cyanoethylation. Keratin from the human stratum corneum has 8–9 nmoles/g, protein which cannot be cyanoethylated. Direct examination of these proteins failed to detect the presence of N- ε-acetyllysine groups or ε-(γ-glutamyl)lysine groups as the explanation of the blocked lysines. The hair-follicle-free epidermis of several vertebrate species contains transamidases similar to the transglutaminases which form ε-(γ-glutamyl)lysine crosslinks, in fibrin, hair follicle, and seminal fluid. The epidermal transamidase has a molecular weight between 40 000 and 60 000, is stimulated by reducing agents and calcium, and has most of its activity in the highermost levels of the epidermis. A semipurified preparation of the epidermal transamidase was able to form γ dimers and α polymers from Factor XIII-free fibrin. A transamidase substrate, [1,4- 3H 2]putrescine, when injected into mouse skin and studied autoradiographically, was localized in the stratum corneum. [1,4- 14C 2]putrescine incorporation into epidermal proteins was inhibited by 50 mM iodoacetamide. The incorporation of [1,4- 14C 2]putrescine was highest in very insoluble protein fractions. The exact role of transamidases in epidermal metabolism is not yet identified.

The structure of feather keratin

When feather rachis is pressed in steam the keratin molecules are partially denatured and the x-ray diffraction pattern, which is greatly simplified, indicates that the microfibrils have a helical structure with four units per turn. Quantitative infra-red and x-ray data have been collected which suggest that this simplified pattern originates from about one third of the material in the form of a central core or framework which is resistant to denaturing agents. A model for this core consisting of a helical array of β-crystallites is shown to give quantitative agreement with the observed x-ray pattern over a wide range of angles. Each crystallite contains two sheets symmetrically disposed about the fibre axis and the sheets are distorted to conform to a ruled surface of opposite sense to the primitive helix.

The Organogenesis and Arrangement of Cutaneous Appendages in Birds

Publisher Summary This chapter discusses the organogenesis and arrangement of cutaneous appendages in birds. The skin of the vertebrate embryo, and of birds, is a suitable system for the analysis of the factors of differentiation. It is composed of two distinct tissues of different developmental history, the interplay of which results in the construction of the cutaneous covering with its varied appendages—feathers and scales in birds. One of the constituents of the skin is derived from the mesodermal layer, that is, the dermis. The other component, namely, the epidermis, stems from the ectodermal layer. During early ontogeny, the ectodermal layers become epithelial in their histological structure, whereas the mesodermal component acquires a mesenchymal conformation. The organized superposition of epithelium and mesenchyme gives rise to the skin as such and its derivatives. The epidermis all over the embryo appears to be a neutral covering and carries only the necessary information for the realization of minute cytological features. It also contains the necessary biochemical equipment for the synthesis of the keratin molecules. The orientation of feathers is determined by the ectodermal component of skin. The phenotypic traits that depend on the ectoderm can never be overruled by mesodermal influence.

Treatments of Wool in Organic Solvents and Their Effect on the Urea-Bisulfite Test

Treatment of wool in a number of organic solvents reduces both its cystine content and its solubility in urea-bisulfite. Some reagents attack the disulfide bond in such a way that hydrogen sulfide is evolved. As, however, no lanthionine has been found, and the effect due to lysinoalanine is not yet known, it could be claimed that no correlation exists between cystine breakdown and reduction in solubility. The latter is probably due to physical changes of the keratin molecule, and it is affected by the temperature of the treatment and the size of the molecule. Purification of wool by extraction in a Soxhlet apparatus with diethylether and ethanol reduces the solubility and must be avoided. Petroleum ether of either low or high boiling range or extraction in the cold with diethylether and ethanol has no effect on the solubility.

An Investigation of the Reactions of Sulphamic Acid with Keratin Fibre

The conditions for making wool fibre resistant to acid dyes by treatment with sulphamic acid have been investigated and it has been shown that the major chemical reactions are complex. The amino groups, disulphide bonds, and aliphatic hydroxy groups in the keratin molecule all participate in the reaction. Only a proportion of each type of group is blocked by sulphamic acid. The dye‐resistant effect is largely due to ionic repulsion of the dye by acidic groups so introduced.

Mechanical versus chemical keratolysis by dermatophytes

It is suggested that the concept of enzymatic keratolysis by dermatophytes stands in need of revision. Non-keratin containing aqueous extracts from human hair and nails have been shown capable of supporting the growth of the pseudoparasitic phase of certain dermatophytes. Destruction of such structures as hair and nails by an infecting fungus may be due to mechanical disintegration following invasion of the non-fibrillary matrix; the fungus feeding on the non-keratins without need to digest the keratin molecule. L'auteur suggère que soit révisée l'idée que l'on se fait actuellement de la lyse enzymatique de la kératine par les dermatophytes. Des extraits aqueux de cheveux et d'ongles humains, exempts de kératine, permettent la croissance de la phase pseudo-parasitaire de certains dermatophytes. La destruction de l'ensemble complexe qu'est un cheveu ou un ongle par un champignon parasite peut être due à une désintégration mécanique faisant suite à l'envahissement de la matrice non fibrillaire; la nutrition du champignon se faisant aux dépens de matériel étranger à la kératine, ne nécessite pas la lyse de la molécule de ce composé.

STUDIES ON THE ANGORA RABBIT FIBERS

The mechanical behaviour of an Angora rabbit hair in the acidic buffer solutions was studied by means of the methods described in the preceding paper and the following facts are observed:(1) When a stretching and recovery is repeated after a relaxation for 5min. after each cyclic operation, the 30% stretching work decreases with the number of the cycles.(2) This decrement is slightest at the isoelectric point, 4.5 and it appears to increase as the medium becomes more acidic or more alkaline, but the magnitude of the work itself decreases at the same time, and consequently the decrement is also slight and the work becomes nearly constant after 2 or 3 cycles at pH 1.1 and 1.9.(3) The 30% stretching work in the air decreases most significantly.(4) The work of recovery is nearly constant and does not change by the cycling. Its value is also maximum at the isoelectric point, and minimum in the air.(5) The hysteresis is largest in the air and decreases remarkably after the first stretching. In the water it is also a function of pH and the relation is similar to that of the stretching work.(6) The resilience, the ratio of the work of recovery to that of stretching, is on the contrary, larger at the lower pH value and is at minimum in the air.(7) The work of stretching which is decreased by 3 or more cyclic operations, as described above, partly recovers when the hair is relaxed in the water for 24hrs. The effect is most significant in the case of the hair which has been stretched in the air.(8) The work at 50°C is less than that at 30°C. This temperature effect grows larger as pH of the medium approaches the isoelectric point of the hair.These phenomena are explained from the view point of the folding and unfolding of the keratin molecules.

STUDIES ON THE ANGORA RABBIT FIBRES

The mechanical behaviour of an Angora rabbit hair in the acidic buffer solutions was studied by means of the methods described in the preceding paper and the following facts are observed:(1) When a stretching and recovery is repeated after a relaxation for 5min. after each cyclic operation, the 30% stretching work decreases with the number of the cycles.(2) This decrement is slightest at the isoelectric point, 4.5 and it appears to increase as the medium becomes more acidic or more alkaline, but the magnitude of the work itself decreases at the same time, and consequently the decrement is also slight and the work becomes nearly constant after 2 or 3 cycles at pH 1.1 and 1.9.(3) The 30% stretching work in the air decreases most significantly.(4) The work of recovery is nearly constant and does not change by the cycling. Its value is also maximum at the isoelectric point, and minimum in the air.(5) The hysteresis is largest in the air and decreases remarkably after the first stretching. In the water it is also a function of pH and the relation is similar to that of the stretching work.(6) The resilience, the ratio of the work of recovery to that of stretching, is on the contrary, larger at the lower pH value and is at minimum in the air.(7) The work of stretching which is decreased by 3 or more cyclic operations, as described above, partly recovers when the hair is relaxed in the water for 24hrs. The effect is most significant in the case of the hair which has been stretched in the air.(8) The work at 50°C is less than that at 30°C. This temperature effect grows larger as pH of the medium approaches the isoelectric point of the hair.These phenomena are explained from the view point of the folding and unfolding of the keratin molecules.