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The newt, a group of urodele amphibians, has outstanding ability to repeatedly regenerate various body parts, even in the terrestrial life-stage. In this animal, when the limb is amputated, a cell mass named the blastema appears on the stump and eventually gives rise to a new functional limb. Erythrocytes (red blood cells) in most non-mammalian vertebrates, including the newt, preserve their nucleus throughout their life-span, although physiological roles of such nucleated erythrocytes, other than oxygen delivery, are not known. Here we report novel behavior of erythrocytes in the newt. We identified an orphan gene Newtic1, whose transcripts significantly increased in the blastema. Newtic1 was expressed in a subset of erythrocytes that formed a novel clump (EryC). EryC formed a complex with monocytes and was circulating throughout the body. When the limb was amputated, EryCs were newly generated in the stump and accumulated into a distal portion of the growing blastema. Our data suggested that the newt erythrocytes carried multiple secretory molecules including growth factors and matrix metalloproteases, and were capable of delivering these molecules into the blastema as a form of EryCs. This study provides insight into regulations and roles of nucleated erythrocytes, that are independent of oxygen delivery.

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Freshwater organisms are increasingly exposed to elevated salinity in their habitats, presenting physiological challenges to homeostasis. Amphibians are particularly vulnerable to osmotic stress and yet are often subject to high salinity in a variety of inland and coastal environments around the world. Here, we examine the physiological responses to elevated salinity of rough-skinned newts (Taricha granulosa) inhabiting a coastal stream on the Pacific coast of North America and compare the physiological responses to salinity stress of newts living in close proximity to the ocean with those of newts living farther upstream. Although elevated salinity significantly affected the osmotic (body weight, plasma osmolality), stress (corticosterone), and immune (bactericidal ability) responses of newts, animals found closer to the ocean were generally less reactive to salt stress than those found farther upstream. Our results provide possible evidence for some physiological tolerance in this species to elevated salinity in coastal environments. As freshwater environments become increasingly saline and more stressful, understanding the physiological tolerances of vulnerable groups such as amphibians will become increasingly important to our understanding of their abilities to respond, to adapt, and, ultimately, to survive.

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The aim of our study was to verify whether environmental concentrations of nonylphenol influenced the adrenal gland of Triturus carnifex. Newts were exposed to 19 µg/L nominal concentration of nonylphenol throughout the periods of December-January and March-April, corresponding to different stages of the chromaffin cell functional cycle. The morphological features of the steroidogenic and chromaffin tissues, and the serum levels of ACTH, aldosterone, corticosterone, norepinephrine and epinephrine were evaluated. Nonylphenol did not influence ACTH serum levels. During the two periods examined, the steroidogenic tissue had the same reaction: the quantity of cytoplasmic lipids, and the corticosteroid serum levels, decreased, suggesting the inhibition of synthesis and release of corticosteroids. During the two periods examined, the chromaffin tissue reacted differently to nonylphenol. During December-January, the numeric ratio of norepinephrine granules to epinephrine granules, and the epinephrine serum levels, increased, suggesting the stimulation of epinephrine release. During March-April, the numeric ratio of norepinephrine granules to epinephrine granules did not change, and the norepinephrine serum levels decreased, suggesting the inhibition of norepinephrine release. Our results show that nonylphenol influences the activity of the newt adrenal gland; considering the physiological role of this gland, our results suggest that nonylphenol may contribute to amphibian decline.

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When specimens of the newt Triturus carnifex, under anaesthesia by submersion in a 0.2% chlorbutol solution for 25 min, are isolated in a respiratory chamber at 18 degrees C containing water with only 1.3 ppm of oxygen, they consume the oxygen completely in about 3 hr, but they can stay alive for many more hours and wake up with no apparent exterior consequences. Hypoxia induces rapid onset of hepatic steatosis and melanosis, as well as a controlled haemolytic process involving a pool of red blood cells of the same order of size as that held as a reserve in the spleen by animals in an aerial habitat. At the origin of the phenomena is an intense response by the hypophysis, histologically detectable 1 hr from the onset of treatment and confirmed 2 hr later by a highly significant increase in the plasma thyroidstimulating hormone (TSH) concentration compared with the controls (41.5 +/- 13.7 microU/L vs. 15.5 +/- 6.2; P < 0.005). The thyroid follicles react by reabsorbing their colloid, but instead of an increase in the plasma free T3 and T4 concentrations, fT3 falls significantly (1.5 +/- 0.3 pg/mL vs., the 2.4 +/- 0.7; P < 0.05), whereas fT4 remains stationary (4.0 +/- 0.5 pg/mL vs. 4.6 +/- 0.8; N.S.). After 6 hr, the plasmatic TSH concentration is still higher than in the controls (27.0 +/- 3.0 microU/L vs. 15.5 +/- 6.2; P < 0.05), whereas fT3 and fT4 remain stable (1.5 +/- 0.3 and 4.4 +/- 0.5 pg/mL, respectively). If T3 or T4 labelled with 125I is administered prior to hypoxia, after 6 hr of treatment the radioactivity is found to be limited exclusively to the liver and kidney; the thyroid, gall bladder and gut result negative, and this does not agree with hypotheses of hormone inactivation by deiodination, sulphation or glucuronidation. This apparently peculiar endocrine path has not been observed in previous studies on hypoxia in vertebrates, because the experiments were always designed to analyse plasma hormone levels after at least 24 hr of hypoxia or during chronic treatments, losing the most interesting phases of the endocrine response. The possibility that the hypoxic newt possesses alternative or complementary metabolic pathways to anaerobic glycolysis to sustain steatogenesis and melanogenesis and maintain the same cardiac activity as the controls is briefly discussed.

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Transition of hemoglobin (Hb) from larval to adult types during the metamorphosis in a salamander Hynobius retardatus has been reported to occur almost independently of thyroid activity, in contrast to the case with many amphibians. In order to obtain further information on the mechanism of the transition in H. retardatus, larval and adult globin cDNAs were cloned, and the globin gene expression was analyzed in normally developing and metamorphosis-arrested animals. Northern hybridization and RT-PCR revealed that larval globin genes were initially expressed 5 days before hatching, and unexpectedly remained expressed even in juveniles 2 years old. The adult globin gene was expressed 19 days after hatching, much earlier than the initiation of morphological metamorphosis. Furthermore, the pattern of globin gene expression in metamorphosis-arrested larvae was almost identical to that in normal controls, suggesting that the transition occurs independently of thyroid hormones. In larvae recovering from anemia, precocious Hb transition, which occurs in Xenopus laevis and Rana catesbeiana, did not occur in H. retardatus. In situ hybridization convincingly demonstrated that the erythropoietic sites are the ventral blood island and the dorsolateral plate at the prehatching stage. During the ontogeny they changed to the liver, kidney, and spleen and were finally restricted to the spleen. Single erythroid cells expressed concurrently larval and adult globin genes, as demonstrated by double in situ hybridization. Thus the transition occurred within a single erythroid cell population, a unique characteristic of H. retardatus.

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Changes in plasma concentrations of androgens, estradiol-17 beta, and progesterone were examined throughout the year in Hynobius nigrescens. The highest mean concentration of androgens (312.7 ng/ml) was found in males in the process of spermiation and having just entered the breeding pond in early spring. Plasma estradiol-17 beta concentration was at its base level in females both before and after the animals had entered the pond. Plasma progesterone concentrations were low throughout the year except during the aquatic phase in March when females possessed some ova within the coelom and many ova in the oviducts and ovisacs after the completion of ovulation. During the terrestrial phase, or nonbreeding season, plasma concentrations of androgens and estradiol-17 beta were in approximate correspondence with the development of sex accessory structures. This is the first documentation of plasma concentrations of sex steroids in hynobiid salamanders.

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In the newt, Cynops pyrrhogaster, parathyroidectomy (PTX) brought about a marked decrease in the concentration of plasma calcium. The animals recovered from the hypocalcemia by 15 days after the operation if the pituitary gland was left intact. After PTX, no significant changes in the plasma sodium concentration were observed. Experiments were then conducted to obtain direct evidence that endogenous prolactin (PRL) is involved in this recovery process. Recovery of the calcium level after PTX was blocked by administration of an antiserum raised against newt PRL. In newts deprived of both the pituitary and parathyroid glands, no recovery from hypocalcemia was observed. Administration of newt or ovine PRL to parathyroidectomized-hypophysectomized newts significantly elevated the blood calcium level. After PTX, the concentration of immunoassayable PRL in the blood rose to 10 times the value in sham-operated animals. These results indicate the involvement of PRL in calcium homeostasis in newts with a shortage of parathormone.

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The micronucleus test using erythrocytes of Pleurodeles waltl larvae (Amphibian, Salamandridae) was used to detect the genotoxic activity of various substances. Most of them were known (or suspected) to be mutagenic or carcinogenic in mammals. Larvae were reared in water containing either a test chemical (experimental group) or in control water and the levels of micronucleated red blood cells (RBCs) compared between the two groups. Aroclor 1254, butylated hydroxyanisole, phenobarbital and 12-O-tetradecanoyl-phorbol-13-acetate produced negative results, while acridine orange, benzo[alpha]pyrene, epsilon-caprolactam, cyclophosphamide, diethyl sulphate, epichlorhydrin, ethidium bromide, ethyl methanesulphonate, ethylene dibromide (dibromoethane), N-ethyl-N'-nitro-N-nitrosoguanidine, N-ethyl-N-nitrosourea, hexamethylphosphoramide, 3-methyl cholanthrene, pyrene and o-toluidine gave positive responses. The results were compared with published data from other tests used to detect the clastogenic or mutagenic properties of chemicals. We suggest that the newt micronucleus test could be used to monitor aquatic pollution, and/or for the quality control of drinking water. Pleurodele larvae may also prove to be of value for the detection of carcinogenic/clastogenic substances dissolved in water.

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A model micronucleus test system using peripheral blood erythrocytes from larvae of Pleurodeles waltl is described. The most suitable larval stage for testing chemical treatments was determined. Larvae were reared in water containing one of the 4 compounds: benzo[a]pyrene (BaP), ethyl methanesulphonate (EMS), diethyl sulphate (DES) and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG). Response curves as a function of treatment duration over a period of 16 days were plotted for 3 different concentrations of the 4 compounds in order to optimize conditions for a low dose micronucleus test. This model can be used as a monitoring system for the detection of fresh water pollution and can also be employed for clastogen screening of chemical compounds. The test is sensitive, reliable and easy to use.

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Active or potentially active gene loci are preferentially sensitive to DNAase I digestion. Cedar and colleagues used this DNAase I sensitivity to label active sequences preferentially by nick translation of nuclei and chromosomes. Using biotinylated nucleotides and immunofluorescence detection, we have looked for compartmentalization of DNAase I-sensitive sequences by nick translation of nuclei maintained as three-dimensional structures. Labeled sequences in mouse L cells are preferentially localized at the nuclear periphery in both permeabilized nuclei and frozen sections. In newt and chicken nucleated erythrocytes, labeled regions are at borders of condensed chromatin masses along interchromatin channels communicating with the nuclear periphery. Control experiments indicate that nick translation of nuclei preferentially labels highly expressed genes and that the preferential localization of DNAase-I sensitive regions is probably not a consequence of fixation artifacts or selective nuclear permeability to the labeling reagents.

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Sex steroid levels in the plasma of the female Pleurodeles waltl were determined by radioimmunoassay of blood samples taken monthly from each newt during an 18-month period. Estradiol-17 beta levels were relatively high (greater than 1 ng/ml) but androgen levels were higher (2-30 ng/ml). Androgen and estradiol-17 beta levels fluctuated according to season with two annual peaks, in autumn and in March. Testosterone was the principal androgen present in this plasma. Dihydrotestosterone levels were relatively low but showed slight fluctuations during the year. 4-Androstenedione and estrone levels were very low and relatively constant throughout the seasons. Fluctuations of androgen and estradiol-17 beta levels were synchronous between the several newts studied during the year. The ratio [E2-17 beta]/[Androgen] was stable during the major part of the year (0.2-0.3) except in July and August, when it reached the value of 1.0. The seasonal variations in the steroid levels were well correlated to the morphological modifications of sexual characteristics during the year.

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Seasonal changes in the plasma levels of androgens (testosterone plus dihydrotestosterone) and 17 beta-estradiol in diploid and triploid adult newts, Pleurodeles waltl were studied. In both male and female diploid individuals, large variations were reported with highest levels being found during breeding periods. In triploid newts seasonal variations were also found, similar to the diploid ones, but the plasma concentrations of the 17 beta-estradiol and androgens in triploid females and androgens in triploid males were lower throughout the year than those reported for the diploids. This difference is discussed in relation to the genetic sexual constitution.

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Sex steroid levels in the plasma of the male Pleurodeles waltl were determined by radioimmunoassay of blood samples taken monthly from each newt during an 18-month period. Androgen levels varied according to season with annual peaks in October-November and in March. The position of these two peaks was the same in the 2 years studied. Testosterone was the principal androgen measured, while the levels of dihydrotestosterone and 4-androstenedione were lower but fluctuated in parallel with that of testosterone. The 17 beta-estradiol levels were very low or undetectable. Androgen levels during the year were synchronous between the several newts studied. The cyclical variations in the steroid levels agreed with the morphological modifications of sexual characteristics during the annual cycle.

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Improved electrophoretic resolution revealed two albumin-like proteins in Taricha granulosa plasma (bisalbuminemia). The Taricha proteins were compared to mammalian, avian and reptilian serum albumins regarding molecular weight, amino acid composition, isoelectric character, solubility and the binding of hemin and dyes. The results indicate that although the two Taricha proteins have demonstrated hemoglobin-binding ability, they possess traits that characterize them to be true serum albumins.

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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.

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A minimum of 20 aquatic, adult Notophthalmus viridescens were captured approximately every other month during 1980 in order to investigate seasonal changes in body size and blood parameters. Hemoglobin concentration, hematocrit, mean cell hemoglobin, sex, weight, snout-to-tail length, snout-to-vent length, and the presence of trypanosomes on blood smears were determined. Environmental variables measured included temperature (range 5-26 degrees C), oxygen concentration (range 5.7-12.2 mg/L), and pH (range 6.5-7.5) of water at the pond's edge. Three anemic specimens (less than 0.03 gHb/100 ml blood) were found. The only significant (P less than 0.05) seasonal effects were that the mean hematocrit of the July sample was greater than that of the March sample and the incidence of trypanosome infection of the March collected newts was lower than those collected in May, July, and August. Significant (P less than 0.01) linear relationships were found within the body size and blood parameters but not between them. Females had significantly (P = 0.000) higher hematocrits than males. The hemoglobin concentration and hematocrit did not correlate with the assumed seasonal oxygen demands of the newt.

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Blood plasma from a Urodele amphibian, Pleurodeles waltii, has been found to contain very-low density, low-density and high-density lipoproteins (VLDL, LDL and HDL, respectively). HDL and LDL predominated (concentration range 72-114 and 51-101 mg/dl) with lesser quantities of VLDL (23-51 mg/dl). Following isolation by density gradient ultracentrifugation, the physicochemical properties (morphology, particle size, hydrated density, electrophoretic mobility, and chemical composition) of the three major classes resembled, but were not identical with, those of the corresponding fractions in normal human plasma. The protein moieties of VLDL and LDL consisted mainly of polypeptides of high molecular weight (Mr greater than 60,000), which in their solubility were akin to human apo-B; smaller amounts of another protein migrated to the position of human apo-C-III in basic polyacrylamide gels. The major HDL apolipoproteins were of Mr 31,000 and 27,000, with minor amounts of 13,000, 61,000 and 76,000 components; in alkaline, urea-containing polyacrylamide gels, these major proteins migrated faster than the major components (apo-AI and apo-AII) of human HDL. Immunochemically, lipoproteins with pre-beta-, beta- and alpha-mobility were detected in the VLDL, LDL and HDL, respectively. Salamander VLDL and LDL were found to share an antigenic site(s) with the LDL of trout, chicken and guinea-pig, suggesting the presence of an apo-B-like protein. The chemical compositions and immunochemical reactions of salamander lipoproteins indicate that their structure and metabolism resemble that of other amphibia, reptiles and fish rather more closely than that of most mammals and birds.

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