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Isolation of a vegetalizing inducing factor after extraction with acid ethanol. Concentration-dependent inducing capacity of the factor.

A vegetalizing factor has been isolated from chicken embryos by an improved method. The factor is extracted with acid/ethanol and finally purified by four consecutive steps of reversed phase HPLC. The molecular mass is about 25 kDa. The factor dissociates after reduction with dithiothreitol into two subunits of about 13 kDa. The factor was tested on Triturus alpestris by the implantation method, and on isolated ectoderm of Xenopus laevis in solution. The factor induces as the crude fractions all types of mesodermal tissues dependent on the concentration of the factor.

Acids↗

The vegetalizing factor from chicken embryos: its EDF (activin A)-like activity.

The erythroid differentiation capacity of the HPLC-purified mesoderm- and endoderm-inducing vegetalizing factor from chicken embryos and of recombinant erythroid differentiation factor (EDF = activin A), an evolutionary highly conserved member of the TGF-beta protein superfamily have been compared. Both factors stimulate the synthesis of hemoglobin in erythroleukemia cells in the same concentration range. The EDF-activity of the mesoderm-inducing HPLC-fractions is inhibited by follistatin, an EDF-binding protein. The factor induces in ectoderm of Triturus taeniatus all kinds of mesodermal organs. The wide spectrum of organs is very likely to be induced by secondary interactions. At higher concentration (15 ng/ml), notochord- and endoderm-like tissues are induced in a high percentage.

Activins↗

The toxicity of 2,4-dichlorophenoxyacetic acid to the adult crested newt.

The toxicity of the herbicide Agroxone 5, a commercial formulation of the iso-octyl ester of 2,4-dichlorophenoxyacetic acid (2,4-D), in the adult crested newt (Triturus cristatus carnifex) was tested after percutaneous exposure. The compound was lethal to all the animals within 72 hr at concentrations of 100, 125, and 150 ppm. Males appeared to be more susceptible than females to the toxic effects of 2,4-D, since the LT50 values at 75 ppm were 102 and 132 hr for male and female newts, respectively. At 50 ppm, all the males died within 31 days of exposure, whereas the females were still alive at the end of the 3-month experiment. Only one male died at 25 ppm after 21 days. Vacuolar degeneration of liver parenchyma and necrosis of kidney tubules were found at lethal concentrations of 2,4-D. The severity of the lesions was related to the duration of exposure more than to the concentration of the herbicide.

2,4-Dichlorophenoxyacetic Acid↗

Toxicity of 2-methyl-4-chlorophenoxyacetic acid to the adult crested newt.

The toxicity of the herbicide Agroxone 3, a commercial formulation of the sodium salt of 2-methyl-4-chlorophenoxyacetic acid (MCPA), in the adult newt (Triturus cristatus carnifex) was tested after percutaneous exposure. At the concentration of 3200 ppm, the LT50 values were 17 and 21 hr for male and female newts. At 1600 ppm, the LT50 values were 35 and 45.5 hr. At 800 ppm, most of the animals were still alive after the 3 months of experimentation. There were no deaths at 400 and 200 ppm. Severe neuromuscular disorders were observed in animals exposed to lethal concentrations.

2-Methyl-4-chlorophenoxyacetic Acid↗

Lectin binding patterns in amphibian skin epithelium.

Seven lectins (PNA, DBA, WGA, UEA-I, RCA, SBA, Con A) were used to localize glycoconjugates in the skin of 10 species of Amphibia, 7 anurans (Bufo marinus, Bufo bufo, Rana ridibunda, Rana pipiens, Hyla arborea, Pelobates syriacus and Xenopus laevis) and 3 urodeles (Salamandra salamandra, Triturus vulgaris and Ambystoma mexicanum). It was found that every lectin has a specific binding pattern in the skin of each species. No common pattern could be established, either among frogs or toads, nor for a particular lectin. Each lectin bound specifically and selectively to a particular epithelial component, which differed from one species to the other. A number of lectins showed selective binding to mitochondria-rich cells, but, again, a pattern in positivity could not be found. It is concluded that lectin histochemistry does correlate with cellular function. Our data can be applied in studies of epithelium and skin development, and of changes that occur during adaptation to the environment by amphibian species.

Animals↗

Lithium secretion in kidneys of amphibians and reptiles under hydrated conditions.

Renal lithium transport was studied at different hydration levels in five species of anuran amphibians (Bufo bufo, B. danatensis, B. viridis, Rana ridibunda, and R. temporaria), two species of urodeles (Triturus vulgaris and T. cristatus) and four species of reptiles (lizards Eremias multiocellata, Lacerta vivipara, Trapelus sanguinolentus, and Teratoscincus scincus). Under dehydration conditions, Li+ was reabsorbed in the kidneys of amphibians ans reptiles, but to a lesser degree than in mammalian kidneys: the ratio of lithium clearance (CLi) to glomerular filtration rate (GFR)--fractional lithium excretion--in dehydrated animals was in the range 0.5-0.8. The transition to the hydrated state resulted in a cessation of net renal lithium reabsorption. Under condition of high hydration, all the animals studied, except for urodeles, showed net renal secretion of Li+, i.e., CLi exceeded GFR. The ratio CLi/GFR was 1.2-1.3 in hydrated anurans and 1.7-2.3 in hydrated lizards. In urodeles, this ratio was approximately unity. It is suggested that renal lithium secretion in hydrated amphibians and reptiles reflects fluid secretion in the proximal tubule, which is additional to the glomerular filtration mechanism of fluid delivery to nephron under water loading.

Amphibians↗

Satellite DNA is transcribed on lampbrush chromosomes.

During the lampbrush stage of oogenesis there is widespread transcription, and it has been estimated that the total amount of DNA transcribed may be an order of magnitude greater than that required to produce the necessary functional RNA for the oocyte. We therefore considered it likely that some of the transcribed sequences have little, if any, translational significance, and may include both middle repetitive and highly repeated, or satellite, sequences. Satellite DNA is generally defined as rapidly reannealing DNA which has a short basic sequence that is repeated millions of times in the genome, usually in tandem arrays. The short repeated length, coupled with the organisation of satellite sequences in high order molecular weight tandem arrays in heterochromatic regions, have been put forward as reasons for supposing that this type of DNA is not normally transcribed. We report here that we have looked for and found evidence of transcription of satellite DNA on lampbrush loops in oocytes of the crested newt, Triturus cristatus carnifex.

Animals↗

Presence of angiotensin converting enzyme (ACE) activity in serum of amphibian: comparison with ACE activity of mammalian serum.

The occurrence of angiotensin converting enzyme (EC 3.4.15.1; ACE) was demonstrated for the first time in serum of newt (Triturus carnifex) and frog (Rana esculenta). The enzymatic activity was evidenced following hydrolysis of N-[3-(2-furyl) acryloyl]L-phenylalanyl glycyl glycine (FAPGG), a synthetic substrate of ACE. The serum enzyme liberated N-[3-(2-furyl) acryloyl]L-phenylalanine (FAP) from FAPGG. The properties of the amphibian serum enzymes were compared with those of swine. The amphibian serum FAPGG hydrolysing activities were inhibited by typical ACE inhibitors, captopril and lisinopril. The optimum of pH was 8.3 at 10 and 37 degrees C and the temperature optimum was 45 degrees C. The values were similar to those of swine serum. The FAPGG Michaelis-Menten constants (K(m)) at 37 degrees C of amphibian serum enzymes (0.337 mM and 0.282 mM for frog and newt, respectively) were lower than that of swine (1.305 mM), but close to human serum enzyme. The K(m) values obtained at 10 degrees C were lower than those at 37 degrees C (0.152, 0.086, and 1.029 mM for frog, newt, and swine serum, respectively). Amphibian sera hydrolysed bullfrog synthetic angiotensin I to produce angiotensin II. Captopril (50 microM) inhibited the production of angiotensin II.

Angiotensin I↗

Comparative phylogeography and postglacial colonization routes in Europe.

The Quaternary cold periods in Europe are thought to have heavily influenced the amount and distribution of intraspecific genetic variation in both animals and plants. The phylogeographies of 10 taxa, including mammals (Ursus arctos, Sorex spp., Crocidura suaveolens, Arvicola spp.), amphibians (Triturus spp.), arthropods (Chorthippus parallelus), and plants (Abies alba, Picea abies, Fagus sylvatica, Quercus spp.), were analysed to elucidate general trends across Europe. Only a small degree of congruence was found amongst the phylogeographies of the 10 taxa, but the likely postglacial colonization routes exhibit some similarities. A Brooks parsimony analysis produced an unrooted area phylogram, showing that: (i) the northern regions were colonized generally from the Iberic and Balkanic refugia; and (ii) the Italian lineages were often isolated due to the presence of the Alpine barrier. The comparison of colonization routes highlighted four main suture-zones where lineages from the different refugia meet. Some of the intraspecific genetic distances among lineages indicated a prequaternary divergence that cannot be connected to any particular cold period, but are probably related mainly to the date of arrival of each taxon in the European continent. As a consequence, molecular genetics so far appears to be of limited use in dating Quaternary events.

Animals↗

Stage-dependent genetically-based deformities of the regenerating newt limb from 4-nitroquinoline-N-oxide mutagenesis: potential embryonic regulation of cancer.

The effect of the mutagenic carcinogen 4-nitroquinoline-N-oxide (4NQO) on limb regeneration was studied in adult Triturus cristatus newts. Delayed limb regeneration was observed when a 10- to 15-micrograms crystal of 4NQO was implanted at the late dedifferentiation or late bud stage. Additionally, 4NQO administration at these stages caused developmental deformities in skeletal elements of the subsequent regenerates. In contrast, 4NQO implanted at the wound healing stage did not affect skeletal morphogenesis. These critical stages of limb regeneration affected by carcinogen exposure (but not other manipulations) represent mechanisms distinct from disruption of basement membrane deposition. Secondary regeneration, initiated by reamputation of the 4NQO-treated abnormal regenerates 4-5 mm proximal to the site of carcinogen implantation, produced normal regenerates, setting a limit on the diffusion distance of 4NQO in the limb. Distal reamputation through regenerates at the level of the abnormality resulted in regeneration of the original bone deformity, suggesting that the teratogenic effect of 4NQO is mediated via heritable mutational events in blastema cells. These results extend a previously published conclusion that 4-NQO is mutagenic in non-regenerative tissue to include regenerative tissue as well. However, in spite of mutagenic activity, squamous carcinomas were not induced by 4NQO exposure at any stage of regeneration. Therefore, the resistance of regenerative tissue to 4'-NQO carcinogenesis is not due to resistance to mutations, but rather to other mechanisms perhaps similar to those which regulate malignant cells in murine embryos.

4-Nitroquinoline-1-oxide↗

Abnormal limb regeneration in adult newts exposed to the fungicide Maneb 80. A histological study.

The effects of the fungicide Maneb 80 (manganese ethylenebisdithiocarbamate, 80% active ingredient) on the regenerating limb of the adult crested newt, Triturus cristatus carnifex, was studied. Female newts were exposed percutaneously to 5 ppm Maneb 80. One group of control newts was exposed to the inert ingredients of Maneb 80 (sodium lignin sulfonate and n-butylnaphthalene sulfonate), and another control group was kept in tap water. The limbs were examined histologically at weekly intervals throughout the regeneration period and at the end of the experiment (10-12 wk postamputation). The regenerating limbs of all the animals exposed to Maneb 80 showed growth retardation and skeletal abnormalities. Histological examination provided evidence that vascular disturbances are important for the genesis of the developmental abnormalities induced by Maneb 80. The inert ingredients had a promoting effect on limb growth and had no teratogenic effects under our experimental conditions. There were no histological differences between the two control groups.

Abnormalities, Drug-Induced↗

Studies on amphibian yolk 1. The ultrastructure of the yolk platelet.

The yolk platelets of mature eggs and young embryonic cells of all amphibian species studied (Rana pipiens, Triturus pyrrhogaster, Diemictylus viridescens, Rana nigromaculata, and Bufo vulgaris) have a superficial layer of fine particles or fibrils (ca. 50 A in diameter), a central main body with a crystalline lattice structure, and an enclosing membrane approximately 70 A in thickness. Electron micrographs of the main body reveal hexagonal net (spacing ca. 70 A), square net (spacing ca. 80 A), and parallel band (spacing from 35 to 100 A but most frequent at ca. 70 A) patterns. The crystalline structure is believed to be a simple hexagonal lattice made of closely packed cylindrical rods. Each rod is estimated to be about 80 A in diameter and 160 A in length.

Amphibians↗

AN ELECTRON MICROSCOPE STUDY OF THE ENDOPLASMIC RETICULUM IN NEWT NOTOCHORD CELLS AFTER DISTURBANCE WITH ULTRASONIC TREATMENT AND SUBSEQUENT REGENERATION.

Ultrasonic treatment of the tails of Triturus alpestris tadpoles, at intensities of 8 to 15 watts/cm(2), at 1 megacycle/sec., for 5 minutes, disrupted the epidermis and caused pycnosis in individual cells of the muscle and neural tube, but caused no damage to the notochord that could be detected by light microscopy. Electron microscopy showed that this ultrasonic treatment disordered nearly all the endoplasmic reticulum (ER) of the notochord cells into irregularly rounded vesicles, but within 3 hours after treatment some parallel arrays of normal endoplasmic reticulum were seen near, and continuous with, the outer nuclear membrane. In addition, a re-ordering of the previously disordered ER took place throughout the cytoplasm, in some cases. A classification was made of the state of the ER as shown in electron micrographs of material fixed immediately, 3, and 24 hours after treatment. This showed that more than half the total endoplasmic reticulum in notochord cells was normal again by 24 hours after treatment.

Animals↗

MEASUREMENTS OF SOME CELLULAR CHANGES DURING THE FIXATION OF AMPHIBIAN ERYTHROCYTES WITH OSMIUM TETROXIDE SOLUTIONS.

On average, 15 per cent of the total haemoglobin present in the blood of the newt Triturus cristatus was extracted during 45 minutes of fixation in Palade-Caulfield fixative. This extraction was reduced with fixatives buffered at pH 6.2 instead of pH 7.4. The addition of Ca(++) ions to a final concentration of 0.01 M in the fixative completely suppressed haemoglobin extraction. The effect of the pH, and the presence or absence of Ca(++) ions in the fixative, on the rate of haemoglobin extraction has been determined. During Palade-Caulfield fixation the average projected area of newt erythrocytes increased by 37 per cent, and after dehydration and embedding in Epon the average area was 25 per cent greater than that of the unfixed cell. Fixatives buffered at pH 6.2 and containing 0.01 M Ca(++) ions caused cellular shrinkage, with the average projected area decreasing by 10 per cent in the fixative. This shrinkage continued during dehydration, and the final average area of the erythrocytes in Epon was 26 per cent less than that of the unfixed cells. Similar measurements with erythrocytes of Amphiuma tridactylum showed that after Palade-Caulfield fixation the average cellular area was increased by 45 per cent, and after dehydration and embedding in Araldite it was 36 per cent greater than that of the unfixed cell. The average nuclear area increased by 35 per cent during fixation but after embedding it was 26 per cent greater than that of the unfixed nuclei. With a fixative at pH 6.2 containing 0.01 M Ca(++) ions, both the nucleus and the whole cell shrank during fixation. The nuclear area decreased by 20 per cent and the cellular area by 22 per cent. After dehydration and embedding in Araldite, the average nuclear area had decreased by 35 per cent and the cellular area by 40 per cent. It has been shown that OsO(4) fixation lowers the isoelectric points of haemoglobins and other proteins. This finding has been used in the interpretation of the observed cellular changes resulting from fixation.

Animals↗

An investigation by electron microscopy of the nucleoside phosphatase activity of amphibian and mammalian erythrocytes.

Amphibian and mammalian blood was washed in isotonic saline, fixed in glutaraldehyde, and then stained in the ATPase medium of Wachstein and Meisel. The blood cells were subsequently postfixed in osmium tetroxide, embedded in epoxy resins, and studied by electron microscopy. The plasma membranes of amphibian erythrocytes, from the newt Triturus cristatus and the frog Rana esculenta, were stained after incubation in media containing ATP or ADP as substrates, but were unstained after incubation in media containing AMP or sodium beta-glycerophosphate. The addition of 0.001 M ouabain to ATP-containing media did not inhibit the staining of the plasma membranes, but the omission of Mg(++) ions from the medium inhibited staining. The plasma membranes of rat and rabbit erythrocytes were never stained after incubation in any of the media used.

Adenine Nucleotides↗

Ultrastructure and cytochemistry of metabolic DNA in Tipula.

A DNA body is present in the females of the fly Tipula oleracea and is formed in contact with the sex chromosomes in the oogonial interphases. At each oogonial mitosis, the DNA body follows the chromosomes to one anaphase group and is included in one of the telophase nuclei. The body increases appreciably in size during the interphase of meiosis. All oocytes have the body, but only a few nurse cells possess it. The DNA body synthesizes its DNA at a different time than the chromosomes, as is shown by incorporation of tritiated thymidine, and contains 59% of the DNA of the nucleus, as is disclosed by spectrophotometric measurements. At late diplotene the DNA body disintegrates, releasing its DNA into either the nucleus or the cytoplasm. When studied in the electron microscope, the DNA body appears composed of a tight mass of intertwined fibrils. Demonstration that the main mass of the body is composed of DNA is obtained from cytochemical tests which reveal that the DNA body is Feulgen positive, stains green with azure B, incorporates H(3)-thymidine, and after digestion with DNase is Feulgen negative. The DNA of the body is complexed with histone, like the DNA of the chromosomes, as is revealed by an intense alkaline fast green staining. Electron microscope examination of oocytes reveals that one side of the DNA body is in close contact with the nuclear envelope and that the other side possesses an outer shell composed mainly of particles 150 to 250 A in diameter. Between the outer shell and the chromosomes there is a band of low electron opacity, 4000 to 7000 A thick. In the light microscope, this light band together with the outer shell is Feulgen negative and stains violet with azure B; this is confirmation of the presence of RNA. In the oocytes the nucleoli are found inside the DNA body. These nucleoli have a nucleolonema composed mainly of particles 150 to 250 A. The nucleoli are Feulgen negative, alkaline fast green negative, stain violet with azure B, and do not stain with azure B after RNase digestion, thus confirming their RNA content. The presence of the nucleoli inside the DNA body and of a band of RNA between the body and the chromosomes is indicative of a high RNA synthetic activity. Since the DNA of the body is complexed with histone, as in the chromosomes, and the nucleoli are located inside the body, the simplest interpretation of the DNA body is that it represents hundreds of copies of the operons of the nucleolar organizing region or neighboring regions. The situation found in Tipula has several basic features in common with the polytene chromosomes of other Diptera and with the hundreds of nucleoli present in Triturus oocytes. In all three cases, genes seem to be copied hundreds of times but are kept in different types of packages. A DNA body like the one in Tipula oleracea is found in other species of Diptera and in the Coleoptera. There is no indication, from the present investigation, that the DNA body is in any way associated with a virus.

Cell Division↗

DNA synthesis and mitosis in erythropoietic cells.

Studies of newt (Triturus or Diemictylus viridescens) erythropoietic cells showed that DNA synthesis and mitosis normally occur throughout most of the developmental process. Mitotic divisions were found in all immature precursor stages from the proerythroblast to the highly hemoglobinized reticulocyte. Mitoses were absent in mature erythrocytes. Radioautographic examination of thymidine-(3)H incorporation into DNA revealed that all erythroid cells except the mature erythrocyte were labeled. Microphotometric measurements of Feulgen-stained smears showed that all immature stages were undergoing DNA synthesis whereas the mature erythrocyte was inactive. The results obtained from three independent methods clearly demonstrate that (a) no loss of DNA or of chromosomes occurs during erythrocytic development and (b) highly hemoglobinized and, therefore, well-differentiated cells normally do undergo DNA synthesis and mitosis.

Animals↗

Spheroidal and ring nucleoli in amphibian oocytes. Patterns of uridine incorporation and fine structural features.

In maturing oocytes of the newt Triturus viridescens, the nucleoli undergo a series of morphological changes that are very similar to those described by Callan for the axolotl, Ambystoma mexicanum. The nucleoli first assume the form of spheroids which then become extended into ring or necklace shapes that are DNase-sensitive; in mature oocytes the nucleoli revert to a spheroidal form. Short term in vitro incorporation studies with uridine-(3)H on both species show that RNA synthesis occurs in a restricted, eccentric portion of the spheroidal nucleoli, thereby producing an asymmetrical pattern of labeling. In the ring forms, however, the localization of the radioactivity suggests that synthesis takes place symmetrically throughout their entire length. The changes in nucleolar morphology apparently reflect the fact that the component DNA has undergone a redistribution from a localized region in the spheroidal nucleoli to an extended circle in the rings; the patterns of uridine-(3)H incorporation, therefore, parallel the distribution of DNA in both the spheroidal and the ring nucleoli. Ultrastructurally, the nucleoli contain a fibrillar component that corresponds in position to that of the DNA. The typical spheroidal nucleolus consists of a fibrillar core situated eccentrically and surrounded by a hull of granular, ribonucleoprotein material. The ring nucleoli are composed of a central fibrous region that is ensheathed all around its circumference by a layer of similar granular material. This granular substance is thicker at intervals along the length of the rings, representing the "beads" of the necklaces.

Animals↗