Regional specific differentiation of integumentary organs: SATB2 is involved in α- and ß-keratin gene cluster switching in the chicken.

BACKGROUND: Animals develop skin regional specificities to best adapt to their environments. Birds are excellent models in which to study the epigenetic mechanisms that facilitate these adaptions. Patients suffering from SATB2 mutations exhibit multiple defects including ectodermal dysplasia-like changes. The preferential expression of SATB2, a chromatin regulator, in feather-forming compared to scale-forming regions, suggests it functions in regional specification of chicken skin appendages by acting on either differentiation or morphogenesis. RESULTS: Retrovirus mediated SATB2 misexpression in developing feathers, beaks, and claws causes epidermal differentiation abnormalities (e.g. knobs, plaques) with few organ morphology alterations. Chicken ß-keratins are encoded in 5 sub-clusters (Claw, Feather, Feather-like, Scale, and Keratinocyte) on Chromosome 25 and a large Feather keratin cluster on Chromosome 27. Type I and II α-keratin clusters are located on Chromosomes 27 and 33, respectively. Transcriptome analyses showed these keratins (1) are often tuned up or down collectively as a sub-cluster, and (2) these changes occur in a temporo-spatial specific manner. CONCLUSIONS: These results suggest an organizing role of SATB2 in cluster-level gene co-regulation during skin regional specification.

Structure of Keratin.

Keratins, as a group of insoluble and filament-forming proteins, mainly exist in certain epithelial cells of vertebrates. Keratinous materials are made up of cells filled with keratins, while they are the toughest biological materials such as the human hair, wool and horns of mammals and feathers, claws, and beaks of birds and reptiles which usually used for protection, defense, hunting and as armor. They generally exhibit a sophisticated hierarchical structure ranging from nanoscale to centimeter-scale: polypeptide chain structures, intermediated filaments/matrix structures, and lamellar structures. Therefore, more and more attention has been paid to the investigation of the relationship between structure and properties of keratins, and a series of biomimetic materials based on keratin came into being. In this chapter, we mainly introduce the hierarchical structure, the secondary structure, and the molecular structure of keratins, including α- and ß-keratin, to promote the development of novel keratin-based biomimetic materials designs.

Immunolocalization of epidermal differentiation complex proteins reveals distinct molecular compositions of cells that control structure and mechanical properties of avian skin appendages.

The epidermal differentiation complex (EDC) is a cluster of genes that encode structural proteins of skin derivatives with variable mechanical performances, from the scales of reptiles and birds to the hard claws and beaks, and to the flexible but resistant corneous material of feathers. Corneous proteins with or without extended beta-regions are produced from avian genomes, and include the largely prevalent corneous beta proteins (CßPs, formerly indicated as beta-keratins), and minor contribution from histidine-rich proteins, trichohyalin-like proteins (scaffoldin), loricrin, and other proteins rich in cysteine or other types of amino acids. The light-microscopic and ultrastructural immunolocalization of major and minor EDC-proteins in avian skin (feather CßPs, EDKM, EDWM, EDMTFH, EDDM, and scaffoldin) suggests that each specific appendage consists of a particular mix of these proteins in addition to the main proteins containing a peculiar beta-region of 34 amino acids, indicated as feather/scale/claw/beak CßPs (fCßPs, sCßPs, cCßPs, bCßPs). This indicates that numerous proteins of the EDC are added to the variable meshwork of intermediate filament keratins to produce avian epidermis with different mechanical and functional properties. Although the specific roles for these proteins are not known they likely make an important contribution to the final material properties of the different skin appendages of birds. The highest number of sauropsid CßPs is found in birds, suggesting a relation to the evolution of feathers, and additional epidermal differentiation proteins have contributed to the evolutionary adaptations of avian skin.

Structure and topology of the linkers in the conserved lepidosaur ß-keratin chain with four 34-residue repeats support an interfilament role for the central linker.

The ß-keratin chain with four 34-residue repeats that is conserved across the lepidosaurs (lizards, snakes and tuatara) contains three linker regions as well as a short, conserved N-terminal domain and a longer, more variable C-terminal domain. Earlier modelling had shown that only six classes of structure involving the four 34-residue repeats were possible. In three of these the 34-residue repeats were confined to a single filament (Classes 1, 2 and 3) whereas in the remaining three classes the repeats lay in two, three or four filaments, with some of the linkers forming interfilament connections (Classes 4, 5 and 6). In this work the members of each class of structure (a total of 20 arrangements) have been described and a comparison has been made of the topologies of each of the linker regions. This provides new constraints on the structure of the chain as a whole. Also, analysis of the sequences of the three linker regions has revealed that the central linker (and only the central linker) contains four short regions displaying a distinctive dipeptide repeat of the form (S-X)2,3 separated by short regions containing proline and cysteine residues. By analogy with silk fibroin proteins this has the capability of forming a ß-sheet-like conformation. Using the topology and sequence data the evidence suggests that the four 34-residue repeat chain adopts a Class 4a structure with a ß-sandwich in filament 1 connected through the central linker to a ß-sandwich in filament 2.

Folding Keratin Gene Clusters during Skin Regional Specification.

Regional specification is critical for skin development, regeneration, and evolution. The contribution of epigenetics in this process remains unknown. Here, using avian epidermis, we find two major strategies regulate ß-keratin gene clusters. (1) Over the body, macro-regional specificities (scales, feathers, claws, etc.) established by typical enhancers control five subclusters located within the epidermal differentiation complex on chromosome 25; (2) within a feather, micro-regional specificities are orchestrated by temporospatial chromatin looping of the feather ß-keratin gene cluster on chromosome 27. Analyses suggest a three-factor model for regional specification: competence factors (e.g., AP1) make chromatin accessible, regional specifiers (e.g., Zic1) target specific genome regions, and chromatin regulators (e.g., CTCF and SATBs) establish looping configurations. Gene perturbations disrupt morphogenesis and histo-differentiation. This chicken skin paradigm advances our understanding of how regulation of big gene clusters can set up a two-dimensional body surface map.

Immunolocalization of corneous proteins including a serine-tyrosine-rich beta-protein in the adhesive pads in the tokay gecko.

Adhesive pads of geckos contain many thousands of nanoscale spatulae for the adhesion and movement along vertical or inverted surfaces. Setae are composed of interlaced corneous bundles made of small cysteine-glycine-rich corneous beta proteins (CBPs, formerly indicated as beta-keratins), embedded in a matrix material composed of cytoskeletal proteins and lipids. Negatively charged intermediate filament keratins (IFKs) and positively charged CBPs likely interact within setae, aside disulphide bonds, giving rise to a flexible and resistant corneous material. Using differernt antibodies against CBPs and IFKs an updated model of the composition of setae and spatulae is presented. Immunofluorescence and ultrastructural immunogold labeling reveal that one type of neutral serine-tyrosine-rich CBP is weakly localized in the setae while it is absent from the spatula. This uncharged protein is mainly present in the thin Oberhautchen layer sustaining the setae, although with a much lower intensity with respect to the cysteine-rich CBPs. These proteins in the spatula likely originate a positively charged or neutral contact surface with the substrate but the influence of lipids and cytoskeletal proteins present in setae on the mechanism of adhesion is not known. In the spatula, protein-lipid complexes may impart the pliability for the attachment and adapt to irregular surfaces. The presence of cysteine-glycine medium rich CBPs and softer IFKs in alpha-layers sustaining the setae forms a flexible base for compliance of the setae to substrate and improved adhesion.

Corneous beta proteins of the epidermal differentiation complex (EDC) form large part of the corneous material of claws and rhamphothecae in turtles.

The presence of specific protein types in claws and beaks of turtles is poorly known. The present immunological study describes the localization of some of the main corneous beta proteins (CBPs) coded in the epidermal differentiation complex of turtles. Three antibodies here utilized revealed that glycine-, cysteine-, tyrosine-, and valine-rich CBPs are present in differentiating keratinocytes of the beak and of the dorsal (unguis) and ventral (sub-unguis) sides of the claw in different species, semi-aquatic and terrestrial. These proteins provide mechanical resilience to the horny material of claws and beaks through the formation of numerous -S-S- bonds and also hydrophobicity that contributes to preserve wearing of the horny material. The thicker corneous layer of the unguis is made of elongated and partially merged corneocytes, and no or few cells desquamate superficially. Unknown junctional proteins may contribute to maintain corneocytes connected one to another. In contrast, corneocytes of the sub-unguis show an elongated but lenticular shape and form a looser corneous layer whose cells remain separate and desquamate superficially. This suggests that other specific corneous proteins are present in the unguis in comparison with the sub-unguis to determine this different compaction. The wearing process present in the sub-unguis creates a loss of tissue that may favor the slow by continuous apical migration of corneocytes from the unguis into the initial part of the sub-unguis. Beak corneocytes form a compact corneous layer like the unguis but numerous superficial cells desquamate on both outer (epidermal) and inner (oral) sides.

Review: mapping epidermal beta-protein distribution in the lizard Anolis carolinensis shows a specific localization for the formation of scales, pads, and claws.

The epidermis of lizards is made of multiple alpha- and beta-layers with different characteristics comprising alpha-keratins and corneous beta-proteins (formerly beta-keratins). Three main modifications of body scales are present in the lizard Anolis carolinensis: gular scales, adhesive pad lamellae, and claws. The 40 corneous beta-proteins present in this specie comprise glycine-rich and glycine-cysteine-rich subfamilies, while the 41 alpha-keratins comprise cysteine-poor and cysteine-rich subfamilies, the latter showing homology to hair keratins. Other genes for corneous proteins are present in the epidermal differentiation complex, the locus where corneous protein genes are located. The review summarizes the main sites of immunolocalization of beta-proteins in different scales and their derivatives producing a unique map of body distribution for these structural proteins. Small glycine-rich beta-proteins participate in the formation of the mechanically resistant beta-layer of most scales. Small glycine-cysteine beta-proteins have a more varied localization in different scales and are also present in the pliable alpha-layer. In claws, cysteine-rich alpha-keratins prevail over cysteine-poor alpha-keratins and mix to glycine-cysteine-rich beta-proteins. The larger beta-proteins with a molecular mass similar to that of alpha-keratins participate in the formation of the fibrous meshwork present in differentiating beta-cells and likely interact with alpha-keratins. The diverse localization of alpha-keratins, beta-proteins, and other proteins of the epidermal differentiation complex gives rise to variably pliable, elastic, or hard corneous layers in different body scales. The corneous layers formed in the softer or harder scales, in the elastic pad lamellae, or in the resistant claws possess peculiar properties depending on the ratio of specific corneous proteins.

Immunolocalization of sulfhydryl oxidase in reptilian epidermis indicates that the enzyme participates mainly to the hardening process of the beta-corneous layer.

Reptilian skin is tough and scaled representing an evolutionary adaptation to the terrestrial environment. The presence of sulfhydryl oxidase during the process of hardening of the corneous layer in reptilian epidermis has been analyzed by immunocytochemistry and immunoblotting. Sulfhydryl oxidase-like immunoreactivity of proteins in the 50-65 kDa range of molecular weight is mainly observed in the transitional and pre-corneous layers of crocodilians, chelonian, and in the forming beta-layer of lepidosaurians. The ultrastructural localization of the enzyme by immunogold in lizard epidermis during renewal and resting stages shows that the labeling is mainly distributed in the cytoplasm and along the accumulating beta-packets of differentiating beta-cells while it appears very low to undetectable in differentiating alpha-cells of the lacunar, clear, mesos, and alpha-layers. The labeling however becomes absent or undetectable also in the fully mature beta-layer. The study shows that an oxidative enzyme is likely responsible of the cross-linking of the numerous cysteines present in the main proteins accumulated in corneocytes of reptilian epidermis, known as corneous beta-proteins (beta-keratins). This process of disulphide bond formation is probably largely responsible for the formation of hard beta-corneous layers in reptilian scales, a difference with alpha-corneous layers where substrate proteins of transglutaminase appear predominant.

Fatores associados à prática do aleitamento materno exclusivo por pelo menos seis meses no estado de Pernambuco / Factors associated with breastfeeding practice for at least six months in the state of Pernambuco, Brazil

INTRODUÇÃO: Apesar do consenso científico sobre os benefícios que a amamentação proporciona à mãe, à criança, à família e ao próprio meio ambiente, além da recomendação para que sua prática seja realizada de forma exclusiva nos seis primeiros meses de vida, essa conduta está longe de ser alcançada. OBJETIVO: Analisar os fatores associados à amamentação exclusiva (AME) por pelo menos seis meses, em contraponto ao desmame total até o segundo mês de vida no estado de Pernambuco. MÉTODO: Estudo caso-controle reunindo 124 casos (AME por pelo menos seis meses) pareados por idade e sexo com 248 controles (desmame total até o segundo mês). Casos e controles foram oriundos da III Pesquisa Estadual de Saúde e Nutrição. Foram selecionadas como variáveis de exposição: idade e escolaridade materna, renda familiar, zona de moradia, consultas pré-natais, tipo de parto e profissional que o assistiu e orientação sobre amamentação no pré-natal. Foi aplicada regressão logística nas variáveis que apresentaram um valor de p < 0,2 nas análises bivariadas, adotando para a inclusão no modelo final o nível de significância p < 0,05. RESULTADOS: Dos 8 agrupamentos de variáveis consideradas como possíveis preditoras do AME por pelo menos 6 meses, mantiveram-se como fatores associados a idade materna entre 20 - 35 anos, sendo a odds ratio (OR) 2,5 e o intervalo de confiança de 95% (IC95%) 1,4 - 4,5; e a escolaridade de 5 - 8 anos de estudo (OR 2,1; IC95% 1,2 - 3,6). CONCLUSÃO: O estudo mostra que ainda são necessárias mobilizações dos poderes públicos e estímulo às pesquisas em prol do sucesso do AME e da saúde materno-infantil. .

INTRODUCTION: Despite the scientific consensus on the benefits that breastfeeding provides for the mother, the baby, the family and the environment, and also the recommendation to breastfeed exclusively for six months, this practice is far from being achieved. OBJECTIVE: To analyze the factors associated with exclusive breastfeeding (EBF) for at least six month, as opposed to weaning up to the second month of life in the state of Pernambuco, Brazil. METHODS: A case-control study of 124 cases (EBF for at least six months) matched for age and sex with 248 controls (weaning up to the second month of life). Cases and controls were drawn from the III State Health and Nutrition Survey. The exposure variables selected were maternal age and education, per capita income, housing zone, prenatal consultations, type of delivery, professional who assisted the delivery, and prenatal breastfeeding guidance. Logistic regression was applied to variables that showed a p-value < 0.2 in the bivariate analysis, and the variables with p-value < 0.05 were included in the final model. RESULTS: Of the eight groups of variables considered as possible predictors of EBF for at least six months, two remained as associated factors: maternal age between 20 - 35 years old, with odds ratio (OR) 2.5 and 95% confidence interval 95%CI 1.4 - 4.5; and maternal education of 5 - 8 years of schooling (OR 2.1; 95%CI 1.2 - 3.6). CONCLUSION: The study shows that mobilization of the public sector and stimulus to research is still needed for the success of EBF and for mother and child health. .

Immunolocalization of specific beta-proteins in pad lamellae of the digits in the lizard Anolis carolinensis suggests that cysteine-rich beta-proteins provides flexibility.

Knowledge of beta-protein (beta-keratin) sequences in Anolis carolinensis facilitates the localization of specific sites in the skin of this lizard. The epidermal distribution of two new beta-proteins (betakeratins), HgGC8 and HgG13, has been analyzed by Western blotting, light and ultrastructural immunocytochemistry. HgGC8 includes 16 kDa members of the glycine-cysteine medium-rich subfamily and is mainly expressed in the beta-layer of adhesive setae but not in the setae. HgGC8 is absent in other epidermal layers of the setae and is weakly expressed in the beta-layer of other scales. HgG13 comprises members of 17-kDa glycine-rich proteins and is absent in the setae, diffusely distributed in the beta layer of digital scales and barely present in the beta-layer of other scales. It appears that the specialized glycine-cysteine medium rich beta-proteins such as HgGC8 in the beta-layer, and of HgGC10 and HgGC3 in both alpha- and betalayers, are key proteins in the formation of the flexible epidermal layers involved in the function of these modified scales in adaptation to contact and adhesion on surfaces.

Identification of a feather ß-keratin gene exclusively expressed in pennaceous barbule cells of contour feathers in chicken.

Feathers are elaborate skin appendages shared by birds and theropod dinosaurs that have hierarchical branching of the rachis, barbs, and barbules. Feather filaments consist of ß-keratins encoded by multiple genes, most of which are located in tandem arrays on chromosomes 2, 25, and 27 in chicken. The expansion of the genes is thought to have contributed to feather evolution; however, it is unclear how the individual genes are involved in feather formation. The aim of the present study was to identify feather keratin genes involved in the formation of barbules. Using a combination of microarray analysis, reverse-transcription polymerase chain reaction, and in situ hybridization, we found an uncharacterized keratin gene on chromosome 7 that was expressed specifically in barbule cells in regenerating chicken feathers. We have named the gene barbule specific keratin 1 (BlSK1). The BlSK1 gene structure was similar to the gene structure of previously characterized feather keratin genes, and consisted of a non-coding leader exon, an intron, and an exon with an open reading frame (ORF). The ORF was predicted to encode a 98 aa long protein, which shared 59% identity with feather keratin B. Orthologs of BlSK1 were found in the genomes of other avian species, including turkey, duck, zebra finch, and flycatcher, in regions that shared synteny with chromosome 7 of chicken. Interestingly, BlSK1 was expressed in feather follicles that generated pennaceous barbules but not in follicles that generated plumulaceous barbules. These results suggested that the composition of feather keratins probably varies depending on the structure of the feather filaments and, that individual feather keratin genes may be involved in building different portions and/or types of feathers in chicken.

Immunolocalization of alpha-keratins and associated beta-proteins in lizard epidermis shows that acidic keratins mix with basic keratin-associated beta-proteins.

The differentiation of the corneous layers of lizard epidermis has been analyzed by ultrastructural immunocytochemistry using specific antibodies against alpha-keratins and keratin associated beta-proteins (KAbetaPs, formerly indicated as beta-keratins). Both beta-cells and alpha-cells of the corneous layer derive from the same germinal layer. An acidic type I alpha-keratin is present in basal and suprabasal layers, early differentiating clear, oberhautchen, and beta-cells. Type I keratin apparently disappears in differentiated beta- and alpha-layers of the mature corneous layers. Conversely, a basic type II alpha-keratin rich in glycine is absent or very scarce in basal and suprabasal layers and this keratin likely does not pair with type I keratin to form intermediate filaments but is weakly detected in the pre-corneous and corneous alpha-layer. Single and double labeling experiments show that in differentiating beta-cells, basic KAbetaPs are added and replace type-I keratin to form the hard beta-layer. Epidermal alpha-keratins contain scarce cysteine (0.2-1.4 %) that instead represents 4-19 % of amino acids present in KAbetaPs. Possible chemical bonds formed between alpha-keratins and KAbetaPs may derive from electrostatic interactions in addition to cross-linking through disulphide bonds. Both the high content in glycine of keratins and KAbetaPs may also contribute to increase the hydrophobicy of the beta- and alpha-layers and the resistance of the corneous layer. The increase of gly-rich KAbetaPs amount and the bonds to the framework of alpha-keratins give rise to the inflexible beta-layer while the cys-rich KAbetaPs produce a pliable alpha-layer.

Transition from embryonic to adult epidermis in reptiles occurs by the production of corneous beta-proteins.

The adaptation of the epidermis in amniote vertebrates to life on land took place by a drastic change from an embryonic epidermis made of two-four periderm layers to a terrestrial-proof epidermis. This transition occurred by the increase in types and number of specialized corneous proteins coded by genes of the Epidermal Differentiation Complex. The prevalent types of corneous proteins produced in the reptilian epidermis contain a beta-sheet region of high amino acid homology which allows their polymerization into a meshwork of filaments forming the hard corneous material of scales and claws. The present immunogold ultrastructural study shows that this transition occurs with the synthesis of glycine-rich corneous beta-proteins (formerly indicated as beta-keratins) that are added to the initial framework of acidic intermediate filaments produced in the embryonic epidermis of lizards, snake, alligator and turtle. These corneous beta-proteins are accumulated in the transitional and definitive layers of reptilian epidermis formed underneath the transitory two-four layered embryonic epidermis. In the more specialized reptiles capable of shedding the epidermis as a single unit, such as lizards and snakes, special glycine-cysteine rich beta-proteins are initially produced in a single layer immediately formed beneath the embryonic epidermis, the oberhautchen. The latter layer allows the in ovo shedding of the embryonic epidermis in preparation for hatching, and in the following shedding cycles of the adult epidermis. The production of specialized corneous-specific beta-proteins in addition to intermediate filament keratins was probably an essential addition for terrestrial life during the evolution of reptiles into different lineages, including birds. The increase of glycine and cysteine in epidermal proteins enhanced the hydrophobicity, insolubility and mechanical strength of the stratum corneum in these amniotes.

Immunocytochemistry suggests that the prevalence of a sub-type of beta-proteins determines the hardness in the epidermis of the hard-shelled turtle.

The corneous layer of the epidermis in hard-shelled turtles largely derives from the accumulation of beta-proteins as indicated by microscopic, in situ hybridization, and immunocytochemical and Western blotting analysis. The expression of mRNAs of one of the most common type of beta-proteins shows higher expression in upper spinosus and pre-corneous keratinocytes of growing scutes. Two beta-proteins of 14-16 kDa, indicated as Tu2 and Tu17 and representing two subtypes of beta-proteins co-accumulate in the thick corneous layer of the epidermis in hard-shelled turtle. The two beta-proteins apparently mix in differentiating and mature corneocytes although Tu2 appears more prevalent than Tu17. The specific role of the different subtypes in the formation of the hard corneous material of the carapace and plastron is not clear. It is hypothesized that the relative amount of beta-proteins belonging to the two subclasses in relation to the alpha-keratin meshwork present in keratinocytes contributes to the formation of a variably resistant and inflexible corneous layer. Tu17 may have a more globular structure than Tu2 and is likely present in denser areas of the corneous layer containing also alpha-keratin. The increase of cysteine-glycine-rich beta-proteins in the matrix located among alpha-keratin filaments may allow the formation of a hard corneous material, probably through increase of cross-bridge formation and hydrophobicity.

Immunolocalization of specific keratin associated beta-proteins (beta-keratins) in the adhesive setae of Gekko gecko.

The previous identification of 21 proteins in the digital setae transcriptome of Gekko gecko, 2 alpha-keratins of 52-53kDa and 19 beta-proteins (beta-keratins) of 10-21kDa, has indicated that most of setal corneous proteins are cysteine-rich. The production of specific antibodies for two of the main beta-protein subfamilies expressed in gecko setae has allowed the ultrastructural localization of two beta-proteins indicated as Ge-cprp-9 (cysteine-rich) and Ge-gprp-6 (glycine-rich). Only Ge-cprp-9, representing most of the 16 cysteine-rich beta-proteins, is present in the oberhautchen, setae and in the terminal spatula where adhesion takes place, supporting the previous expression study. Instead, the glycine-rich beta-proteins (Ge-gprp-6), representing the 3 glycine-rich beta-proteins of digital epidermis is only present in the stiff beta-layer of the digital scales and in the thin beta layer of the pad lamella sustaining the setae. Ge-cprp-9 is representative for most of the remaining 15 cys-rich proteins (Ge-cprp 1-16) and may have a structural and functional role in the process of adhesion. Most of the cysteine-rich setal proteins have a net positive charge and it is here hypothesized that these proteins may induce the formation of dipoles at the surface interface between the spatula and the substrate, enhancing the van der Waals forces and therefore adhesion to the substrate. The selection and improvement of these proteins during the evolution of geckos may have represented a successful factor for the survival and ecological adaptations of these climbing lizards.

Immunolocalization of keratin-associated beta-proteins (beta-keratins) in pad lamellae of geckos suggest that glycine-cysteine-rich proteins contribute to their flexibility and adhesiveness.

The epidermis of digital pads in geckos comprises superficial microornamentation from the oberhautchen layer that form long setae allowing these lizards to climb vertical surfaces. The beta-layer is reduced in pad lamellae but persists up to the apical free margin. Setae are made of different proteins including keratin-associated beta-proteins, formerly indicated as beta-keratins. In order to identify specific setal proteins the present ultrastructural study on geckos pad lamellae analyzes the immunolocalization of three beta-proteins previously found in the epidermis and adhesive setae of the green anolis. A protein rich in glycine but poor in cysteine (HgG5-like) is absent or masked in gecko pad lamellae. Another protein rich in glycine and cysteine (HgGC3-like) is weakly present in setae, oberhautchen and beta-layer. A glycine and cysteine medium rich beta-protein (HgGC10-like) is present in the lower part of the beta-layer but is absent in the oberhautchen, setae, and mesos layer. The latter two proteins may form intermolecular bonds that contribute to the flexibility of the corneous material sustaining the setae. The pliable alpha-layer present beneath the thin beta-layer and in the hinge region of the pad lamellae also contains HgGC10-like proteins. Based on the possibility that some HgGC3-like or other cys-rich beta-proteins are charged in the setae it is suggested that their charges influence the mechanism of adhesion increasing the induction of dipoles on the substrate and enhancing attractive van der Waals forces.

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.

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.

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.