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PubMed1976

Quantitative and comparative ultrastructure of the vertebrate cornea. I. Urodele Amphibia.

The cornea of the urodele amphibian Triturus c. cristatus was studied ultrastructurally in order to provide the basis for a comparison among corneas throughout the vertebrate phylum. The cornea of this salamander consists of relatively thick epithelium and basement membrane and thin Descemet's membrane, unlike the mammalian corneas. The outermost epithelial cells contain Ruthenium Red stainable extracellular filaments and intracellular vesicles which are thought to play a role in the process of lubricating the corneal surface. Occluding junctions have been observed in the apical region of the superficial epithelial cells and are considered as barriers to the intercellular passage of material. A thin substantia propria (stroma) consists of about 40 collagenous highly organized lamellae. The thicknesses of the basement membrane, Descemet's membrane and the epithelium are believed to represent the primitive situation in the process of corneal evolution.

Animals↗

PubMed1985

Embryonic induction and cation concentrations in amphibian embryos.

Explanted ectoderm from early gastrulae of Triturus alpestris was treated with the Na-K ionophore gramicidin (10(-9) to 10(-5) M) and the Ca-ionophore A 23187 (10(-7) to 10(-5) M). The ectoderm developed almost exclusively to atypical epidermis as in the control explants. When the ectoderm was treated with ouabain (10(-4) M), intracellular Na+ increased about 4.4-fold and K+ was reduced by half. Mesenchyme cells in small number differentiated in about 40% of the ouabain-treated explants. The time course of total Na+ and K+ ion concentrations was measured over a period of 72 h in ectoderm of T. alpestris after induction with vegetalizing factor and in control explants. In the first 15 h after explantation, no significant differences between control and induced explants were found. Thereafter, the steady state concentration of K+ decreased in the induced explants, whereas the steady-state concentration of Na+ slightly increased. The membrane resting potential recorded intracellularly of ectoderm sandwiches from early gastrula stages was found to be -41.3 mV in control and -59.3 mV in induced explants. From the specific conductances and permeabilities of non-induced and induced cells it is concluded that the induction process leads to a differentiation of the cell membrane, which acquires the characteristics of ionic selectivity. Ectoderm from Ambystoma mexicanum forms neural or neuroid tissue, mesenchyme and melanophores after explantation in salt solution in up to 50% of the explants without any additions. Isolated Ambystoma ectoderm is therefore not suitable for test experiments.

Ambystoma↗

PubMed1985

Effect of concanavalin A and vegetalizing factor on the outer and inner ectoderm layers of early gastrulae of Xenopus laevis after treatment with cytochalasin B.

Neural (archencephalic) structures have been evoked in the competent ectoderm (consisting of both ectodermal layers) of Xenopus laevis by treatment with Concanavalin A (Con A), which probably acts on the plasma membrane. The size of the neural structures is increased when the ectoderm is incubated in Cytochalasin B prior to the Con A treatment. The results indicate that Cytochalasin B could have an influence on the binding of Con A to receptors on the plasma membrane. On the other hand, Cytochalasin B seems to have an inhibitory effect on the action of the vegetalizing factor, which could be correlated with the decline of endocytotic processes and internalization. In further series, it could be shown that the isolated superficial ectoderm, in contrast to the inner ectoderm layer, does not react to Con A treatment with the differentiation of neural structures. Studies with FITC-Con A indicate that the marker binds less to the outer ectoderm than to the inner ectoderm layer. However, by xenoplastic combinations of the outer ectoderm layer of X. laevis as reacting tissue and chordamesoderm of Triturus vulgaris as inducer, it could be demonstrated that the superficial layer, which is normogenesis does not come into contact with the inducing chordamesoderm but forms the ependymal part of the brain only, is also able to form archencephalic brain structures under in vitro conditions.

Animals↗

PubMed1978

Changes of nucleosome frequency in nucleolar and non-nucleolar chromatin as a function of transcription: an electron microscopic study.

The morphology of nucleolar and non-nucleolar (lampbrush chromosome loops) chromatin was studied in the electron microscope during states of reduced transcriptional activity in amphibian oocytes (Xenopus laevis, Triturus alpestris, T. cristatus). Reduced transcriptional activity was observed in maturing stages of oocyte development and after treatment with an inhibitor, actinomycin D. Strands of nucleolar chromatin appear smooth and thin, and contain only few, if any, nucleosomal particles in the transcribed units. This is true whether they are densely or only sparsely covered with lateral ribonucleoprotein fibrils. This smooth and non-nucleosomal character is also predominant in the interspersed, apparently nontranscribed rDNA spacer regions. During inactivation, however, nucleolar chromatin frequently and progressively assumes a beaded appearance in extended fibril-free--that is, apparently nontranscribed--regions. In either full-grown oocytes or late after drug treatment, most of the nucleolar chromatin is no longer smooth and thin, but rather shows a beaded configuration indistinguishable from inactive non-nucleolar chromatin. In many chromatin strands, transitions of fibril-associated regions of smooth character into beaded regions without lateral fibrils are seen. Similarly, in the non-nucleolar chromatin of the retracting lampbrush chromosome loops, reduced transcriptional activity is correlated with a change from smooth to beaded morphology. Here, however, beaded regions are also commonly found interspersed between the more or less distant bases of the lateral fibrils, the putative transcriptional complexes. In both sorts of chromatin, detergents (in particular Sarkosyl) that remove most of the chromatin proteins including histones from the DNA axis but leave the RNA polymerases of the transcriptional complexes attached were used to discriminate between polymerases and nucleosomal particles. The results suggest that nucleosomes are absent in heavily transcribed chromatin regions but are reformed after inactivation. In contrast to the findings with inactivated nucleolar genes, in lampbrush chromosome loops the beaded nucleosomal configuration appears to be assumed also in regions within transcriptional units that, perhaps temporarily, are not involved in transcription.

Animals↗

PubMed1988

Evaluation of the carcinogenic risk of the phenoxyherbicide MCPA to an urodele amphibian.

To evaluate the carcinogenic potential of the phenoxyherbicide 2-methyl-4-chlorophenoxy-acetic acid (MCPA) in the crested newt, Triturus cristatus carnifex, a long-term study has been carried out exposing the animals by the percutaneous route. Two hundred adult newts were divided into one control and three experimental groups of 20 females and 30 males each. The control group was kept in tap water and the experimental groups were kept for 4 days a week in an aqueous solution of Agroxone 3, a commercial formulation of MCPA, at concentrations equivalent to 100, 200, and 400 ppm of the active ingredient. Treatment was continued for 1 year, after which all the animals were kept under observation for approximately another year. Surviving female newts were killed 22-24 months after the beginning of experimentation, whereas the male newts were killed after 28 months, at the end of 18 weeks of exposure to the tumour promoter 12-O-tetradecanoylphorbol-13-acetate. Under experimental conditions, there was no carcinogenic activity of MCPA. Putative preneoplastic nodules of the liver and tumor-like lesions of the lower jaw were occasionally observed among the animals that survived more than 22 months after the beginning of experimentation. However, no significant differences in frequency between control and experimental groups were found.

2-Methyl-4-chlorophenoxyacetic Acid↗

PubMed1985

Mobilization of ferritin iron in erythroblasts by chelating agents.

Intracellular ferritin in newt (Triturus cristatus) erythroblasts was accessible to the chelating effects of EDTA and pyridoxal phosphate. EDTA (0.5-1 mM) promoted release of radioactive iron from ferritin of pulse-labelled erythroblasts during chase incubation, but its continuous presence was not necessary for ferritin iron mobilization. Brief exposure to EDTA was sufficient to release 60-70% of ferritin 59Fe content during ensuing chase in EDTA-free medium. EDTA also suppressed cellular iron uptake and utilization for heme synthesis, but these activities were restored upon its removal. Pyridoxal-5'-phosphate (0.5-5 mM) also stimulated loss of radioactive iron from ferritin; however, ferritin iron release by pyridoxal phosphate required its continued presence. Unlike EDTA, pyridoxal phosphate did not interfere with iron uptake or its utilization for heme synthesis. Chelator-mobilized ferritin iron accumulated initially in the hemolysate as a low-molecular-weight component and appeared to be eventually released into the medium. No radioactive ferritin was found in the medium of chelator-treated cells, indicating that secretion or loss of ferritin was not responsible for decreasing cellular ferritin 59Fe content. Moreover, there was no transfer of radioactive iron between the low-molecular-weight component released into the medium and plasma transferrin. These results indicate that chelator-released ferritin iron is not available for cellular utilization in heme synthesis and that ferritin iron released by this process is not an alternative or complementary iron source for heme synthesis. Correlation of these data with effects of succinylacetone inhibition of heme synthesis and with previous studies indicates that the main role of erythroid cell ferritin is absorption and storage of excess iron not used for heme synthesis.

Animals↗

PubMed1986

Cobalt-dependent stimulation of sodium transport in the amphibian skin and nephron.

One to ten millimolar CoCl2, when applied to the outer surface of the apical membrane of the frog skin (Rana temporaria), reversibly increased the potential differences and short-circuit current. These observations suggest that Co2+ may control the gating system of the sodium channel. In the kidney of the newt (Triturus vulgaris), proximal reabsorption is increased under the influence of 0.5 mM CoCl2 injected into the lumen. When CoCl2 (0.5 mM) was injected into the lumen of the distal tubule of the newt kidney, Na+ and Cl- reabsorption was stimulated simultaneously Ca2+ transport was inhibited. The data obtained suggested that Co2+ may affect the state of the sodium and calcium channels of nonexcitable membranes of amphibia, and thus may be involved in the regulation of the function of the renal tubules.

Animals↗

PubMed1994

Multiple sources of the pituitary pars intermedia innervation in amphibians: a DiI retrograde tract-tracing study.

Afferent projections to the pituitary pars intermedia were studied using the DiI tract-tracing technique in two amphibian species, the urodelan Triturus carnifex, and the anuran Rana esculenta. After DiI crystal application into the pituitary intermediate lobe, in both species cells were retrogradely labeled in the preoptic nucleus, in the supra- and retro-chiasmatic hypothalamus and in the brainstem (especially in the area indicated as locus coeruleus). The findings are discussed in relation to data on the neurochemical nature of the innervation of the pars intermedia in amphibians.

Animals↗

PubMed1984

Ferritin is not a required intermediate for iron utilization in heme synthesis.

Pulse-chase analysis of newt (Triturus cristatus) erythroblasts has shown that ferritin is not a primary source of iron for heme synthesis. During chase incubation with and without non-radioactive plasma iron in the medium, no transfer of 59Fe from ferritin to hemoglobin was detected although the integrity of heme synthesis was maintained. In puromycin-inhibited cells where iron uptake was drastically curtailed, heme synthesis continued to occur, though at reduced levels; incorporation of 59Fe from the plasma appeared initially in heme and hemoglobin without any prior labelling of ferritin. These results indicate that ferritin is neither an obligatory iron intermediate in heme synthesis nor a cytosolic transport molecule involved in mobilization of iron from the transferrin-receptor complex. The most likely role for erythroid ferritin is storage of excess iron.

Animals↗

PubMed1978

Distribution and homologies of subunits in the LDH isoenzyme pattern of urodeles.

1. The subunit distribution and homologies of LDH isoenzymes are investigated in the species Triturus vulgaris, cristatus, alpestris and helveticus, and Ambystoma mexicanum by means of immunoprecipitation and starch gel electrophoresis. 2. Fresh tissue extracts contain the following patterns: (a) Trit. vulgaris--Two isoenzymes: the M4 and H4 tetramers. No hybrid formation is observed between H and M subunits. (b) Trit. cristatus--Two isoenzymes: the M4 and H4 tetramers. Occasional hybrid formation between H and M subunits takes place. (c) Trit. alpestris--Six isoenzymes: the M4, H4, H3H', H2H'2, HH'3 and H'4 tetramers. No hybrid formation between the M and the H and H' subunits is detected. (d) Trit. helveticus--Six isoenzymes: the M4, H4, H3H', H2H'2, HH'3, and H'4 tetramers; the H' subunit is more positively charged than the M subunit, which leads to pattern reversal in heart and skeletal muscle tissues. No hybrid formation between the M and the H and H' subunits is observed. (e) Ambyst. mex.--Eleven isoenzymes in heart, eye and brain, six isoenzymes in all other tissues tested. The presence of two M subunits, which form hybrids with the H subunit in a restricted way, is suggested. 3. In tissue extracts of the tested species the tendency of all present LDH subunits to form hybrids with each other without restriction is increased after prolonged storage at 2 degrees C. 4. The most acidic of the major isoenzymes (LDH1) in Trit. vulgaris, cristatus and helveticus tends to split into a series of sub-bands which migrate faster to the anode than the original main band.

Ambystoma↗

PubMed1983

Lipovitellin-phosvitin crystals with orthorhombic features: thin-section electron microscopy, gel electrophoresis, and microanalysis in teleost and amphibian yolk platelets and a comparison with other vertebrates.

Yolk-platelet crystals in the teleosts Pelvicachromis pulcher and Noemacheilus barbatulus and the amphibians Xenopus laevis, Rana temporaria, R. esculenta, and Triturus sp. have been studied by electron diffraction and imaging using a standardized processing (glutaraldehyde-osmium tetroxide fixation, glutaraldehyde-urea embedding, thin-section staining), by X-ray microanalysis, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of their constituents. The crystal lattice is orthorhombic having--following standardized processing--in three amphibians a = 9.0 nm, b = 17.6 nm, c = 19.2 nm, and in the two teleosts a = 8.9 nm, b = 17.6 nm, c = 20.0 nm (averages). These values are very close to X-ray data from wet crystals (Xenopus laevis). Crystal images in the three axial projections point to the presence of space group P212121 (or an approximation of it since the lipovitellin dimers cannot be fully equivalent in some cases), to differences between the phosvitins of the two teleosts, and to a highly unusual stain exclusion from large crystal constituents interpreted as representing lipovitellin dimers. Microanalysis in ultrathin cryosections and other preparations revealed K and Cl to be the prominent ions in the crystals (and in the superficial layer of the platelet). Gel electrophoresis (including data of cyclostomes) showed considerable molecular variations despite a closely similar crystal architecture, emphasizing a physiological significance of the architecture, which may have remained conserved for nearly 400 million years according to paleontologic views.

Amphibians↗

PubMed1979

The identification of globin messenger ribonucleic acid in newt erythropoietic cells.

Polyadenylated [poly(A)+]-RNA isolated from newt (Triturus cristatus) erythropoietic cells contained two main species sedimenting at 9S and 25S, and minor amounts of a 15-20S component. The 9S poly(A)+-RNA fraction induced synthesis of newt haemoglobin and globins in frog oocytes and in an mRNA-dependent rabbit reticulocyte lysate, confirming its identity as newt globin mRNA. Translation of 9S globin mRNA in reticulocyte lysate was concentration-dependent, the patterns of globin synthesis suggesting both preferential utilization and unequal amounts of the different globin mRNA subspecies. Globin mRNA activity was also evident in the 25S poly(A)+-RNA fraction whose localization in polyribosomes excluded its function as a nuclear globin mRNA precursor. Denaturation in formamide and estimation of its relative methyl content indicated that the 25S poly(A)+-RNA fraction contained equimolar amounts of 9S globin mRNA and 26S rRNA. Translation of the 25S fraction in reticulocyte lysate was less efficient than that of comparable amounts of 9S globin mRNA and induced a pattern of globin synthesis similar to that obtained with subsaturating amounts of 9S mRNA. The 25S mRNA-rRNA complex was considered to be a non-physiological aggregate generated by extraction of RNA in the presence of buffers of moderate to high ionic strength.

Animals↗

PubMed1992

A newt ribozyme: a catalytic activity in search of a function.

We analyzed the cleavage properties and the transcription regulation of the newt (Triturus vulgaris meridionalis) self-cleaving RNA. In vitro self-cleavage of model oligoribonucleotides occurs within a double hammerhead structure. In addition, an entire ribozyme molecule, as well as its catalytic domain, "trans-cleaves" in vitro appropriate oligoribonucleotide substrates. Signals encoded within the ribozyme DNA sequences regulate the ribozyme transcription, which is RNA polymerase II dependent. Finally, the deduced secondary structure of the self-cleaving RNA appears to be conserved in evolutionarily distant newt species. These features suggest that the newt ribozyme could play some role in the cell, possibly related to its cleavage properties.

Animals↗

PubMed1993

Histone H4 acetylation and transcription in amphibian chromatin.

Lampbrush chromosomes from oocytes of the amphibian Triturus cristatus have been used to examine the role of histone acetylation in transcription by indirect immunofluorescence with antisera to H4 acetylated at specific lysine residues. Electrophoresis on acid-urea-Triton gels and Western blotting have confirmed the specificity of these antisera and defined the order in which particular lysine residues are acetylated in amphibian cells. As in mammals, lysine 16 is acetylated first, followed by 8 and/or 12 and then 5. With lampbrush chromosomes from immature (previtellogenic) oocytes, antisera to H4 acetylated at lysines 8, 12, and 16 labeled fluorescent foci at the bases of transcription loops. Antisera to H4 acetylated at lysine 5 labeled weakly (i.e., the tri- and tetraacetylated isoforms must be rare). Loops showed weak labeling of the chromatin axis but intense fluorescence at particular points, which probably represent incompletely decondensed chromatin. The RNP matrix of loops, including the RNP-rich sphere bodies and the dense matrix of "marker" loops, was not labeled. Treatment of immature oocytes with butyrate for 12 h to inhibit histone deacetylation did not affect immunolabeling, suggesting that turnover of H4 acetates is slow. In contrast, in chromosomes from mature oocytes, in which loops have retracted and transcription is low, butyrate caused an increase in labeling with all antisera, followed by the appearance of vestigial loops, weakly labeled, but with regions of intense fluorescence. These loops contain RNP and are presumably transcriptionally active. We conclude that H4 acetates turn over more rapidly in mature than immature oocytes and that histone hyperacetylation precedes, and possibly induces, loop formation and transcriptional activation.

Acetylation↗

PubMed1967

Octagonal nuclear pores.

Negative staining of isolated nuclear envelopes by phosphotungstate shows that the nuclear pores are octagonal rather than circular. Pores of the same shape and approximately the same width, 663 +/- 5 A, were demonstrated in the newt, Triturus, the frog, Rana, and the starfish, Henricia. The outer and inner diameters of the annulus associated with each pore are respectively greater and less than the width of the pore itself. For this reason surface views of the envelope, unless negatively stained, fail to show the true dimensions of the pores.

Animals↗

PubMed1971

Electrical coupling between fat cells in newt fat body and mouse brown fat.

White fat from the newt, Triturus pyrrhogaster, fat body, and brown fat from the interscapular fat pad of newborn mice have been tested for the presence of low-resistance intercellular junctions. 42 pairs of amphibian fat cells and 15 pairs of mammalian brown fat cells were found to be "electrically coupled." In most of these cases intracellular deposition of a dye, Niagara Sky Blue: 6B, was used to supplement and confirm direct observations of impalements. Coupling was often difficult to find in both preparations, but the mechanical disturbance of the tissue during the preparative procedures may have uncoupled many cells. The fact that, in both types of fat, coupling was observed between cells separated by one or more other cells suggests that coupling may be more widespread in vivo. Electron microscopy (provided by Dr. J. -P. Revel and Mrs. K. Wolken) of the brown fat revealed frequent intercellular junctions resembling "gap junctions" but possibly lacking the substructure usually visible with colloidal lanthanum infiltration. The results are discussed in relation to current ideas about the exchange of regulatory molecules via low-resistance junctions and about the control of brown fat by hormones and nerves.

Adipose Tissue↗

PubMed1974

Wave of stiffness propagating along the surface of the newt egg during cleavage.

In the eggs of the newt, Cynops (Triturus) pyrrhogaster, change in stiffness of the cortex was measured in various regions at the time of the cleavage. Measurements were performed by Mitchison and Swann's cell elastimeter method with a modification, in which two fine pipettes were attached to the surface of one egg at the same time, in order to compare the rigidity of two regions. The stiffness of the cortex changed very little before the start of the first cleavage. However, just before the appearance of the first cleavage furrow, the stiffness increased rapidly at the animal pole region, which later returned to the former level. As the cleavage furrow progressed, a wave of high stiffness travelled meridionally as a belt along the surface from the animal pole region toward the vegetal region. At second cleavage, the cycle of change in stiffness was repeated.

Animals↗

PubMed1976

Regulation of transcription of genes of ribosomal rna during amphibian oogenesis. A biochemical and morphological study.

Natural changes in the transcription of rRNA genes were studied in nucleoli from three oogenic stages of the newt Triturus alpestris with electron microscope, auto-radiographic, and biochemical techniques. From determinations of the uridine triphosphate pool sizes and [3H]uridine uptake, phosphorylation, and incorporation into 28S and 18S rRNAs in vivo it was estimated that the rate of rRNA synthesis was about 0.01% in previtellogenic oocytes and 13% in mature oocytes when compared to midvitellogenesis. Spread preparations of nucleoli showed significant morphological changes in the transcriptional complexes. The total number of lateral fibrils, i.e., ribonucleoproteins containing the nascent rRNA precursor, were drastically decreased in stages of reduced synthetic activity. This indicates that rRNA synthesis is regulated primarily at the level of transcription. The resulting patterns of fibril coverage of the nucleolar chromatin axes revealed a marked heterogeneity. On the same nucleolar axis occurred matrix units that were completely devoid of lateral fibrils, matrix units that were almost fully covered with lateral fibrils, and various forms of matrix units with a range of lateral fibril densities intermediate between the two extremes. Granular particles that were tentatively identified as RNA polymerase molecules were not restricted to the transciptional complexes. They were observed, although less regularly and separated by greater distances, in untranscribed spacer regions as well as in untranscribed gene intercepts. The results show that the pattern of transcriptional control of rRNA genes differs widely in different genes, even in the same genetic unit.

Animals↗