Keratinization in the epidermis of amphibians and the lungfish: comparison with amniote keratinization

Keratinization in the epidermis of amphibians and the lungfish has been studied by electron microscopy, autoradiography and immunocytochemistry to determine whether histidine-rich proteins, filaggrin and loricrin are present. In the lungfish and amphibian tadpoles, anti-keratin antibodies (AE1 and AE3) stain the whole epidermis but not the AE2 antibody, a marker for keratinization. In adult epidermis, the AE2 antibody mainly stains keratinized layers, AE1 mainly stained basal cells, less suprabasal cells and no pre-keratinized and keratinized layers, and AE3 stains all epidermal layers. This staining pattern resembles that of amniote epidermis. Little tritiated histidine is taken up in toad epidermis at 4-6 h post-injection but 24 h after injection the radioactivity is most concentrated in the replacement layer beneath the corneus. This indicates that protein synthesis takes place in the epidermis but, due to the metabolic conversion that takes place in 24 h, it is unlikely that histidine-rich proteins are formed. Neither filaggrin-like nor loricrine-like immunoreactivities are present in amphibian and lungfish epidermis. This indicates absence of histidine-rich matrix proteins and corneous cell envelope proteins and only mucus is present among keratin filaments. Filaggrine-like and loricrin-like proteins are characteristic of amniotes epidermis and might have originated in basic amniotes (cotylosaurs).     

Abnormal Epidermal Keratinization in the repeated epilation mutant mouse

Repeated epilation (Er) is a radiation-induced, autosomal, incomplete dominant mutation in mice which is expressed in heterozygotes but is lethal in the homozygous condition. Many effects of the mutation occur in skin: the epidermis in Er/Er mice is adhesive (oral and nasal orifices fuse, limbs adhere to the body wall), hyperplastic, and fails to undergo terminal differentiation. Skin from fetal +/+, Er/+ and Er/Er mice at ages pre- and postkeratinization examined by light, scanning, and transmission electron microscopy showed marked abnormalities in tissue architecture, differentiation, and cell structure; light and dark basal epidermal cells were separated by wide intercellular spaces, joined by few desmosomes, and contained phagolysomes. The numbers of spinous, granular, and superficial layers were highly variable within any given region and among various regions of the body. In some areas, 2-8 layers of granular cells, containing large or diminutive keratohyalin granules, extended to the epidermal surface; in others, the granular layers were covered by several layers of partially keratinized or nonkeratinized cells. In rare instances, a single or small group of cornified cells was present among the granular layers but was not associated with the epidermal surface. Both the granular and nonkeratinized/partially keratinized upper epidermal layers Er/Er skin gave positive immunofluorescence with antiserum to the histidine-rich, basic protein, filaggrin. Proteins in epidermal extracts from +/+, Er/+ and Er/Er mice were separated and identified by radio- and immunolabeling techniques. The Er/Er extract was missing a 26.5- kdalton protein and had an altered ratio of bands in the keratin region. The 26.5-kdalton band was histidine-rich and cross-reacted with the antiserum to rat filaggrin. Several high molecular weight bands present in both Er/Er and +/+ extracts also reacted with the antiserum. These are presumed to be the precursors of filaggrin and to account for the immunofluorescence om Er/Er epidermis even though the product protein is absent. The morphologic and biochemical data indicated that the genetic defect has a general and profound influence on epidermal differentiation, including alteration of two proteins (filaggrin and keratin) important in normal terminal differentiation, tissue architecture, and cytology. Identification of epidermal abnormalities at early stages of development (prekeratinization) and defective structure of other tissues and gross anatomy suggest that the mutation is responsible for a defect in same regulatory step important in many processes of differentiation and development.     

Inhibition of the Transsulfuration Pathway Affects Growth and Feather Follicle Development in Meat-Type Chickens

Cysteine is a nonessential amino acid in poultry nutrition. Poultry diets are deficient in cysteine, but the bird’s cysteine need is met through the transsulfuration pathway (TSP) where homocysteine is converted to cysteine: a process catalyzed by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH). Cysteine is also a major component of keratinized protein found in feathers, but the extent to which cysteine is involved in feather and skin development in poultry is unknown. We randomly assigned chicks to control and treatment (control diet plus 100?mg/kg body weight of propargylglycine which is an inhibitor of CTH) diets. The thickness of skin layers, primary feather follicle parameters, growth, and mRNA expression of CBS and CTH were measured. Inhibition of TSP corresponded with the upregulation of liver mRNA of both CBS and CTH and reduction in growth from 35 to 40 days of age. The epidermis thickness, feather follicle length, and diameter were reduced from 10 to 40 days of age. Incorporation of cysteine into keratinized protein may be more sensitive to the level of available cysteine than into nonkeratinized proteins. Thus, disruption of the TSP could affect the thermoregulatory ability of the bird.     

Immunohistochemical analysis of stratum corneum components in oral squamous epithelia

The generation of a stratum corneum in squamous epithelia involves marked changes in morphology and in the expression of cell products. We have examined the expression of some of the components involved in this process in oral squamous epithelia with different terminal differentiation patterns by use of immunofluorescent techniques. Involucrin and transglutaminase are involved in formation of cornified envelopes consistently seen in the stratum corneum. Both components were present in keratinized oral epithelia (palatal epithelium and hyperkeratinized buccal epithelium). The nonkeratinized normal buccal epithelium stained positive as well. Filaggrin, a protein derived from a precursor present in keratohyalin granules, is proposed to aggregate keratin filaments in the cornified layer. Although the staining differed markedly in quantity, this component was likewise detected in both keratinized and nonkeratinized epithelia. The staining patterns for different keratin polypeptides, however, showed qualitative differences between the different epithelia. Thus, it seems that the keratin composition shows differentiation-specific characteristics, whereas the presence of other important components needed to generate a stratum corneum is not as closely related to the terminal differentiation pattern of oral epithelia.     

Protein and carbohydrate histochemistry in relation to the keratinization in the epidermis of Barbus sophor (Cyprinidae, Pisces)

The distribution of protein and carbohydrate constituents in the epidermis of Barbus sophor is described in order to give a better understanding of its cellular organization and physiology.
Various cytochemical techniques show the keratinized nature of the outer free margins of the polygonal cells in the most-superficial layer. These contain appreciable amounts of cysteine bound sulphydryl groups, basic proteins, protein bound NH₂ groups, ribonucleic acid and calcium and give a strong Papanicolaou's reaction. Absence of cystine bound disulphide groups suggests that the cornified layer in B. sophor is probably mechanically weak as adjacent keratin chains remain unbonded. The polygonal cells showing keratinization at the outer free margins remain metabolically active and are not sloughed off at the surface. This is in contrast to the keratinized epidermis of other teleosts so far reported in which the keratinized cells are dead and are sloughed off at the surface.
In addition to keratinization the polygonal cells undergo mucogenesis synthesizing sulphated acid mucopolysaccharides.
The presence of eosinophilic granular cells in the epidermis is interesting. The possible role of these cells in the protection of the epidermis has been discussed. The epidermis on the inner surface of the scale is very thin so it may not have much protective significance in these areas.     

Investigation of the effect of various buffer solutions used for microinjection of gene-engineering constructs on preimplantation development of mouse embryos

We studied the effects of some buffer solutions used for microinjection in mammalian zygotes on preimplantation development of (CBA x C57BL)F1 mouse embryos in vitro. The rate of embryo survival was estimated according to their capacity to develop to the stages of blastocyst and blastocyst hatched from zona pellucida. The results obtained suggested a reduced rate of survival of zygotes to the blastocyst stage after the injection into a pronucleus of the buffers Tris-HCl with EDTA, Tris-HCl with MgCl2 and NaCl, and medium M2 (p < 0.05) and to the stage of blastocyst hatched from zone pellucida after injection of a Dulbecco solution, as compared to the control. No differences were found in the survival rate of zygotes injected with different buffer solutions.     

Correlation of specific keratins with different types of epithelial differentiation: monoclonal antibody studies

We have prepared three monoclonal antibodies against human epidermal keratins. These antibodies were highly specific for keratins and, in combination, recognized all major epidermal keratins of several mammalian species. We have used these antibodies to study the tissue distribution of epidermis-related keratins. In various mammalian epithelia, the antibodies recognized seven classes of keratins defined by their immunological reactivity and size. The 40, 46 and 52 kilodalton (kd) keratin classes were present in almost all epithelia; the 50 kd and 58 kd keratin classes were detected in all stratified squamous epithelia, but not in any simple epithelia; and the 56 kd and 65-67 kd keratin classes were unique to keratinized epidermis. Thus the expression of specific keratin classes appeared to correlate with different types of epithelial differentiation (simple versus stratified; keratinized versus nonkeratinized).     

Keratinization of fish skin with special reference to the catfish Bagarius bagarius

Summary Histochemical reactions indicating keratinization have previously been demonstrated in parts of the epidermis of Bagarius bagarius. Fluorescence histochemistry and electron microscopy have now confirmed these results. Elevated areas of the epidermis are capped by a layer of dead cells with altered contents. On the outer aspect of these cells a dense layer, 18 nm thick, beneath the plasma membrane corresponds to the resistant envelope found in keratinized cells in tetrapod vertebrates. In Bagarius this layer does not extend to all faces of the keratinized cells, but a similar envelope has been detected in two other sites of piscine keratinized epidermis investigated, namely in the breeding tubercles of Phoxinus phoxinus and in the teeth of Lampetra fluviatilis. In the elevated areas of Bagarius-epidermis, the epithelial cells undergo progressive changes in cytoplasmic organization as they become more superficial. The second tier from the surface is sealed by tight junctions and is separated from the overlying keratinized cells by a sub-corneal space resembling that found in keratinized amphibian epidermis. Histochemical evidence of a high lipid content in the outer layers of the epidermis correlates with the presence of lipid inclusions and lamellated membranous profiles in the material studied by electron microscopy. Histochemical results show that the fin skin of Blennius pholis is not keratinized, but secretes a cuticle, histochemically reactive for both proteins and glycoproteins.     

Structure of the sensory innervation of the anal canal in the pig. A light- and electron-microscopical study

The sensory innervation of the anal canal of the pig was investigated by light and electron microscopy. The distribution of the different types of sensory nerve endings correlates with the histology of different zones: (1) After the rectal mucosa there was a zone lined with nonkeratinized stratified squamous epithelium. (2) A middle zone was lined with keratinized stratified squamous epithelium. Here the dermis already showed a papillary and reticular layer. (3) The last zone showed hairy skin with a high hair density. The following nerve endings were found: Free nerve endings reached the stratum superficiale in nonkeratinized squamous epithelium and the stratum granulosum in the keratinized squamous epithelium. Dermal free nerve endings were found in all zones near the epithelium and two different types were identified as those derived from C-fibers and those from A-delta-fibers. Merkel nerve endings showed different features depending on their location. Few Merkel-like cells were found in the epithelium of the anal crypts. Typical Merkel Tastscheiben were located at the base of epithelial ridges or pegs in zones 2 and 3. The number of Merkel cells varied up to 200. The myelinated afferent fiber supplied 10-15 Merkel cells. Merkel cells were also found regularly in the outermost layer of the external rooth sheath of hair follicles at about the same level as perifollicular nerve endings. Lamellated corpuscles were found in the dermis of all zones except the cranial part of zone 1, where the anal crypts are located. Generally they consisted of a central nerve terminal which may be branched. Each terminal was surrounded by an inner core of concentrically arranged lamellae of the terminal Schwann cell and one or several inner cores were included in a capsule of perineural cells. The size of the corpuscle, the regularity of the inner core and the number of capsular layers depended on the location of the corpuscle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression and role of E- and P-cadherin adhesion molecules in embryonic histogenesis. II. Skin morphogenesis

Expression and the role of E- and P-cadherin in the histogenesis of the surface epidermis and hair follicles were examined using the upper lip skin of the mouse. P-cadherin is expressed exclusively in the proliferating region of these tissues, that is in the germinative layer of the surface epidermis, the outer root sheath and the hair matrix. E-cadherin is coexpressed in these layers but this molecule was also detected in non-proliferating regions such as the intermediate layer of the surface epidermis and the immature regions of the inner root sheath. Neither P- nor E-cadherin was detected in fully keratinized layers such as the horny layer of the surface epidermis, the outermost layer of the outer root sheath and the mature hair fibres. These two cadherins were not detected in dermal cells. We cultured pieces of the upper lip skin in vitro in the absence or presence of a monoclonal antibody to E-cadherin (ECCD-1) or to P-cadherin (PCD-1). In control cultures, skin morphogenesis normally occurred in a pattern whereby the hair follicles grew and dermal cells were condensed to form the dermal sheath. A mixture of ECCD-1 and PCD-1, however, induced abnormal morphogenesis in the skin in several respects. (1) The cuboidal or columnar arrangement of basal epithelial cells was distorted. (2) Hair follicles were deformed. (3) Condensation of dermal cells was suppressed, causing a homogeneous distribution of these cells. These results suggest that cadherins present in epidermal cells are involved not only in maintaining the arrangement of these cells but also in inducing dermal condensation.     

The 50- and 58-kdalton keratin classes as molecular markers for stratified squamous epithelia: cell culture studies

The keratins are a highly heterogeneous group of proteins that form intermediate filaments in a wide variety of epithelial cells. These proteins can be divided into at least seven major classes according to their molecular weight and their immunological reactivity with monoclonal antibodies. Tissue-distribution studies have revealed a correlation between the expression of specific keratin classes and different morphological features of in vivo epithelial differentiation (simple vs. stratified; keratinized vs. nonkeratinized). Specifically, a 50,000- and a 58,000-dalton keratin class were found in all stratified epithelia but not in simple epithelia, and a 56,500- and a 65-67,000-dalton keratin class were found only in keratinized epidermis. To determine whether these keratin classes can serve as markers for identifying epithelial cells in culture, we analyzed cytoskeletal proteins from various cultured human cells by the immunoblot technique using AE1 and AE3 monoclonal antikeratin antibodies. The 56,500- and 65-67,000-dalton keratins were not expressed in any cultured epithelial cells examined so far, reflecting the fact that none of them underwent morphological keratinization. The 50,000- and 58,000-dalton keratin classes were detected in all cultured cells that originated from stratified squamous epithelia, but not in cells that originated from simple epithelia. Furthermore, human epidermal cells growing as a monolayer in low calcium medium continued to express the 50,000- and 58,000-dalton keratin classes. These findings suggest that the 50,000- and 58,000-dalton keratin classes may be regarded as "permanent" markers for stratified squamous epithelial cells (keratinocytes), and that the expression of these keratin markers does not depend on the process of cellular stratification. The selective expression of the 50,000- and 58,000-dalton keratin classes, which are synthesized in large quantities on a per cell basis, may explain the high keratin content of cultured keratinocytes.     

On the existence of non-cycling germinative cells in human epidermis in vivo and cell cycle aspects of psoriasis

Abstract. This report deals with the controversies of whether all germinative epidermal cells in human epidermis are in the cycling state and whether stimulated hyperproliferation of psoriatic epidermis is due to a shortening of the cell cycle time or to a recruitment of non-cycling germinative epidermal cells. Experiments were performed on human subjects in vivo . Continuous infusion of [³H]thymidine for 8½ days indicated that 40% of germinative epidermal cells reside in the non-cycling state. Proliferative stimulation by tape stripping indicated recruitment of non-cycling (G₀) germinative epidermal cells in both normal and psoriatic skin, and a prolongation (rather than a shortening) of cell cycle traverse in activated psoriatic epidermal cells.     

Investigation of the Effect of Various Buffer Solutions Used for Microinjection of Gene-Engineering Constructs on Preimplantation Development of Mouse Embryos

We studied the effects of some buffer solutions used for microinjection in mammalian zygotes on preimplantation development of (CBA × C57BL) F₁ mouse embryos in vitro. The rate of embryo survival was estimated according to their capacity to develop to the stages of blastocyst and blastocyst hatched from zona pellucida. The results obtained suggested a reduced rate of survival of zygotes to the blastocyst stage after the injection into a pronucleus of the buffers Tris-HCl with EDTA, Tris-HCl with MgCl₂ and NaCl, and medium M2 (p < 0.05) and to the stage of blastocyst hatched from zone pellucida after injection of a Dulbecco solution, as compared to the control. No differences were found in the survival rate of zygotes injected with different buffer solutions.     

Effects of phosphate on the activity, stability and regulatory properties of phosphoenolpyruvate carboxylase from the C4 plant Cynodon dactylon

The extraction of phosphoenolpyruvate carboxylase, PEPC (EC 4.1.1.31) from leaves of Cynodon dactylon (L.) Pers. with phosphate buffer (pH 7.4, 105 mM) was advantageous in comparison to the usual extraction with Tris-HCl buffer (pH 7.4, 100 mM); a higher activity was obtained, which was most evident at low substrate (phosphoenolpyruvate) concentrations. The PEPC activity was stable under dilution or in storage for at least 48 h at room temperature. The effects of phosphate buffer were not due to inhibition of phosphatase(s) action during the extraction, since they were also observed when the phosphates were added after the extraction with Tris-HCl. The phosphate-extracted enzyme was less responsive to both L-malate inhibition and activation by glucose-6-phosphate. The effects of phosphates might be due to preferential exclusion from the enzymic protein domain and, therefore, to a confinement of the enzyme to a fraction of the total volume. This revised version was published online in August 2006 with corrections to the Cover Date.     

Ultrastructure of the epidermis of the lizard (Lacerta vivipara) at the resting stage of the sloughing cycle

The ultrastructure of the epidermis of the lizard ( Lacerta vivipara ) one day after sloughing is described. The non-keratinized layers of the epidermis are essentially similar in structure to those of amphibians and mammals. The cells of the basal layer are not however separated from each other by the large spaces described in the amphibian (Farquhar & Palade, 1965). The middle layers of the epidermis at this stage of the sloughing cycle produce neither the characteristic mucous granules found in amphibians nor the keratohyalin granules of mammals. A small number of granules corresponding in size and location to the "Odland bodies" of both mammalian and amphibian epidermis are, however, present. The intermediate layer cells also contain a number of bodies similar in appearance to those described by Farquhar & Palade as lysosomes in amphibian skin. These structures are both osmium iodide and acid phosphatase positive. Unlike the condition in amphibians and mammals, the cytoplasm of cells in the layer immediately beneath the keratinized strata is honeycombed with small vesicles, and contains large irregular vacuoles of uncertain content. Certain nonkeratinizing elements within the epidermis are tentatively interpreted as nerve terminations. Two morphologically distinct keratinized strata can be distinguished, the inner stratum consisting of flattened cells similar to those of the stratum corneum of mammalian epidermis; individual cell outlines cannot be distinguished in the outer stratum, which has a structure similar to that of avian feather keratin. A shallow surface zone of the outer keratinized stratum has been identified as the Oberhautchen. This consists of longitudinally disposed leaflets or laminae which are responsible for the sculptured pattern of the epidermal surface. The observations reported here provide a basis for analysis of changes occurring at other stages of the sloughing cycle.     

The characterization of human epidermal filaggrin. A histidine-rich, keratin filament-aggregating protein

Filaggrin is a histidine-rich, cationic protein that aggregates with keratin filaments in vitro and may function as the keratin matrix protein in the terminally differentiated cells of the epidermis. This protein has been previously isolated from rodent epidermis. In this investigation, a similar protein from human skin was identified, isolated and characterized by biochemical and immunologic techniques. Indirect immunofluorescence of human skin using antiserum to rat filaggrin gave positive immunofluorescence of keratohyalin granules and the stratum corneum. This indicated the presence of a human filaggrin in the epidermis in a localization similar to that of the rodent. The protein was isolated from human epidermis and purified by ion-exchange chromatography and preparative gel electrophoresis. The purified protein crossreacts with antibody to rat filaggrin and migrates as a doublet of molecular weight (Mr) approximately 35 000 on SDS-polyacrylamide gels. It is relatively rich in polar amino acids such as histidine, arginine, serine and glycine, but is poor in nonpolar amino acids. Unlike rodent filaggrin, the human protein contains ornithine. This protein aggregates with human keratin filaments, forming compact macrofibrils in a manner analogous to that of rodent filaggrin. Thus, a human epidermal protein has been isolated which has many of the characteristics of rodent filaggrin and may function as the human keratin matrix protein.     

Endogenous beta-galactoside-binding lectin expression is suppressed in retinol-induced mucous metaplasia of chick embryonic epidermis

The fate of endogenous beta-galactoside-binding lectin of chick embryo (14K type) was investigated during the course of skin differentiation. Lectin (14K) was found in keratinized epidermis and was localized mainly in the basal and intermediate cells. However, the protein lectin in the epidermis disappeared when the cultured skin was treated with vitamin A and mucous metaplasia was observed. The synthesis of lectin mRNA was also strongly suppressed by vitamin A in a concentration-dependent manner. On the other hand, in the dermis, in which the lectin was localized in the extracellular matrix, lectin expression was scarcely affected by vitamin A. These results indicated that the lectin was expressed in the keratinized epidermis but that its expression was suppressed in vitamin A-induced mucous-secreting epithelium. The suppression may be a result of a transition of the epidermal regulatory system to one of mucous-secreting epithelium. This is the first finding that 14K lectin expression might be regulated during the course of the epidermal differentiation.     

Assay of N-myristoyl transferase by selective adsorption of myristoyl-coenzyme A on acidic alumina

We have developed a simple and rapid method for detecting the enzyme myristoyl-CoA:protein N-myristoyl transferase. The enzyme catalyzes the transfer of the myristoyl moiety of myristoyl-CoA to the amino-terminal glycine residue of a peptide (protein). Incorporation of the [14C]myristate into the peptide is quantified after separation of the [14C]myristoyl-peptide from unreacted [14C]myristoyl-CoA by selective adsorption of [14C]myristoyl-CoA on acidic alumina. Optimal assay concentrations were 200 microM synthetic peptide, 1 microM [14C]myristoyl-CoA, 10 mM Tris-HCl/1 mM dithiothreitol/0.1 mM ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid/aprotinin (10 micrograms/ml) buffer, pH 7.4, and 1-10 micrograms protein.