Immunolocalization of keratin-associated beta-proteins (beta-keratins) in scales of the reptiles Sphenodon punctatus indicates that different beta-proteins are present in beta- and alpha-layers

The present ultrastructural immunocytochemical study analyzes the localization of keratin-associated beta-proteins (beta-keratins) in the epidermis of the ancient reptile Sphenodon punctatus, a relict species adapted to mid-cold conditions. The epidermis comprises two main layers, indicated as beta- and alpha-keratin layers. The beta-layer contains small beta-proteins (beta-keratins) identified by using three different antibodies while the alpha-layer is poorly or not labeled for these proteins. Using other two antibodies directed against specific amino acid sequences identified in beta-proteins of lizard it results that a high-glycine beta-protein (HgG5) is specific for the beta-layer. Another antibody that recognizes glycine–cysteine medium-rich beta-proteins (HgGC10) immuno-stains beta- and alpha-layers. This pattern of distribution suggests that both beta- and alpha-layers contain beta-proteins of different types that associate and replace intermediate-filament alpha-keratins during the terminal differentiation of keratinocytes. Therefore the different epidermal layers of the epidermis in S. punctatus, characterized by a specific cytology, material properties and consistency appear to derive from the prevalent type of beta-proteins synthesized in each epidermal layer and not from the alternation between beta- and alpha-keratins. The present observations are discussed in comparison to previous results from lizard epidermis and indicate that beta-keratins correspond to keratin-associated proteins that through their internal beta-pleated region are capable to form filaments in addition to intermediate filaments keratins.     

Distribution of specific keratin-associated beta-proteins (beta-keratins) in the epidermis of the lizard Anolis carolinensis helps to clarify the process of cornification in lepidosaurians

The epidermis of different scales in the lizard Anolis carolinensis expresses specific keratin-associated beta-proteins (beta-keratins). In order to localize the sites of accumulation of different beta-proteins, we have utilized antibodies directed against representative members of the main families of beta-proteins, the glycine-rich (HgG5), glycine-cysteine rich (HgGC3), glycine-cysteine medium-rich (HgGC10), and cysteine-rich (HgC1) beta-proteins. Immunoblotting and immunocytochemical controls confirm the specificity of the antibodies made against these proteins. Light and ultrastructural immunocytochemistry shows that the glycine-rich protein HgG5 is present in beta-layers of different body scales but is scarce in the oberhautchen and claws, and is absent in alpha-layers and adhesive setae. The cysteine-glycine-rich protein HgGC3 is low to absent in the oberhautchen, beta-layer, and mesos-layer but increases in alpha-layers. This beta-protein is low in claws where it is likely associated with the hard alpha-keratins previously studied in this lizard. The glycine-cysteine medium-rich HgGC10 protein is low in the beta-layer, higher in alpha-layers, and in the oberhautchen. This protein forms a major component of setal proteins including those of the adhesive spatula that allow this lizard to stick on vertical surfaces. HgC1 is poorly localized in most epidermis analyzed including adhesive setae and claws and appears as a minor component of the alpha-layers. In conclusion, the present study suggests that beta- and alpha-layers of lizard epidermis represent regions with different accumulation of glycine-rich proteins (mainly for mechanical resistance and hydrophobicity in the beta-layer) or cysteine-glycine-rich proteins (for both resistance and elasticity in both alpha- and beta-layers).     

Proteolytic modification of acidic and basic keratins during terminal differentiation of mouse and human epidermis

Keratins from the living cell layers of human and neonatal mouse epidermis (prekeratins) have been compared to those from the stratum corneum (SC keratins). Human and mouse epidermis contained four prekeratins, two of each keratin subfamily: type II basic (pI 6.5-8.5; human 68 kDa, 60.5 kDa and mouse 67 kDa, 60 kDa) and type I acidic (pI 4.7-5.7; human 57 kDa, 51 kDa and mouse 58 kDa, 53 kDa,). While all four were present in equal amounts in adult human epidermis, two (67 kDa basic, 58 kDa acidic) were more prominent in neonatal mouse epidermis. Preliminary results with cell fractions (basal, spinous and granular) indicated that quantitative differences were a function of morphology, basal cells containing the smaller member of each subfamily and granular cells the larger. Mouse stratum corneum extracts contained four keratins (three in human): type II neutral-acidic (pI 5.7-6.7; human 65 kDa and mouse 64 kDa, 62 kDa) and type I acidic (pI 4.9-5.4; human 57.5 kDa, 55 kDa and mouse 58.5 kDa, 57.5 kDa). In both species, one-dimensional and two-dimensional peptide mapping (with V8 protease and trypsin respectively) indicated that while all four prekeratins were distinct gene products, similarities existed in the type II basic and the type I acidic keratin subfamilies. A strong homology also existed between type II SC keratins and the larger basic (type II) prekeratin (human 68 kDa and mouse 67 kDa) and between type I SC keratins and the larger acidic (type I) prekeratin (human 57 kDa and mouse 58 kDa). These results indicate a precursor-product relationship within each keratin subfamily, between SC keratins and the prekeratins abundant in the adjacent granular layer. This differentiation-related keratin processing was similar in mouse and human epidermis, and may represent a widespread phenomenon amongst keratinising epithelia.     

Immunolocalization of keratin‐associated beta‐proteins (beta‐keratins) in the regenerating lizard epidermis indicates a new process for the differentiation of the epidermis in lepidosaurians

The process of keratinocyte differentiation was analyzed in the regenerating epidermis of the lizard Anolis carolinensis, where the genes coding for beta‐proteins (beta‐keratins) are known. The regenerating epidermis forms all epidermal layers found in normal scales (Oberhäutchen‐, beta‐, mesos‐, and alpha‐layer). Three specific proteins representing the larger families of beta‐proteins, glycine‐rich (HgG5, 28% glycine, 3.6% cysteine), glycine‐cysteine medium‐rich (HgGC10, 13% glycine, 14.5% cysteine), and glycine‐cysteine rich (HgGC3, 30.4% glycine, 8.7% cysteine) have been immunolocalized at the ultrastructural level. HgG5 is only present in differentiating beta‐cells, a weak or no labeling is observed in Oberhäutchen and is absent in alpha‐cells. The protein is located in the pale corneous material forming the compact beta‐layer but is absent in mature Oberhäutchen cells. HgGC10 is present among beta‐packets in Oberhäutchen and beta‐cells but disappears in more compact and electron‐pale corneous material. The labeling disappears in mesos‐cells and is present with variable intensity in alpha‐cells, whereas lacunar and clear‐cells are low labeled to unlabeled. HgGC3 is sparse or absent in beta‐cells but is lightly present in the darker corneous material of differentiating and mature alpha‐cells, lacunar‐cells, and clear‐cells. The study suggests that while glycine‐rich proteins (electron‐pale) are specifically used for building the resistant and hydrophobic beta‐layer, cysteine–glycine rich proteins (electron‐denser) are used to form the pliable corneous material present in the Oberhäutchen and alpha‐cells. The differential accumulation of beta‐proteins on the alpha‐keratin cytoskeleton scaffold and not the alternance of beta‐ with alpha‐keratins allow the differentiation of different epidermal layers. © 2012 Wiley Periodicals, Inc.     

Wound keratins in the regenerating epidermis of lizard suggest that the wound reaction is similar in the tail and limb

The keratin cytoskeleton of the wound epidermis of lizard limb (which does not regenerate) and tail (which regenerates) hase been studied by qualitative ultrastructural, immunocytochemical, and immunoblotting methods. The process of re-epithelialization is much shorter in the tail than in the limb. In the latter, a massive tissue destruction of bones, and the shrinkage of the old skin over the stump surface, delay wound closure, maintain inflammation, reduce blastemal cell population, resulting in inhibition of regeneration. The expression of special wound keratins found in the newt epidermis (W6) or mammalian epidermis (K6, K16, and K17) is present in the epidermis of both tail and limb of the lizard. These keratins are not immunolocalized in the migrating epithelium or normal (resting) epidermis but only after it has formed the thick wound epithelium, made of lacunar cells. The latter are proliferating keratinocytes produced during the cyclical renewal or regeneration of lizard epidermis. W6-immunolabeled proteic bands mainly at 45-47 kDa are detected by immunoblotting in normal, regenerating, and scarring epidermis of the tail and limb. Immunolabeled proteic bands at 52, 62-67 kDa (with K6), at 44-47, 60, 65 kDa (with K16), and at 44-47 kDa (with K17) were detected in normal and regenerating epidermis. It is suggested that: (1) these keratins constitute normal epidermis, especially where the lacunar layer is still differentiating; (2) the wound epidermis is similar in the limb and tail in terms of morphology and keratin content; (3) the W6 antigen is similar to that of the newt, and is associated with tonofilaments; (4) lizard K6 and K17 have molecular weights similar to mammalian keratins; (5) K16 shows some isoforms or degradative products with different molecular weight from those of mammals; (6) K17 increases in wound keratinocytes and localizes over sparse filaments or small bundles of short filaments, not over tonofilaments joined to desmosomes; and (7) failure of limb regeneration in lizards may not depend on the wound reaction of keratinocytes.     

Ultrastructural localization of alpha-keratins in the regenerating epidermis of the lizard Podarcis muralis during formation of the shedding layer

In the epidermis of lizards, alpha- and beta-keratins are sequentially produced during a shedding cycle. Using pre- and post-embedding immunocytochemistry this study shows the ultrastructural distribution of 3 alpha-keratin antibodies (AE1, AE2, AE3) in the renewing epidermis and in the shedding complex of the regenerating tail of the lizard Podarcis muralis. The AE1 antibody that recognizes acidic low MW keratins is confined to tonofilament bundles in basal and suprabasal cells but is not present in keratinizing beta- and alpha-cells. The AE2 antibody that recognises higher MW keratins weakly stains pre-keratinized cells and intensely keratinized alpha-layers. A weak labeling is present in small electrondense areas within the beta-layer. The AE3 antibody, that recognizes low and high MW basic keratins, immunolabels tonofilament bundles in all epidermal layers but intensely the alpha-keratinizing and keratinized layers (mesos, alpha-, lacunar and clear). Keratohyalin-like granules, present in the clear cells of the shedding layer, are negative to these antibodies so that the cornified clear layer contains keratins mixed with non-keratin material. The AE3 antibody shows that the mature beta-layer and the spinulated folds of the oberhautchen are labeled only in small dense areas among the prevalent electron-pale beta-keratin material. Therefore, some alpha-keratin is still present in the beta-layer, and supports the idea that alpha-keratins (basic) function as scaffold for beta-keratin deposition.     

Characterization of keratins and associated proteins involved in the corneification of crocodilian epidermis

Crocodilian keratinocytes accumulate keratin and form a corneous cell envelope of which the composition is poorly known. The present immunological study characterizes the molecular weight, isoelectric point (pI) and the protein pattern of alpha- and beta-keratins in the epidermis of crocodilians. Some acidic alpha-keratins of 47-68 kDa are present. Cross-reactive bands for loricrin (70, 66, 55 kDa), sciellin (66, 55-57 kDa), and filaggrin-AE2-positive keratins (67, 55 kDa) are detected while caveolin is absent. These proteins may participate in the formation of the cornified cell membranes, especially in hinge regions among scales. Beta-keratins of 17-20 kDa and of prevalent basic pI (7.0-8.4) are also present. Acidic beta-keratins of 10-16 kDa are scarce and may represent altered forms of the original basic proteins. Crocodilian beta-keratins are not recognized by a lizard beta-keratin antibody (A68B), and by a turtle beta-keratin antibody (A685). This result indicates that these antibodies recognize specific epitopes in different reptiles. Conversely, crocodilian beta-keratins cross-react with the Beta-universal antibody indicating they share a specific 20 amino acid epitope with avian beta-keratins. Although crocodilian beta-keratins are larger proteins than those present in birds our results indicate presence of shared epitopes between avian and crocodilian beta-keratins which give good indication for the future determination of the sequence of these proteins.     

Immunolocalization of keratin‐associated beta‐proteins in developing epidermis of lizard suggests that adhesive setae contain glycine–cysteine‐rich proteins

The localization of specific keratin‐associated beta‐proteins (formerly referred to as beta‐keratins) in the embryonic epidermis of lizards is not known. Two specific keratin‐associated beta‐proteins of the epidermis, one representing the glycine‐rich subfamily (HgG5) and the other the glycine‐cysteine medium‐rich subfamily (HgGC10), have been immunolocalized at the ultrastructural level in the lizard Anolis lineatopus. The periderm and granulated subperiderm are most immunonegative for these proteins. HgG5 is low to absent in theOberhäutchen layer while is present in the forming beta‐layer, and disappears in mesos‐ and alpha‐layers. Instead, HgGC10 is present in the Oberhäutchen, beta‐, and also in the following alpha‐layers, and specifically accumulates in the developing adhesive setae but not in the surrounding cells of the clear layer. Therefore, setae and their terminal spatulae that adhere to surfaces allowing these lizards to walk vertically contain cysteine–glycine rich proteins. The study suggests that, like in adult and regenerating epidermis, the HgGC10 protein is not only accumulated in cells of the beta‐layer but also in those forming the alpha‐layer. This small protein therefore is implicated in resistance, flexibility, and stretching of the epidermal layers. It is also hypothesized that the charges of these proteins may influence adhesion of the setae of pad lamellae. Conversely, glycine‐rich beta‐proteins like HgG5 give rise to the dense, hydrophobic, and chromophobic corneous material of the resistant beta‐layer. This result suggests that the differential accumulation of keratin‐associated beta‐proteins over the alpha‐keratin network determines differences in properties of the stratified layers of the epidermis of lizards. J. Morphol. 2013. © 2012 Wiley Periodicals, Inc.     

Immunolocalization of acidic and basic fibroblast growth factors during mouse odontogenesis.

Acidic and basic fibroblast growth factors (aFGF and bFGF), are both known to bind to extracellular matrix components, particularly proteoheparin sulfates, and to regulate in vitro proliferation, differentiation and morphology of cells of neuroectodermal and mesodermal origins. Their patterns of distribution were studied during mouse odontogenesis by means of indirect immunofluorescence and immunoperoxidase histochemistry on frozen fixed sections and after Bouin's fixative and paraffin embedding. Localization of aFGF on frozen fixed sections was observed in the oral epithelium, dental lamina and oral mesenchyme (day-12 of gestation), the stellate reticulum and oral epithelium (day-14), the stratum intermedium and at the basal and apical poles of preameloblasts at bell stage. After birth aFGF epitopes were localized within the predentin-dentin area, the stratum intermedium and at the secretory pole of ameloblasts. There was no staining with anti-aFGF antibodies after Bouin's fixative and paraffin embedding. In contrast, using this protocol, intense stainings were found with anti-bFGF antibodies predominantly within dental and peridental basement membranes and mesenchyme: staining of the dental basement membranes was transient (bud and cap stage) and discontinuous; a preferential concentration of bFGF epitopes in the condensed dental mesenchyme of incisors (cap stage) and the dental papillae mesenchymal cells of molars (bell stage) was observed in the posterior and the cervical part of tooth germs. An intense immunostaining of the stellate reticulum with anti-bFGF antibodies was also found on paraffin sections from bud to bell stage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution and characterization of proteins associated with cornification in the epidermis of gecko lizard

The distribution and molecular weight of epidermal proteins of gecko lizards have been studied by ultrastructural, autoradiographic, and immunological methods. Setae of the climbing digital pads are cross-reactive to antibodies directed against a chick scutate scale beta-keratin but not against feather beta-keratin. Cross-reactivity for mammalian loricrin, sciellin, filaggrin, and transglutaminase are present in alpha-keratogenic layers of gecko epidermis. Alpha-keratins have a molecular weight in the range 40-58 kDa. Loricrin cross-reactive bands have molecular weights of 42, 50, and 58 kDa. Bands for filaggrin-like protein are found at 35 and 42 kDa, bands for sciellin are found at 40-45 and 50-55 kDa, and bands for transglutaminase are seen at 48-50 and 60 kDa. The specific role of these proteins remains to be elucidated. After injection of tritiated histidine, the tracer is incorporated into keratin and in setae. Tritiated proline labels the developing setae of the oberhautchen and beta layers, and proline-labeled proteins (beta-keratins) of 10-14, 16-18, 22-24 and 32-35 kDa are extracted from the epidermis. In whole epidermal extract (that includes the epidermis with corneous layer and the setae of digital pads), beta-keratins of low-molecular weight (10, 14-16, and 18-19 kDa) are prevalent over those at higher molecular weight (34 and 38 kDa). In contrast, in shed epidermis of body scales (made of corneous layer only while setae were not collected), higher molecular weight beta-keratins are present (25-27 and 30-34 kDa). This suggests that a proportion of the small beta-keratins present in the epidermis of geckos derive from the differentiating beta layer of scales and from the setae of digital pads. Neither small nor large beta-keratins of gecko epidermis cross-react with an antibody specifically directed against the feather beta-keratin of 10-12 kDa. This result shows that the 10 and 14-16 kDa beta-keratins of gecko (lepidosaurian) have a different composition than the 10-12 kDa beta-keratin of feather (archosaurian). It is suggested that the smaller beta-keratins in both lineages of sauropsids were selected during evolution in order to build elongated bundles of keratin filaments to make elongated cells. Larger beta-keratins in reptilian scales produce keratin aggregations with no orientation, used for mechanical protection.     

Immunolocalization and characterization of beta-keratins in growing epidermis of chelonians

Beta-keratins constitute most of the corneous material of carapace and plastron of turtles. The production of beta-keratin in the epidermis of a turtle and tortoise (criptodirians) and of a species of pleurodiran turtle was studied after injection of tritiated proline during the growth of carapace, plastron and claws. Growth mainly occurs near hinge regions along the margins of scutes and along most of the claws (growing regions). Proline incorporation occurs mainly in the growing centers, and is more specifically associated with beta-keratin synthesis. Proline-labeled bands of protein at 12-14 kDa and 25-27 kDa, and 37 kDa, in the molecular weight range of beta-keratins, were isolated from the soft epidermis of turtles 3 h after injection of the labeled amino acid. After extraction of epidermal proteins, an antibody directed against a chicken beta-keratin was used for immunoblotting. Bands of beta-keratin at 15-17 kDa, 22-24 kDa, and 36-38 kDa appear in all species. Beta-keratin is present in the growing and compact stratum corneum of the hard (shell) and soft (limbs, neck and tail) epidermis. This was confirmed using a specific antibody against a turtle beta-keratin band of 15-16 kDa. The latter antibody recognized epidermal protein bands in the range of 15-16 kDa and 29-33 kDa, and labels beta-keratin filaments. This result indicates that different forms of beta-keratins are produced from low molecular weight precursors or that larger aggregate form during protein preparation. The present study shows that beta-keratin is abundant in the scaled epidermis of tortoise but also in the soft epidermis of pleurodiran and cryptodiran turtles, indicating that this form of hard keratin is constitutively expressed in the epidermis of chelonians.     

Low‐cysteine alpha‐keratins and corneous beta‐proteins are initially formed in the regenerating tail epidermis of lizard

During tail regeneration in lizards, the stratified regenerating epidermis progressively gives rise to neogenic scales that form a new epidermal generation. Initially, a soft, un‐scaled, pliable, and extensible epidermis is formed that is progressively replaced by a resistant but non‐extensible scaled epidermis. This suggests that the initial corneous proteins are later replaced with harder corneous proteins. Using PCR and immunocytochemistry, the present study shows an upregulation in the synthesis of low‐cysteine type I and II alpha‐keratins and of corneous beta‐proteins with a medium cysteine content and a low content in glycine (formerly termed beta‐keratins) produced at the beginning of epidermal regeneration. Quantitative PCR indicates upregulation in the production of alpha‐keratin mRNAs, particularly of type I, between normal and the thicker regenerating epidermis. PCR‐data also indicate a higher upregulation for cysteine‐rich corneous beta‐proteins and a high but less intense upregulation of low glycine corneous protein mRNAs at the beginning of scale regeneration. Immunolabeling confirms the localization of these proteins, and in particular of beta‐proteins with a medium content in cysteine initially formed in the wound epidermis and later in the differentiating corneous layers of regenerating scales. It is concluded that the wound epidermis initially contains alpha‐keratins and corneous beta‐proteins with a lower cysteine content than more specialized beta‐proteins later formed in the mature scales. These initial corneous proteins are likely related to the pliability of the wound epidermis while more specialized alpha‐keratins and beta‐proteins richer in glycine and cysteine are synthesized later in the mature and inflexible scales. J. Morphol. 278:119–130, 2017. ©© 2016 Wiley Periodicals,Inc.     

Comparative immunolocalization of keratin‐associated beta‐proteins (beta‐keratins) supports a new explanation for the cyclical process of keratinocyte differentiation in lizard epidermis

Lizard epidermis is made of beta‐ and alpha‐layers. Using Western blot tested antibodies, the ultrastructural immunolocalization of specific keratin‐associated beta‐proteins in the epidermis of different lizard species reveals that glycine‐rich beta‐proteins (HgG5) localize in the beta‐layer, while glycine–cysteine‐medium‐rich beta‐proteins (HgGC10) are present in oberhautchen and alpha‐layers. This suggests a new explanation for the formation of different epidermal layers during the shedding cycle in lepidosaurian epidermis instead of an alternance between beta‐keratins and alpha‐keratins. It is proposed that different sets of genes coding for specific beta‐proteins are activated in keratinocytes during the renewal phase of the shedding cycle. Initially, glycine–cysteine‐medium‐rich beta‐proteins with hydrophilic and elastic properties accumulate over alpha‐keratins in the oberhautchen but are replaced in the next cell layer with glycine‐rich hydrophobic beta‐proteins forming a resistant, stiff, and hydrophobic beta‐layer. The synthesis of glycine‐rich proteins terminates in mesos and alpha‐cells where these proteins are replaced with glycine–cysteine‐rich beta‐proteins. The pattern of beta‐protein deposition onto a scaffold of intermediate filament keratins is typical for keratin‐associated proteins and the association between alpha‐keratins and specific keratin‐associated beta‐proteins during the renewal phase of the shedding cycle gives rise to epidermal layers possessing different structural, mechanical, and texture properties.     

Immunolocalization of hepatic estrogen and progesterone receptors in the female lizard Uromastyx acanthinura

The hormonal regulation of hepatic synthesis of vitellogenin during the annual reproductive cycle was performed for the first time in the deserticole, oviparous, diurnal and herbivorous Uromastyx acanthinura, a lizard belonging to the Agamidae family. In order to elucidate what kind of estrogen receptor is involved in this process, an immunohistochemical study was performed. Changes were obtained in the labeling and cellular distribution of the estrogen and progesterone receptors according to the period of the reproductive cycle and the experimental administration of 17β-estradiol. Only the ERβ subtype was present; it was found in all phases of the cycle with a variable localization: nuclear and cytosolic during vitellogenesis, mainly cytosolic in the female with egg retention (luteal phase) and strictly cytosolic in females at sexual rest. The progesterone receptors were present only at the luteal phase and during sexual rest and disappeared completely from females after 17β-estradiol treatment in sexual rest. Our data suggested that mediation of action of the 17β-estradiol in the vitellogenin synthesis in the lizard U. acanthinura occured via ERβ. PRA and PRB could both be necessary for the negative effect of progesterone on the hepatic synthesis of vitellogenin.     

Ultrastructural and immunocytochemical detection of keratins and extracellular matrix proteins in lizard skin cultured in vitro

The present study shows the localization of epidermal and dermal proteins produced in lizard skin cultivated in vitro. Cells from the skin have been cultured for up to one month to detect the expression of keratins, actin, vimentin and extracellular matrix proteins (fibronectin, chondroitin sulphate proteoglycan, elastin and collagen I). Keratinocytes and dermal cells weakly immunoreact for Pan-Cytokeratin but not with the K17-antibody at the beginning of the cell culture when numerous keratin bundles are present in keratinocyte cytoplasm. The dense keratin network disappears after 7-12 days in culture, and K17 becomes detectable in both keratinocytes and mesenchymal cells isolated from the dermis. While most epidermal cells are lost after 2 weeks of in vitro cultivation dermal cells proliferate and form a pellicle of variable thickness made of 3-8 cell layers. The fibroblasts of this dermal equivalent produces an extracellular matrix containing chondroitin sulphate proteoglycan, collagen I, elastic fibers and fibronectin, explaining the attachment of the pellicle to the substratum. The study indicates that after improving keratinocyte survival a skin equivalent for lizard epidermis would be feasible as a useful tool to analyze the influence of the dermis on the process of epidermal differentiation and the control of the shedding cycle in squamates.     

Immunocytochemical analysis of beta keratins in the epidermis of chelonians,lepidosaurians, and archosaurians

Beta (beta) keratins are present only in the avian and reptilian epidermises. Although much is known about the biochemistry and molecular biology of the beta keratins in birds, little is known for reptiles. In this study we have examined the distribution of beta keratins in the adult epidermis of turtle, lizard, snake, tuatara, and alligator using light and electron immunocytochemistry with a well-characterized antiserum (anti-beta(1) antiserum) made against a known avian scale type beta keratin. In lizard, snake, and tuatara epidermis this antiserum reacts strongly with the beta-layer, more weakly with the oberhautchen before it merges with the beta-layer, and least intensely with the mesos layer. In addition, the anti-beta(1) antiserum reacts specifically with the setae of climbing pads in gekos, the plastron and carapace of turtles, and the stratum corneum of alligator epidermis. Electron microscopic studies confirm that the reaction of the anti-beta(1) antiserum is exclusively with characteristic bundles of the 3-nm beta keratin filaments in the cells of the forming beta-layer, and with the densely packed electron-lucent areas of beta keratin in the mature bet- layer. These immunocytochemical results suggest that the 3-nm beta keratin filaments of the reptilian integument are phylogenetically related to those found in avian epidermal appendages.     

Cornification in reptilian epidermis occurs through the deposition of keratin‐associated beta‐proteins (beta‐keratins) onto a scaffold of intermediate filament keratins

The isolation of genes for alpha‐keratins and keratin‐associated beta‐proteins (formerly beta‐keratins) has allowed the production of epitope‐specific antibodies for localizing these proteins during the process of cornification epidermis of reptilian sauropsids. The antibodies are directed toward proteins in the alpha‐keratin range (40–70 kDa) or beta‐protein range (10–30 kDa) of most reptilian sauropsids. The ultrastructural immunogold study shows the localization of acidic alpha‐proteins in suprabasal and precorneous epidermal layers in lizard, snake, tuatara, crocodile, and turtle while keratin‐associated beta‐proteins are localized in precorneous and corneous layers. This late activation of the synthesis of keratin‐associated beta‐proteins is typical for keratin‐associated and corneous proteins in mammalian epidermis (involucrin, filaggrin, loricrin) or hair (tyrosine‐rich or sulfur‐rich proteins). In turtles and crocodilians epidermis, keratin‐associated beta‐proteins are synthesized in upper spinosus and precorneous layers and accumulate in the corneous layer. The complex stratification of lepidosaurian epidermis derives from the deposition of specific glycine‐rich versus cysteine‐glycine‐rich keratin‐associated beta‐proteins in cells sequentially produced from the basal layer and not from the alternation of beta‐ with alpha‐keratins. The process gives rise to Oberhäutchen, beta‐, mesos‐, and alpha‐layers during the shedding cycle of lizards and snakes. Differently from fish, amphibian, and mammalian keratin‐associated proteins (KAPs) of the epidermis, the keratin‐associated beta‐proteins of sauropsids are capable to form filaments of 3–4 nm which give rise to an X‐ray beta‐pattern as a consequence of the presence of a beta‐pleated central region of high homology, which seems to be absent in KAPs of the other vertebrates. J. Morphol., 2013. © 2012 Wiley Periodicals, Inc.     

Immunocytochemical localization of sulfhydryl oxidase in mammalian epidermis suggests that the enzyme cross‐links keratins in the granular and transitional corneous layers

The epidermis of representative mammalian species including humans has been examined for the presence of sulfhydryl oxidase, an enzyme likely involved in the oxidation of corneous proteins containing sulfhydryl groups in the epidermis. A database search indicates that the enzyme shares common sequences in numerous mammalian species so that an antibody against the human sulfhydryl oxidase 2 has been utilized on other species. The immunofluorescent study on the epidermis of the platypus (monotreme), red kangaroo (marsupials), hamster and human (placentals) reveals a prevalent labelling in the granular, transitional and lowermost part of the stratum corneous layer. The detailed ultrastructural immunogold study of the human epidermis reveals a diffuse and uneven labelling in the paler component of the composite keratohyalin granules or among keratin filaments of the transitional layer while the labelling disappears in the corneous layer. The study supports the hypothesis of the participation of the enzyme in the oxidative process that determines the formation of stable disulphide groups among keratins and other corneous proteins of the stratum corneum. This process gives rise to the resistant cell corneous envelope of keratinocytes in addition to the isopeptide bonds that derive from the catalytic action of epidermal transglutaminase on several corneous proteins.