Figures of Eberth in the amphibian larval epidermis.

Figures of Eberth are prominent extensive filamentous structures in the basal epidermal cells of larval amphibians. They are compared and contrasted qualitatively and quantitatively in a number of species of the three groups of living amphibians. Fully developed Figures consist of massive skeins of tonofilaments oriented in three dimensions and hinged on hemidesmosomes within the cell. The overall appearance of the Figures is similar in anurans, urodeles and Ichthyophis among the apodans. However, in terms of size and number per unit length of the proximal cell margin, the hemidesmosomes and the thickness or their emergent skeins in anurans and Ichthyophis differ significantly from those parameters in urodeles, a feature that is presumably independent of cell size. Figures are poorly developed or missing in embryos of Typhlonectes, which has no larval stage in its life history. These ubiquitous skeletogenous structures in the aquatic larval amphibians, among other things, could be protective of underlying delicate tissues and act as a stabilizer in bodily movement during swimming. They could also serve as a reserve of cytokeratin for use during later cellular division and sloughing.     

20-Hydroxyecdysone control of the synthesis of putative calcium-binding proteins in the epidermis of Tenebrio molitor

The altered pattern of synthesis of putative calcium-binding proteins (pCaBPs) in the mid-instar epidermis following exposure to 20-hydroxyecdysone (20HE) in vitro was followed through the incorporation of [³⁵S]methionine into newly synthesized polypeptides. pCaBPs were separated from other epidermal polypeptides by Ca²⁺-dependent hydrophobic interaction chromatography, followed by polyacrylamide gel electrophoresis and fluorography. The dominant effect of 20HE is to depress pCaBP synthesis. Of the 17 newly-synthesized pCaBPs consistently detected in total cell lysates, the synthesis of ten was depressed strongly and that of the remaining seven was unaltered by exposure to 20HE. Most newly-synthesized pCaBPs identified were found in the cytosolic fraction of the epidermis. One pCaBP was identified as Tenebrio calmodulin based on its altered electrophoretic mobility in the absence of calcium ions, its isoelectric focusing point, its binding to phenyl-Sepharose and phenothiazine and its binding to antibodies against purified mammalian calmodulin. The synthetic rate of this pCaBP was not affected by 20HE. The distribution of another pCaBP (32 kDa) shifted from the cytosolic to the microsomal fraction on adding Ca²⁺ (or reversed by adding EGTA) to the cell extract before fractionation. The synthesis of this protein was depressed by 20HE. These findings suggest that 20HE influences epidermal behaviour, at least in part, through its ability to modulate the synthetic rate of several pCaBPs.     

Identification of a rapidly labelled 350K histidine-rich protein in neonatal mouse epidermis

The major histidine-rich protein (HRP) found in the stratum corneum of neonatal mouse epidermis (band 2 protein, molecular weight 27,000) is a relatively late product of epidermal differentiation and incorporates labelled amino acids in vivo only after a 6-9 h lag period. A number of putative precursor HRPs in the 70-300 K molecular weight range were initially identified using short pulse labeling times and our previously described methods for isolation of epidermis and extraction of proteins. However, when steps were taken to minimise proteolysis during preparation, a single species of approximately 350 K molecular weight was the most strongly labelled protein following a 1 h in vivo pulse of [3H]-histidine. This protein was stable in sodium dodecyl sulphate dithiothreitol at 100 degrees C and in 4 M urea, suggesting a single covalently linked polypeptide. The kinetics of labelling and the localisation of the 350 K HRP in the lower granular layers suggest that it is a precursor of the stratum corneum HRP. The processing of the 350 K HRP to the stratum corneum species appears to involve a complex series of specific cleavage steps which give rise to a number of HRPs of intermediate molecular weight.     

Polarization of plasma membrane glycoconjugates in amphibian epidermis during metamorphosis

Summary Wheat germ agglutinin (WGA) binding sites have been examined in tadpole epidermal cells at the level of both light and electron microscopy using the WGA-ovomucoid-gold technique. In premetamorphic tadpoles the reaction was observed on the plasma membranes of epithelial cells showing a gradient from inner to outer membranes. These glycoconjugates were polarized during development, and at the end of metamorphic climax they were only located in plasma membranes of stratum corneum. The existence of an apical cell surface coat is needed to facilitate the absorption of water through the adult epidermis. The possible implications of this polarization process are discussed.     

Polarization of plasma membrane glycoconjugates in amphibian epidermis during metamorphosis

Wheat germ agglutinin (WGA) binding sites have been examined in tadpole epidermal cells at the level of both light and electron microscopy using the WGA-ovomucoid-gold technique. In premetamorphic tadpoles the reaction was observed on the plasma membranes of epithelial cells showing a gradient from inner to outer membranes. These glycoconjugates were polarized during development, and at the end of metamorphic climax they were only located in plasma membranes of stratum corneum. The existence of an apical cell surface coat is needed to facilitate the absorption of water through the adult epidermis. The possible implications of this polarization process are discussed.     

Enzyme cytochemical and immunocytochemical studies of flask cells in the amphibian epidermis

The localization of oxidoreductases and transport enzymes in flask cells of the amphibian epidermis was studied at the light-microscopic level. In these cells, the deposition of cytochemical reaction products was very similar to that found in fish epidermal ionocytes, thus demonstrating histochemical similarities between these two types of cells. The present histochemical results revealed high levels of activity of alkaline phosphatase (ALPase), potassium-dependent nitrophenylphosphatase (K+-p-NPPase) and carbonic-anhydrase isozymes (CA-I and CA-II) in the apical region of the flask cells, indicating that enzyme zonation may be the main site of the ion pumping.     

Enzyme cytochemical and immunocytochemical studies of flask cells in the amphibian epidermis

Summary The localization of oxidoreductases and transport enzymes in flask cells of the amphibian epidermis was studied at the light-microscopic level. In these cells, the deposition of cytochemical reaction products was very similar to that found in fish epidermal ionocytes, thus demonstrating histochemical similarities between these two types of cells. The present histochemical results revealed high levels of activity of alkaline phosphatase (ALPase), potassium-dependent nitrophenylphosphatase (K⁺-p-NPPase) and carbonic-anhydrase isozymes (CA-I and CA-II) in the apical region of the flask cells, indicating that enzyme zonation may be the main site of the ion pumping.     

Presence of ranatensin-like and bombesin-like peptides in amphibian brains

Antibodies specific for the carboxyl-terminal regions of bombesin and of ranatensin were used to study the tissue distribution of substances immunochemically similar to these two peptides in three amphibian species. Brain extracts of Rana catesbeiana, Rana pipiens, and Xenopus laevis all contained considerable quantities of both types of immunoreactivity, with measured concentrations as high as several hundred pmol per g tissue. The two antibodies used in this study had very low crossreactivity (less than 1% by RIA) with the other peptide. In addition, gel filtration revealed different elution profiles for the two immunoreactive substances extracted from amphibian brains. Immunocytochemistry revealed differences in localization within nerve fibers and cell bodies and specific absorption by the appropriate peptide. Ranatensin-like peptides were also present in high concentrations in skin of the two Rana species but not in that of Xenopus. Bombesin-like peptides were more abundant in the stomach of all three species. Significant amounts of substance P/phylasaemin-like immunoreactivity also were detected in the brains of all three species. It is concluded that ranatensin-like peptides are not confined to the skin and can be included as central nervous system neuropeptides in amphibians. These two groups of peptides are not species-specific since both are found in brain and stomach of amphibians whose skin contains only ranatensin-like peptide or neither.     

Putative histidin-rich proteins in the epidermis of lizards

In the stratum granulosum of mammalian epidermis, histidin-rich proteins (filaggrins) determine keratin clumping and matrix formation into terminal keratinocytes of the stratum corneum. The nature of matrix, interkeratin proteins in the epidermis of nonmammalian vertebrates, and in particular in that of reptilian, mammalian progenitors are unknown. The present biochemical study is the first to address this problem. During a specific period of the renewal phase of the epidermis of lizards and during epidermal regeneration, keratohyalin-like granules are formed, at which time they take up tritiated histidine. The latter also accumulate in cells of the alpha-keratin layer (soft keratin). This pattern of histidine incorporation resembles that seen in keratohyalin granules of the stratum granulosum of mammalian epidermis. After injection of tritiated histidine, we have analysed the distribution of the radioactivity by histoautoradiography and electrophoretic gel autoradiography of epidermal proteins. Extraction and electrophoretic separation of interfilamentous matrix proteins from regenerating epidermis 3-48 hours post-injection reveals the appearance of protein bands at 65-70, 55-58, 40-43, 30-33, 25-27, and 20-22 kDa. Much weaker bands were seen at 100, 140-160, and 200 kDa. A weak band at 20-22 kDa or no bands at all are seen in the normal epidermis in resting phase and in the dermis. In regenerating epidermis at 22 and 48 hours post-injection, little variation in bands is detectable, but low molecular weight bands tend to increase slightly, suggesting metabolic turnover. Using anti-filaggrin antibodies against rat, human, or mouse filaggrins, some cross-reactivity was seen with more reactive bands at 40-42 and 33 kDa, but it was reduced or absent at 140, 95-100, 65-70, 50-55, and 25 kDa. This suggests that different intermediate degradative proteins of lizard epidermis may share some epitopes with mammalian filaggrins and are different from keratins with molecular weight ranging from 40 to 65-68 kDa. The immunocytochemical observation confirms that a weak filaggrin-like immunoreactivity characterizes differentiating alpha-keratogenic layers in normal and regenerating tail. A weak filaggrin labeling is discernable in small keratohyalin-like granules but is absent from the larger granules and from mature keratinocytes. The present results indicate, for the first time, that histidine-rich proteins are involved in the process of alpha-keratinization in reptilian epidermis. The cationic, interkeratin matrix proteins implicated may be fundamentally similar in both theropsid-derived and sauropsid amniotes.     

Similarities between stratum corneum basic protein and histidine-rich protein II from newborn rat epidermis

The stratum corneum basic protein and histidine-rich protein II were each isolated from newborn rat epidermis and compared by biochemical and immunologic methods. The proteins were indistinguishable by immunodiffusion using antiserum elicited to either protein. The migration of the proteins on SDS-polyacrylamide gel electrophoresis was identical giving a molecular weight of 49 000. These proteins, which have similar but unusual amino acid compositions, give very similar tryptic peptide maps. Both proteins aggregate with keratin filaments to form macrofibrils. These results suggest that histidine-rich protein II and stratum corneum basic protein are the same protein. We suggest that this protein be called histidine-rich basic protein.     

Nucleo-cytoplasmic exchange of non-histone proteins in amphibian embryos

As a basis for understanding the role of non-histone proteins in nuclear differentiation, we have identified one period during embryogenesis when intense accumulation of non-histones occurs in nuclei of Rana pipiens. We then demonstrated, experimentally, the loss of non-histones from nuclei after transplantation into enucleated eggs. ³H-tryptophan or ³H-lysine was injected into blastocoeles of mid-blastulae and into archenterons of late gastrulae; embryos were subsequently studied autoradiographically. Nuclei of animal hemisphere cells from blastulae accumulated only small amounts of ³H-tryptophan within 3 h, whereas a large accumulation occurred in endodermal nuclei of gastrulae as early as 1 h, and after 3 h 95.9% ( ) of the nuclei were densely labelled. Significant accumulation of ³H-lysine occurred in the majority of nuclei of both types within 3 h (blastulae ; gastrulae ). Controls, involving RNase or boiling TCA, demonstrated that the ³H-amino acids have been incorporated mainly into proteins. Endodermal nuclei labelled either with ³H-tryptophan or ³H-lysine after a 3 h incubation were transplanted singly into enucleated eggs. Autoradiograms demonstrated that most non-histones leave the nucleus during its reprogramming in the egg cytoplasm prior to first cleavage; whereas other types of proteins labelled with ³H-lysine remain for the most part in the nucleus. Cytochemical studies indicated that some of the non-histones which leave transplanted nuclei are acidic proteins; whereas some of those proteins which remain in the nucleus are histones.In addition to the above findings, the results of these studies demonstrate the feasibility in the future of studying the nucleocytoplasmic migration of different kinds of macromolecules in a developmental metazoan system and determining their roles in the establishment of nuclear differentiation.     

Studies on the synthesis and degradation of a high molecular weight, histidine-rich phosphoprotein from mammalian epidermis

The synthesis and subsequent fate of the histidine-rich proteins, which form a major component of keratohyalin granules in mammalian epidermis, have been studied in the guinea-pig and new-born rat. In both species the protein first synthesised is of very high molecular weight, approximately 340 000. It is short-lived and breaks down to lower molecular weight proteins 1-2 days after its synthesis. These smaller proteins differ in the two species. In the guinea-pig, the high molecular weight protein breaks down to proteins of molecular weight 250 000 and 200 000, which are themselves unstable and break down to low molecular weight species, probably amino acids. The initial breakdown of the high molecular weight protein coincides with the dispersion of the keratohyalin granules and the transition of the granular cell into the stratum corneum. This high molecular weight histidine-rich protein has been purified to homogeneity, despite its instability to several treatments during purification. The protein is highly phosphorylated, containing 6 mol% of phosphoserine, but is otherwise very basic. The possibility that dephosphorylation of the protein produces highly basic matrix proteins in the stratum corneum is discussed.     

The proteins of living psoriatic epidermis

Psoriatic epidermis has a rapid rate of tunrover and produces a stratum corneum with an abnormal tonofilament composition. One polypeptide chain, (M_r 70 000) is absent or greatly decreased in relative amount and two other chains, (M_r 63 000 and 55 000), which are normally modified in the living cells, persists into the stratum corneum. Increasing the turnover of normal epidermis has been shown to cause the persistence of 63 and 55 kilodalton chains in the stratum corneum but does not affect the relative amount of 70 kilodalton chain. It has, therefore, been suggested that, in psoriasis, the deficiency of the 70 kilodalton chain may occur prior to or simultaneuously with the induction of increased tissue turnover. In the present study, the polypeptide chain composition of living psoriatic epidermis has been examined. It is shown that the relative amounts of 70 kilodalton chain in psoriatic stratum corneum and involved living epidermis from the same site are not significantly different. The abnormality is therefore, already present in the living cells and it appears that, in psoriasis, the synthesis of the 70 kilodalton chain is defective. The uninvolved epidermis of psoriatics is intermediate between normal and involved psoriatic epidermis both in the ability to synthesise the 70 kilodalton chain and to modify the 63 and 55 kilodalton chains. Comparisons of amino acid compositions of proteins containing different proportions of 70 kilodalton chain suggest that it has a considerably higher content of glycine and serine than the other tonofilament chains. These studies suggest that the 70 kilodalton chain may be functionally different from the other tonofilament chains. The defect in its synthesis in psoriasis is a relatively early event and may be involved with the induction of increased tissue turnover or induced by the same abnormal conditions as the increased tissue turnover.     

Presence of rhodopsin-like proteins in the planarian head

The presence of rhodopsin-like protein was detected in the head of the freshwater planarian Dugesia japonica japonica Ichikawa et Kawakatsu by use of anti-frog-rhodopsin rabbit IgG. Two membrane proteins of molecular weights 65000 and 62000 were separated by sodium-dodecyl-sulfate polyacrylamide gel electrophoresis and found to react with the anti-rhodopsin IgG. The antibody may be useful for monitoring regeneration of the planarian eye.     

Presence of non-histone proteins in nucleosomes

It has been established that nucleosomes are made of histones and DNA fragments. The purpose of this work to establish whether some non-histone proteins are also present in these chromatin subunits. We have found that nucleosome preparations contain phosphorylated non-histone proteins and protein kinases by sucrose gradient analysis. In order to establish whether these proteins are actually bound to nucleosomes or if they represent unbound or aggregated proteins, the following experiments were performed. (a) Free non-histone proteins and proteins released from chromatin by DNase overdigestion were analyzed by sucrose gradient centrifugation. No phosphoproteins but some phosvitin kinase activity was found in the part of the gradient which contained the nucleosomes. It could be assumed that part of the phosphoproteins are bound to nucleosomes. (b) A digestion of nucleosomes with DNase I suppressed the phosvitin kinase activity in the 11-S region of the gradient. (c) High ionic strength, which extracted non-histone proteins, suppressed the phosvitin kinase activity in the nucleosome region. Part of phosvitin kinase and of nuclear phosphoproteins are therefore bound to nucleosomes and are released by nuclease digestion and by high ionic strength.     

Two hemoglobin-binding proteins identified in the plasma of the amphibian Taricha granulosa

The plasma of the amphibian newt Taricha granulosa has been shown to be devoid of haptoglobin. Upon hemolysis, Taricha albumin and another protein associate with hemoglobin. The acute-phase response to inflammation observed in birds and mammals appears to be absent in Taricha. Taricha hemoglobin failed to bind to human haptoglobin. Taricha hemoglobin not only failed to dissociate into alpha beta dimers as did human Hb, but formed alpha beta octamers.     

A putative new hydrostatic skeletal function for the epidermis in Monhysterids (Nematoda)

The ultrastructure of the epidermis of two Monhysterid nematodes (Geomonhystera disjuncta and Diplolaimella dievengatensis) is studied in detail. The epidermis is composed of discrete uninucleated cells. The cytoplasmic layer of the epidermis between the cuticle and the somatic muscles is very thin and contains bundles of filaments that attach the muscles to the cuticle. The epidermal chords are voluminous and contain the nuclei and most of the cell organelles. In the chords many large electron-transparent vacuoles are found. It is hypothesized that these vacuoles fulfill a function as a compartmentalised hydrostatic skeleton.