Proteomic analysis of the skin from Chinese fire-bellied newt and comparison to Chinese giant salamander.

Animal skin that directly interfaces with the external environment has developed diverse adaptive functions to a variety of ecological conditions laden with pathogenic infection and physical harm. Amphibians exhibit various adaptations related to their "incomplete" shift from the aquatic to the terrestrial habitat. Therefore, it is very necessary to explore the molecular basis of skin function and adaptation in amphibians. Currently, the studies on the molecular mechanisms of skin functions in anuran amphibians have been reported, but in urodele amphibians are rare. This study identified the skin proteomes of Chinese fire-bellied newt Cynops orientalis by a proteomic method, and compared the results to the skin proteomes of Chinese giant salamander Andrias davidianus obtained previously. A total of 452 proteins were identified in the newt skin by MALDI-TOF/MS, and functional annotation results by DAVID analysis showed that special functions such as wound healing, immune response, defense and respiration, were significantly enriched. Comparison results showed that the two species had a great difference in the aspects of protein kinds and abundance, and the highly expressed proteins may tightly correlate with living conditions. Moreover, the newt skin might have stronger immunity, but weaker respiration than the giant salamander skin to adapt to various living environments. This research provides a molecular basis for further studies on amphibian skin function and adaptation.

Proteomic analysis of the skin of Chinese giant salamander (Andrias davidianus).

The Chinese giant salamander (Andrias davidianus), renowned as a living fossil, is the largest and longest-lived amphibian species in the world. Its skin has developed mucous gland which could secrete a large amount of mucus under the scraping and electric stimulation, and the molting is the degraded skin stratum corneum. Although several proteomic studies have focused on functional proteomes of mammalian and frog skin, the skin proteome of Chinese giant salamander has not yet been carefully studied. To establish the functional skin proteome of Chinese giant salamander, two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS) were applied to detect the composition and relative abundance of the proteins in the skin, mucus and molting. Our findings indicated that 249 proteins were identified in the skin, 155 proteins in the mucus, and 97 proteins in the molting. Furthermore, Gene Ontology (GO) analysis showed that these proteins participated in various physiological activities, including extracellular matrix organization, defense, immune response, wound healing, respiration, etc. In conclusion, the proteomic results provide new insight in the aspects of the proteomes in the skin, mucus and the molting of Chinese giant salamander. BIOLOGICAL SIGNIFICANCE: This was the first study to examine the protein expression abundance in the skin, mucus and molting of Chinese giant salamander by a proteomics approach. Meantime, the identification of a more global proteome in normal skin may provide a basis for characterizing and comparing the skin proteomes from other amphibian species.

Expression of G proteins in the olfactory receptor neurons of the newt Cynops pyrrhogaster: their unique projection into the olfactory bulbs.

We analyzed the expression of G protein α subunits and the axonal projection into the brain in the olfactory system of the semiaquatic newt Cynops pyrrhogaster by immunostaining with antibodies against Gαolf and Gαo , by in situ hybridization using probes for Gαolf , Gαo , and Gαi2 , and by neuronal tracing with DiI and DiA. The main olfactory epithelium (OE) consists of two parts, the ventral OE and dorsal OE. In the ventral OE, the Gαolf - and Gαo -expressing neurons are located in the apical and basal zone of the OE, respectively. This zonal expression was similar to that of the OE in the middle cavity of the fully aquatic toad Xenopus laevis. However, the Gαolf - and Gαo -expressing neurons in the newt ventral OE project their axons toward the main olfactory bulb (MOB) and the accessory olfactory bulb (AOB), respectively, whereas in Xenopus, the axons of both neurons project solely toward the MOB. In the dorsal OE of the newt, as in the principal cavity of Xenopus, the majority of the neurons express Gαolf and extend their axons into the MOB. In the vomeronasal organ (VNO), the neurons mostly express Gαo . These neurons and quite a few Gαolf -expressing neurons project their axons toward the AOB. This feature is similar to that in the terrestrial toad Bufo japonicus and is different from that in Xenopus, in which VNO neurons express solely Gαo , although their axons invariably project toward the AOB. We discuss the findings in the light of diversification and evolution of the vertebrate olfactory system.

Nuclear and mitochondrial multilocus phylogeny and survey of alkaloid content in true salamanders of the genus Salamandra (Salamandridae).

The genus Salamandra represents a clade of six species of Palearctic salamanders of either contrasted black-yellow, or uniformly black coloration, known to contain steroidal alkaloid toxins in high concentrations in their skin secretions. This study reconstructs the phylogeny of the genus Salamandra based on DNA sequences of segments of 10 mitochondrial and 13 nuclear genes from 31 individual samples representing all Salamandra species and most of the commonly recognized subspecies. The concatenated analysis of the complete dataset produced a fully resolved tree with most nodes strongly supported, suggesting that a clade composed of the Alpine salamander (S. atra) and the Corsican fire salamander (S. corsica) is the sister taxon to a clade containing the remaining species, among which S. algira and S. salamandra are sister species. Separate analyses of mitochondrial and nuclear data partitions disagreed regarding basal nodes and in the position of the root but concordantly recovered the S. atra/S. corsica as well as the S. salamandra/S. algira relationship. A species-tree analysis suggested almost simultaneous temporal splits between these pairs of species, which we hypothesize was caused by vicariance events after the Messinian salinity crisis (from late Miocene to early Pliocene). A survey of toxins with combined gas chromatography/mass spectroscopy confirmed the presence of samandarine and/or samandarone steroidal alkaloids in all species of Salamandra as well as in representatives of their sister group, Lyciasalamandra. Samandarone was also detected in lower concentrations in other salamandrids including Calotriton, Euproctus, Lissotriton, and Triturus, suggesting that the presence and possible biosynthesis of this alkaloid is plesiomorphic within the Salamandridae.

Variability of alkaloids in the skin secretion of the European fire salamander (Salamandra salamadra terrestris).

The two major alkaloids, samandarine and samandarone, were identified in the skin secretion of individual specimens from two populations of the European fire salamander (Salamandra salamandra terrestris) by gas chromatography/mass spectrometry. High intraspecific variability in the ratio of both alkaloids was observed, but also in individual specimens over a period of 4 months suggesting separate metabolic pathways of the compounds. Alkaloid synthesis appears to take place also in liver, testes and ovaries, whereas the larvae of the salamanders are entirely free of alkaloids.

Structure-function relationships in the integument of Salamandra salamandra during ontogenetic development.

Morphological, cytological and transport properties of the integument of Salamandra salamandra were investigated during natural ontogenetic development, from birth to adult. Three stages were operationally defined: I, larvae, from birth to metamorphosis; II, metamorphosis (judged externally by the colour change and loss of the gills); and III, post-metamorphosis to adult. Pieces of skin were fixed at various stages for immunocytochemical examinations, and the electrical properties were investigated on parallel pieces. Distinct cellular changes take place in the skin during metamorphosis, and lectin (PNA, WGA and ConA) binding indicates profound changes in glycoprotein composition of cell membranes, following metamorphosis. Band 3 and carbonic anhydrase I (CA I) were confined to mitochondria-rich (MR)-like cells, and were detected only in the larval stage. CA II on the other hand, was detected both in MR-like and in MR cells following metamorphosis. The electrical studies show that the skin becomes more tight (transepithelial resistance increases) upon metamorphosis, followed by manifestation of amiloride-sensitive short-circuit current (I(SC)) indicating that functional Na+ uptake has been acquired. The skin of metamorphosed adults had no finite transepithelial Cl- conductance, and band 3 was not detected in its MR cells. The functional properties of MR-like and MR cells remain to be established.

Ovarian steroid levels in Salamandra salamandra infraimmaculata during the reproductive cycle.

Gonadal steroid levels were determined in the ovary of Salamandra salamandra infraimmaculata during the reproductive cycle in populations from a xeric region in northern Israel. Varying proportions of previtellogenic and vitellogenic oocytes were present throughout the year, and mature oocytes were present in winter and spring. The numbers of mature oocytes were greater between December and April, after parturition. The levels of 17 beta-estradiol and testosterone rose during oocyte vitellogenesis and maturation. Levels of progesterone and 17 alpha-hydroxy progesterone appeared to be related to the level of vitellogenesis. Gravid females contained greater quantities of all four steroids than did nongravid females.

[Appearance and distribution of retinoic acid in the limb regeneration of Cynops orientalis].

Urodeles are the unique vertebrate which can regenerate their limbs in larvae and adult. The pattern formation of blastema cells in the limb regeneration is a central problem. Many evidence have been established to support the hypothesis that RA may be the morphogen in limb development. The limb development and regeneration are so similar morphogenetic processes that we suspect RA may also play an important role in the latter process. In order to give an answer to this question, we tested the limbs in different stages of regeneration with the specific antibody prepared against RA. The results indicated that RA appeared on day six and arrived its peak on day eight after amputation. During this time, the dedifferentiation of the stump is proceeding to the strongest degree and the distribution of RA showed a posterior-anterior gradient. If the stump is treated with RA for 24 hours at this stage, proximal distal duplication of the regenerate can be induced. These results and the fact that the close connection between apical epidermal cap and mesenchymal cells and abundant RA outside the epidermal cells suggest that RA may play an important role in the establishment of positional information of the blastema.

Physicochemical characterization and tissue distribution of esterases in two salamandridae species (Mertensiella luschani and Salamandra salamandra).

1. Tissue- and species-specificity of the electrophoretic patterns of the multiple molecular forms of esterases were observed in the urodele amphibians Mertensiella luschani luschani, M.l. helverseni and Salamandra salamandra. All esterases--distributed into two electrophoretic mobility areas in gonads, muscles and brain and into four areas in liver, stomach and intestine--were characterized as carboxylesterases. 2. M. l. luschani and S. salamandra liver esterases were electrofocused into nine and eleven major bands with pIs ranging from 4.60 to 5.65 and from 4.40 to 6.20, respectively. 3. Two size groups of esterases were observed in liver extracts of the above three subspecies by Sephadex G-200 gel filtration. The mean values of their apparent molecular weights were 70,000 and 230,000 respectively.

Light and electron microscopic study on complex carbohydrates in the testis of Salamandra salamandra L. (Amphibia, Urodela).

The distribution of complex carbohydrates was studied in the testis of the European fire salamander, Salamandra salamandra, by light- and electron-microscopic methods. The basal laminae and fibrous structures in the connective tissue between the lobules are PAS-positive. After alcianblue staining (at pH = 2.8), acid mucopolysaccharides could be demonstrated in steroid hormone-producing cells in the interstitial tissue between lobules containing spermatids, spermatozoa, and lobules after spermiation, as well as in most of the Sertoli cells in lobules after spermiation. In all spermatogenic stages from secondary spermatocytes to mature sperms, dictyosome-like structures and flat vesicles showed a distinct contrast enhancement, as did parts of the acrosome after treatment with the phosphotungstic acid-chromic acid method for electron microscopy.

Seasonal variations of androgens, estrogens, and progesterone in the different lobules of the testis and in the plasma of Salamandra salamandra.

Progesterone, 4-androstenedione, testosterone, dihydrotestosterone, 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-diol), 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol), estrone, and estradiol levels were determined by radioimmunoassay in the different lobules of the testis of Salamandra salamandra throughout the year according to the seasonal cycle. 3 beta-diol levels were not detectable. High levels of steroids were found in the grandular tissue (enlarged pericystic cells after spermiation) and large variations were showed for progesterone, 4-androstenedione, testosterone, 3 alpha-diol, and estrone. In the mature lobule (formed by cysts with mature spermatozoa), only testosterone showed seasonal variations and in the immature lobule (with early stages of meiosis), 3 alpha-diol showed fluctuations. The major estrogen found in the testis of Salamandra was estrone; estradiol stayed at a low level throughout the cycle. The steroids fluctuation seems to be related to the histological evolution of the testis throughout the cycle. The present data were the first on steroid seasonal variations in the testis of an urodele.

Hepatic arginase activity in intra- and extrauterine larvae of the ovoviviparous salamander. Salamandra salamandra (L.) (Amphibia, Urodela).

The hepatic arginase activity of Salamandra salamandra was determined at three different stages of intra- and extrauterine larval development and at fully metamorphosed juveniles. The highest enzymatic activity was found in intrauterine larvae in November, the lowest in intrauterine larvae in June of the following year. These data can be correlated with the ureotelism of intrauterine larvae previously described and are discussed with respect to the metabolism of larval and juvenile fire salamanders.

Ultrahistochemical and autoradiographic evidence of epithelial transport in the uterus of the ovoviviparous salamander, Salamandra salamandra (L.) (Amphibia, Urodela).

The uterine epithelium of pregnant females of the terrestrial ovoviviparous Salamandra salamandra is characterized by a considerable enlargement of its basolateral surface. Chloride and cations (among others sodium), preferentially within the intercellular spaces, can be demonstrated ultrahistochemically. There is indirect evidence of Na+ --K+ -ATPase activity along the basolateral plasma membranes of the epithelial cells using the Sr-technique for demonstration of a K+ -NPPase and 3H-ouabain autoradiography. Preliminary measurements reveal a potential difference across the uterine wall of 15--25 mV, the lumenal (mucosal) surface being negative with respect to the coelomic (serosal) surface, and a short circuit current of 200--300 microA. The possible electrogenic ion transport is ouabain-sensitive. The results are in agreement with the model of a "forward" transporting, i.e. absorptive epithelium. An active transport of solute out of the uterine lumen across the epithelium to the subjacent connective tissue and the blood vessels may be involved in the regulation of an intrauterine milieu appropriate for the development of the offspring.

Ca and P concentrations of the urodele tooth as revealed by electron microprobe analysis.

The electron microprobe analysis has been used to determine the concentrations of Ca and P in different regions of functional teeth in adult Salamandra salamandra (Amphibia, Urodela). Ca and P levels were found to be more elevated in the dentine shaft below the apex than in its basal part and in the pedicel. The concentrations are similar to those measured in teeth of higher vertebrates.

Gut hormones in Salamandra salamandra. An immunocytochemical and electron microscopic investigation.

Histological, cytochemical and immunocytochemical methods were used in light and electron microscopical studies to demonstrate the presence of a neuroendocrine system in the gut of the urodele, Salamandra salamandra. Cytochemical stains capable of detecting peptide-producing endocrine cells demonstrate cells reacting with Masson's silver (argentaffin) method, Grimelius' argyrophil silver method, masked metachromasia method and the lead haematoxylin stain. Using antisera raised to a variety of mammalian gut peptides, cells containing bombesin-, gastrin-, somatostatin-, substance P- and glucagon-like immunoreactivity were indentified; vasoactive intestinal polypeptide- and substance P-like immunoreactivities were found in nerve fibres in the submucous and myenteric plexus. No immunoreactivity was detected from motilin, gastric inhibitory polypeptide, cholecystokinin or secretin. The ultrastructure of the immunoreactive cells and nerves was revealed by the semithin/thin method. All the cells indentified contained numerous electrondense secretory granules, which varied in their characteristic morphological structure from one cell type to another. The evidence collected in this study indicates that a complex neuroendocrine system regulating gut function is present in this amphibian and may have developed prior to the emergence of the phylum.

Electron microscopic localization of 3beta-hydroxysteroid dehydrogenase and NADH-ferricyanide reductase activities in amphibian interrenal cells.

3beta-hydroxysteroid dehydrogenase and NADH-ferricyanide reductase activities were localized at the ultrastructural level in amphibian interrenal (adrenocortical) cells previously fixed in a mixture of formaldehyde and glutaraldehyde. Potassium ferricyanide was used as an electron acceptor. Copper ferrocyanide deposits resulting from 3beta-HSD activity were seen in close association with the external faces of the membranes of the smooth endoplasmic reticulum. Very rare grains of precipitate appeared in mitochondrial cristae. The addition of phenazine methosulfate to the incubation medium had no effect on these localizations. The interrenal cells showed also a strong NADH-ferricyanide reductase activity. The copper ferrocyanide grains were abundant in the mitochondrial cristae and in the hyaloplasm, where they were not preferentially associated with the smooth endoplasmic reticulum.

Electron microscopic localization of Mg2+ -dependent adenosine triphosphatase activity in the amphibian pancreas (Salamandra salamandra L. and Rana esculenta L.).

The ultrastructural distribution of Mg2+ -dependent adenosine triphosphatase (ATPase) activity has been investigated in the salamander and frog pancreas by using glutaraldehyde fixations and a modified Wachstein-Meisel reaction medium. In both species the reaction product (lead phosphate) was found associated with the plasma membrane external side of all islet cell types (B-, A- and D-cells) and of acinar and ductular/centro-acinar cells. Except the apical pole of salamander acinar and centro-acinar cells, usually devoid of reaction, no preferential distribution of enzyme activity depending on endocrine or exocrine cell aspects could be observed. Other specific enzyme localizations included the mitochondria matrices, nucleoles, condensed nuclear chromatin, periaxolemmal spaces in nerve bundles and sometimes the cleft of neuro-glandular junctions. The occurrence of reaction deposits in connective tissue, in the cytoplasm of both islet and exocrine cells and in the nerve fiber axoplasm was considered as a possible diffusion artifact. The reaction intensity, but not its distribution, varied sensibly with the incubation period. 2-iodoacetamide and p-chlormercuribenzoic acid decreased the amount of reaction deposits at the level of all reactive sites and especially in mitochondria. The specificity of Mg2+ -ATPase demonstration in this paper is analysed taking into account several inherent shortcomings of the Wachstein-Meisel incubation medium and of the fixative. The different enzyme localizations, as well as their functional significances are discussed in relation with the findings of other authors.