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Structure elucidation of NeuAc, NeuGc and Kdn-containing O-glycans released from Triturus alpestris oviductal mucins. Characterization of the poly LacdiNAc sequence: HSO3(4)(GalNAcbeta1-4GlcNAcbeta1-3)1-3GalNAcbeta1-4(GlcNAcbeta1-3)0-1GlcNAcbeta1-6GalNAc-ol.

Eggs from Amphibia are surrounded by several layers of jelly that are needed for proper fertilization. Jelly coat is composed of highly glycosylated mucin-type glycoproteins containing up to 60% of carbohydrates, which display a remarkable species-specificity. This material obtained from Triturus alpestris was submitted to reductive beta-elimination and the released oligosaccharide-alditols were further fractionated by HPLC. Structural characterization was performed through a combination of two dimensional (1)H-(1)H and (1)H-(13)C NMR and ESI-MS/MS analysis. Numerous carbohydrate chains are characterized by the presence of the Cad (Sd(a)) determinant, including respectively NeuAc, NeuGc or Kdn as a sialic acid. But the most significant O-glycan sequences which mark the difference between the jelly of T. alpestris and other studies amphibian jellies are polymers of GalNAc(beta 1-4)GlcNAc (LacdiNAc) which form part of the following sequence: HSO(3)(4)(GalNAcbeta 1-4GlcNAcbeta 1-3)(1-3)GalNAcbeta 1-4(GlcNAcbeta 1-3)(0-1)GlcNAcbeta 1-6GalNAc-ol.

Animals↗

Characteristics of changes in the intracellular potential associated with transport of neutral, dibasic and acidic amino acids in Triturus proximal tubule.

(1)Introduction of L-alanine and L-lysine into the lumen of the proximal tubule of Triturus kidney evoked an immediate and sustained depolarization of the peritubular membrane potential (Epm) and a small increase in the transtubular potential (Ett). L-Aspartate had no effect. (2) The alanine-induced depolarization was absolutely dependent on the presence of Na+, whereas the lysine-induced one was partially dependent on Na+. In the absence of Na+, alanine usually evoked a transient hyperpolarization of the Epm, while lysine evoked a diffusion potential-like PD change. (3) Addition of alanine or lysine to the peritubular fluid did not cause any immediate change in the Epm, but the cells depolarized with a marked time delay. The delayed depolarization could be ascribed to the entrance of amino acids into the lumen through the nephrostromes and the paracellular pathways. (4) Cellular uptake of alanine and lysine was partially dependent on Na+, while that of aspartate was completely dependent on Na+. (5) Characteristics of the observed electrical events were explained in terms of the differences in the charge transfer associated with transport of these amino acids across the luminal membrane.

Amino Acids↗

Length polymorphism in rDNA indicates somatic alterations in the genome of Triturus vulgaris.

We have analyzed the EcoRI restriction pattern of ribosomal genes (rDNA) isolated from several organs of single individuals of the newt Triturus vulgaris by Southern blotting and hybridization with corresponding Xenopus probes (r11 and r12). Using length polymorphism of rDNA spacers as a molecular marker, it became evident that in individual newts the pattern of ribosomal genes is not always constant but varies between different tissues of the same animal suggesting the occurrence of genome alterations during ontogenesis.

Animals↗

Effects of permanent deafferentation of the anterior preoptic area on serum aldosterone levels in the crested newt (Triturus cristatus carnifex Laur.).

By cutting off the fiber systems running along the medial forebrain bundle of the urodele amphibian Triturus cristatus, a wide deafferentation of the preoptic area was evoked. This operation elicited a decrease in aldosterone serum level, probably through a reduction of ACTH secretion. At present we are not able to ascertain whether such reduction was prompted by changes in the hypothalamic production of the neurohypophysial hormones or the corticotropin-releasing factor (CRF).

3-Hydroxysteroid Dehydrogenases↗

Sex steroid profile and plasma vitellogenin during the annual reproductive cycle of the crested newt (Triturus carnifex Laur.).

The annual reproductive cycle of the crested newt, Triturus carnifex, has been studied in the field. Temperatures, rainfall, humidity, and photoperiod were recorded throughout the year. Adult male and female newts were sampled monthly; snout vent lengths, crest heights of males, and body ovarian, oviducal, testicular, and abdominal gland weights were recorded. Plasma samples were assayed for androgen, estradiol-17 beta, and progesterone by radioimmunoassay and for vitellogenin by enzyme-linked immunosorbent assay. Air, deep water, water surface, and soil temperatures were low from October to March, but increased in April and May without consistent summer variations. Ovarian and oviducal weights increased in October to reach maximum values between January and March (reproductive period). Crest height and abdominal gland weight in males mirrored the ovarian and oviducal pattern, while testicular weights were maximal in October and November. In females, plasma androgens were high during the reproductive period, and plasma estradiol peaked sharply in March, while plasma progesterone changed little. In the males plasma androgen and estradiol concentrations were similar to those of females, while plasma progesterone was significantly correlated with the cycle in testicular weight. In both sexes androgens showed a significantly negative correlation with air and water surface temperature. Plasma vitellogenin peaked in March but it did not correlate with either ovarian weight or plasma estradiol concentrations. These data support and confirm those previously reported for newts under laboratory conditions. The negative correlation between androgens and temperature suggests that this hormone may trigger the reproductive process. Moreover the correlations between plasma progesterone and testicular weight may indicate that this hormone is involved in male newts reproduction.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgens↗

Immunocytochemical identification of growth hormone- and prolactin-producing cells in the hypophysis of the newt Triturus cristatus carnifex Laur.

Single indirect immunocytochemical methods (immunofluorescence, PAP, and ABC) and double sequential staining (ABC followed by immunofluorescence) were used to localize GH- and PRL-producing cells in the pituitary distal lobe from Triturus cristatus. The following antisera were employed: rabbit anti-ovine PRL, anti-Rana catesbeiana PRL, anti-ovine GH, anti-bovine GH, and monkey anti-rat GH. A cell population corresponding to type-2 acidophils localized in the dorsal and central region, under the intermediate lobe, immunoreacted with GH antisera. Both ovine and bullfrog PRL antisera labeled a large cell population, corresponding to type-1 acidophils, predominantly localized in the ventral anterior two-thirds of the gland. The pattern of localization shown by the two cell types, although consistent with the majority of data on adult amphibians, disproves the findings obtained on the same species by other authors.

Animals↗

Androgen control of male sex behavior in the crested newt (Triturus cristatus carnifex Laur.): castration and sex steroid administration.

Castration significantly lowers serum testosterone in sexually active male Triturus cristatus. Replacement therapy by implants of testosterone in silastic capsules elevates the serum testosterone level to higher values than normal. Sex behavior is depressed by castration and partially reinstated by replacement therapy with testosterone. 5 alpha-dihydrotestosterone was the only testosterone metabolite showing some behavioral effectiveness in castrates; estradiol and 5 beta-dihydrotestosterone failed to elicit sex behavior.

Androgens↗

Molecular organization of ribosomal RNA genes clustered at variable chromosomal sites in Triturus vulgaris meridionalis (Amphibia, Urodela).

The ribosomal RNA genes of Triturus vulgaris meridionalis (Amphibia, Urodela) show the peculiar feature of being clustered not only at the nucleolar organizer, present in the species at a definite chromosome location, but also at "additional ribosomal sites" which are highly variable in number and chromosomal distribution among individuals. The additional ribosomal sites are most often found at specific chromosome regions, such as telomeres, C-bands and centromeres, in virtually all the chromosomes. With increasing numbers of additional clusters, the genomic dosages of ribosomal RNA genes are found to increase over a tenfold range, though not linearly. At a molecular level, the ribosomal DNA repeats differ in size because of discrete variations in the length of the non-transcribed spacers. However, the resulting length heterogeneity of the gene family is rather limited within a single genome as well as within the species. Many of the ribosomal loci appear to be internally homogeneous with respect to the repeat length. Moreover, separate clusters from distant genomic regions can share the same size class of ribosomal repeats even in the same specimen. The nucleolar organizer is mostly endowed with "shorter" ribosomal repeating units, ranging in size from 13.7 X 10(3) to 15.2 X 10(3) base-pairs. The additional ribosomal sites are characterized by the occurrence of "longer" repeats, ranging in size from 16.2 X 10(3) to 19.7 X 10(3) base-pairs. The "shorter" class of ribosomal repeats is always detected in the amplified ribosomal DNA, suggesting that the nucleolar organizer locus is involved in the amplification process in most oocytes. "Longer" ribosomal repeats are also detectable in the amplified ribosomal DNA of a few females.

Animals↗

Extra-ribosomal spacer sequences in Triturus.

We show that, in Triturus vulgaris meridionalis, sequences homologous to the rDNA "non-transcribed" spacer (NTS) are clustered at chromosomal loci where they are not associated with 18 S or 28 S rDNA genes: these sequences are referred to as the extra-ribosomal spacer sequences. Genomic clones containing such extra-ribosomal spacer sequences have been isolated. As shown by restriction mapping, these clones appear to consist mostly of repetitive BamHI fragments that are, in turn, internally repetitious and highly homologous to each other. The structure of the clones was confirmed by nucleotide sequence analysis, which also demonstrates the high degree of conservation between the BamHI elements and the homologous NTS sequences. An intriguing 12 base-pair homology between the extra-ribosomal spacer sequences and a Xenopus NTS enhancer sequence is reported. The possibility that a repetitive octanucleotide motif found within the BamHI elements could act as a recombination hotspot by virtue of its similarity with the Escherichia coli chi sequence is discussed.

Animals↗

Plasma prostaglandin F2 alpha in the male Triturus carnifex (Laur.) during the reproductive annual cycle and effects of exogenous prostaglandin on sex hormones.

Plasma patterns of prostaglandin F2 alpha (PGF2 alpha) and sex hormones (progesterone, androgens and 17 beta-estradiol) have been studied in the male crested newt, Triturus carnifex (Laur.), during the sexual cycle. The effects of exogenous PGF2 alpha on sex steroids have also been observed. In addition, effects of one week's captivity are reported. The patterns of plasma sex hormones, during the annual cycle, are consistent with the results previously reported for the same newt species. PGF2 alpha plasma level peaks in April, is low in summer, and progressively increases during autumn to peak again in December. The April PGF2 alpha peak coincides with a plasma estradiol increase and with an androgens drop. In April-collected newts, moreover, PGF2 alpha treatment induces a significant estradiol increase. These findings lead us to suppose that at the end of the breeding season (April) a PGF2 alpha-dependent estradiol synthesis occurs which could be implied in reproductive period termination. In several vertebrates, including some amphibian species, in fact, chronic administration of estradiol results in a strong inhibition of testicular endocrine tissue activity. The putative role of PGF2 alpha-dependent estradiol production in the gonadal regulation in amphibia living in temperate zones is discussed. The autumn PGF2 alpha increase has been tentatively related to the recovery gonadal processes and secondary sexual character development.

Androgens↗

Plasma prostaglandin F2 alpha and reproduction in the female Triturus carnifex (Laur.).

Plasma patterns of prostaglandin F2 alpha (PGF2 alpha) and sex hormones (progesterone, androgens and 17 beta-estradiol) have been studied in the female crested newt, Triturus carnifex (Laur.), during the annual sexual cycle. The effects of exogenous PGF2 alpha on sex hormones were determined. In addition, the effects of one week's captivity on plasma PGF2 alpha and sex hormones were reported. PGF2 alpha plasma level peaked in April, was low in summer, and progressively increased during the autumn to peak again in December. The April PGF2 alpha coincided with a 17 beta-estradiol rise, and with a progesterone drop. The autumn PGF2 alpha increase was coupled to a 17 beta-estradiol rise, and therefore it has been tentatively related to ovary and oviduct development. In newts collected in April, moreover, a PGF2 alpha-dependent 17 beta-estradiol synthesis could occur, since PGF2 alpha injection induced a significant 17 beta-estradiol plasma increase. These findings led us to suppose that PGF2 alpha intervenes in spring breeding season termination through the induction of a 17 beta-estradiol synthesis as in other amphibian species. PGF2 alpha injection caused a progesterone decrease, probably by inducing corpora lutea lysis. The patterns of plasma sex hormones were consistent with the results reported for the same newt species.

Animals↗

PGF2 alpha, PGE2, and sex steroids from the abdominal gland of the male crested newt Triturus carnifex (Laur.).

Prostaglandin F2 alpha (PGF2 alpha), prostaglandin E2 (PGE2), progesterone, androgens, and 17 beta-estradiol in vitro release by the abdominal gland of the crested newt, Triturus carnifex (Laur.), was studied during the prereproductive, reproductive and postreproductive periods. In addition, the in vitro effects of the PGF2 alpha and/or PGE2 on progesterone, androgens and estradiol release by the abdominal gland were evaluated. PGF2 alpha, PGE2 and progesterone release was higher during the reproductive period, and in the same period, PGE2 treatment induced a progesterone increase. PGF2 alpha induced an increase of abdominal gland estradiol release at the end of the reproductive period. These results seemed to confirm the pheromonal role assigned to progesterone, and suggested a PGE2 stimulatory role in inducing progesterone release, even if pheromonal activity of PGF2 alpha and PGE2 cannot be excluded. In addition, PGF2 alpha-dependent estradiol increase at the end of reproduction could be interpreted as a mechanism for interruption of the abdominal gland activity.

Abdomen↗

Multiple hemoglobins in Triturus cristatus: their degradation by sulfhydryl compounds.

1. Hemolysates of newt (Triturus cristatus) red cells contain four distinct hemoglobin species which have been observed consistently both in individual animals and pooled samples. 2. Hemoglobin heterogeneity in the species arises from existence of multiple hemoglobulins, with no indication of genetic polymorphism. 3. While the relative proportions of the different hemoglobins may vary in different samples, HbII is usually the most abundant, with HbIII and HbIV constituting most of the remainder of the total hemoglobin complement. HbI never exceeds 3-5% of the total hemoglobin. 4. Neither the electrophoretic migration nor the anion exchange properties of the four hemoglobin species are altered by conversion of oxyhemoglobin to the cyanmet derivative, excluding artifacts due to different oxidation states of iron. 5. The average molecular weight of newt total hemoglobulin is 67,182 with no indication of hemoglobin polymers. 6. The use of sulfhydryl-reducing agents (mercaptoethanol, dithiothreitol) as a precaution against aggregation results in extensive degradation of newt hemoglobin through a process similar to "coupled oxidation" by ascorbate. 7. The degradative effects of sulfhydryl-reducing agents on newt hemoglobin suggest that these reagents be used cautiously in any hemoglobin analysis.

Animals↗

Multiple hemoglobins in Triturus cristatus: their study by analytical isoelectrofocusing.

Electrophoretic separation of the hemoglobin of healthy adult Triturus cristatus reveals four components. Isoelectric focusing of the same hemolysates in various commercial ampholytes of different chemical composition and pH range results in the separation of eight individual hemoglobin bands. The bands obtained by electrophoresis are not homogeneous as revealed by individual gel electrofocusing. They finally separate into the same eight components, as in the whole hemolysate. From the above findings it is concluded that this species has not four but eight individual hemoglobin molecular forms. Our results demonstrate lack of hemoglobin polymorphism in this species.

Animals↗

Vitamin A promotes DNA synthesis and mitosis in blastemas of Triturus alpestris.

Vitamin A palmitate orally administered to young, post-metamorphic Triturus alpestris (Amphibia-Urodela), for 4 and 7 days after bilateral forelimb amputation at the middle of the zeugopodium, enhanced the mean DNA content of the blastema cells. Moreover, the number of cells in mitosis was nearly twice as high in vitamin A treated animals than in control ones.

Animals↗

Autoradiographic evaluation of vitamin A effects on Triturus alpestris blastema cells.

Autoradiographic studies on Triturus alpestris regenerates treated with vitamin A palmitate for 4, 7, 11 and 14 days after amputation, reveal a considerable diminution in the percentage of cells duplicating DNA, as judged by measurements of labelling indices. Based on previous observations, that vitamin A promotes DNA-synthesis, mitosis and growth of regenerates, we conclude that the lower labelling indices calculated are due to an acceleration of S-phase processes, resulting in a concomitant reduction in its duration.

Animals↗

Cadmium, copper and zinc-binding protein (metallothionein) in the liver of the water lizard Triturus pyrrhogaster.

A low-Mr copper (Cu) and zinc (Zn)-binding protein was present in the liver of a control water lizard Triturus pyrrhogaster. Cadmium (Cd) accumulated in the liver of the animals after intramuscular injections of Cd salt and was bound to Cu- and Zn-binding protein of a single isoform that had properties characteristic of metallothionein (MT). Effects of Cd loading on several essential elements in the liver and kidney were also determined.

Animals↗

Cardiac activity of calcitonin gene-related peptide in amphibian species (Rana tigrina, Triturus sp).

1. Calcitonin gene-related peptide (CGRP) produced dose-related positive chronotropic and inotropic responses in isolated atria of a newt, Triturus sp. and a frog, Rana tigrina. It was apparent that CGRP was stimulatory on the heart of the amphibian species. 2. In the frog, these dose-related responses were attenuated in the presence of a CGRP antagonist (hCGRP 3 x 10(-8) M). This was indicative of the presence of cardiac CGRP receptors. 3. In the presence of beta-adrenergic (propranolol 10(-6) M) and calcium channel (verapamil 10(-8) M, but not nifedipine 10(-8) M) blocker, the basal AR in the frog were decreased. AR induced by CGRP were increased however. 4. The maximal AR and AT responses of the isolated atria to CGRP were not affected by the presence of propranolol. 5. In the presence of calcium channel blockers (verapamil and nifedipine), the maximal AT response was attenuated. These changes suggest the cardiac effects of CGRP are related to calcium mobilization.

Animals↗