Identification of highly acidic peptides from processing of the skin prepropeptides of Xenopus laevis

The skin secretion of the frog Xenopus laevis has been fractionated by reverse-phase HPLC and the most polar components studied by fast-atom-bombardment mass spectrometry (FAB/MS). Esterification of the hydrophilic peptides with methanol and ethanol was employed to improve the sensitivity of the technique. A number of small, highly acidic peptides have been identified, and alcoholysis of the peptide bonds within a number of these permitted their sequencing by FAB/MS. The sequences confirmed that they originate from acidic spacer regions found in the precursors to peptide hormones, such as caerulein, which have already been found in the secretion. In addition, acidic peptides derived from the spaces of the precursor to the antimicrobial peptides, PGS (or the magainins) have been isolated. The release of these from the preprotein cannot be fully accounted for by documented processing mechanisms, suggesting that a novel type of cleavage site has been identified.     

Development of Xenopus laevis skin glands producing 5-hydroxytryptamine and caerulein

Summary The granular glands in Xenopus laevis skin are known to contain large quantities of biogenic amines and bioactive peptides which closely resemble mammalian brain-gut peptides. We studied the development of glands producing 5-hydroxytryptamine (5-HT) and caerulein using immunohistochemistry, HPLC-fluorometric systems and RIA. The immunoreactivities of 5-HT and caerulein were first detected in the spherical gland rudiments in the stratum spongiosum at St. 58 (Nieuwkoop and Faber stage), or at the beginning of metamorphosis. Both immunoreactivities appeared in the same rudiment at the same time. Some of the gland rudiments have a small lumen filled with both immunoreactive materials at St. 58–59. During the rest of the metamorphic period, the glands grow in size, accumulating immunoreactive materials in the lumen. The concentrations of 5-HT and caerulein in the skin of tadpoles were below 1 ng per mg wet tissue at St. 58–59, increased as metamorphosis proceeded and reached 63 and 134 ng per mg wet tissue at St. 66, or at the end of metamorphosis, respectively. The amphibian granular glands where large quantities of biogenic amines and hormone-like peptides are rapidly synthesized may provide a useful model for the study of the development of amine- and peptide-producing cells including neurons and paraneurons.     

Serum immunoreactive insulin levels in intact and regenerating postmetamorphic Xenopus laevis

In order to confirm the presence of immunoreactive insulin (IRI) in the serum of postmetamorphic Xenopus laevis, radioimmunoassay (RIA) methods were used. The concentration of hormone found in samples of blood serum taken from nonanaesthetized intact male and female animals by the guillotine method was 10.46 +/- 0.76 microU/ml. Significantly higher IRI concentrations were found in our intact animals anaesthetized in MS 222 at pH 3.5 (21.9 microU/ml) compared with intact controls anaesthetized in MS 222 adjusted to pH 7.0 (14.4 microU/ml). During the wound-healing stage subsequent to forelimb amputation in the experimental cases (0 hours to 3 days) anaesthetized in MS 222 pH 7.0, there were intervals of significantly elevated serum IRI followed by a period of decreased IRI concentration compared with the levels in anaesthetized (MS 222 pH 7.0) and nonanaesthetized intact controls. These fluctuations were due, presumably, to stress caused by amputational injury and/or anaesthetic. Serum IRI increased steadily from 3 to 14 days postamputation then remained stable for the balance of the regeneration period (28 days) compared with nonanesthetized intact controls. A positive correlation was found between immunoreactive insulin and glucose levels in the serum of our animals. However, no correlation exists between serum IRI levels and serum osmolality in the data.     

Localization of xenopsin and xenopsin precursor fragment immunoreactivities in the skin and gastrointestinal tract of Xenopus laevis

Summary Xenopsin (Xp) and xenopsin precursor fragment (XPF) are bioactive peptides derived from a single precursor molecule; both were isolated previously from extracts of Xenopus laevis skin. The present immunohistochemical study was undertaken to determine the specific cellular localization of these two peptides in the skin and also in the gastrointestinal tract of adult Xenopus. We report here that Xp-like and XPF-like immuno-reactivities co-exist in the granular glands of the skin and specific granular cells in the lower esophagus and stomach. However, only Xp-like immunoreactivity, not XPF-like immunoreactivity, was detected in tall, thin cells of the duodenum and in club-shaped cells of the large intestine. The immunochemical co-localization of the two peptides in specific cells of the skin, lower esophagus and stomach suggests that the same gene is expressed in each of these cells, and that the precursor molecule undergoes similar post-translational processing. In contrast, the observation that certain cells of the duodenum and large intestine display only one peptide immunoreactivity suggests an alternative phenomenon, possibly involving selective peptide accumulation or expression of a different gene.     

Nerve-independent DNA synthesis and mitosis in regenerating hindlimbs of larval Xenopus laevis

Summary Xenopus laevis larvae at stage 52–53 (according to Nieuwkoop and Faber 1956) were subjected to amputation of both limbs at the thigh level as well as to repeated denervations of the right limb. Results obtained in larvae sacrificed during wound healing (1 after amputation), blastema formation (3 days) and blastema growth (5 and 7 days) showed that denervated right limbs have undergone the same histological modifications observed in innervated left limbs and have formed a regeneration blastema consisting of mesenchymal cells with a pattern of DNA synthesis and mitosis very similar to that in presence of nerves. Also, the patterns of cellular density in regenerating right and left limbs were very similar. On the whole, the data here reported show a highly remarkable degree of nerve-independence for regeneration in hindlimbs of larval Xenopus laevis at stage 52–53 and lend some substance to the hypothesis that, in early limbs, there would exist trophic factors capable of replacing those released by nerves, promoting DNA synthesis and mitosis in blastemal cells. Offprint requests to: S. Filoni     

A novel peptide designated PYLa and its precursor as predicted from cloned mRNA of Xenopus laevis skin

A variety of peptides closely related to mammalian hormones and neurotransmitters are secreted from amphibian skin. Using cDNA clones of mRNA isolated from skin of Xenopus laevis, we have been searching for precursors of some of these constituents. Here we present the sequences of parts of cloned mRNAs which code for precursors of a novel peptide. In the predicted polypeptides, pairs of basic residues flank a sequence of 25 amino acids terminating with glycine, the signal for the formation of a terminal amide. The predicted final product liberated from these precursors would be a peptide comprised of 24 amino acids starting with tyrosine and ending with leucine amide, which has therefore been designated PYLa. This peptide can form an amphipathic helix similar to that found in peptides with cytotoxic, bacteriostatic and/or lytic properties.     

Biosynthesis, processing, and control of release of melanotropic peptides in the neurointermediate lobe of Xenopus laevis

The neurointermediate lobes of dark-adapted toads Xenopus laevis were incubated for 30 min in [3H]arginine and then "chased" for various time periods. By use of this pulse-chase paradigm there were detected 10 trichloroacetic acid (TCA)-precipitable peptides separated on acid-urea polyacrylamide gels and one TCA-soluble peptide separated by high- voltage electrophoresis (pH 4.9) with melanotropic activity. Each of these peptides had a different degree of melanocyte stimulating hormone (MSH) activity as revealed by the Anolis skin bioassay. Three of these TCA-precipitable peptides comigrated with ACTH, beta-lipotrophin, and alpha-MSH on acid-urea gels. Evidence suggesting a precursor-product mode of biosynthesis of the melanotropic peptides is presented. 7 of the 10 TCA-precipitable peptides and the one TCA-soluble peptide with melanotropic activity were released into the medium. The half-time of release of the TCA-precipitable peptides was about 2 h, whereas the half-time of TCA-soluble peptide release was about 30 min. The release of these peptides was inhibited by 5 X 10(-5) M dopamine. Dopamine inhibition of release did not appear to affect the biosynthesis of the melanotropic peptides, but did appear to enhance the degradation of the newly synthesized TCA-soluble peptide in the tissue. White adaptation of the toads greatly decreased the biosynthesis of all of the TCA- precipitable melanotropic peptides.     

Levitide, a neurohormone-like peptide from the skin of Xenopus laevis. Peptide and peptide precursor cDNA sequences

A novel peptide, levitide, less than Glu-Gly-Met-Ile-Gly-Thr-Leu-Thr-Ser-Lys-Arg-Ile-Lys-Gln-NH2 has been isolated from skin secretions of the South African frog Xenopus laevis and sequenced by fast atom bombardment mass spectrometry. Synthetic oligonucleotides were used as probes to screen a X. laevis skin cDNA library for species coding for preprolevitide. Two such clones were detected and their sequences are reported here. Preprolevitide is 88 residues long, exhibits a putative signal sequence at the amino terminus, and contains the levitide peptide at the carboxyl terminus. The levitide precursor shows a striking nucleotide and amino acid (86%) sequence homology with the precursor of xenopsin, a biologically active octapeptide from Xenopus skin, and also encodes a 25-residue amphipathic peptide that is released by processing at a single arginine residue.     

Functional and morphological correlates of connexin50 expressed in Xenopus laevis oocytes

Electrophysiological and morphological methods were used to study connexin50 (Cx50) expressed in Xenopus laevis oocytes. Oocytes expressing Cx50 exhibited a new population of intramembrane particles (9.0 nm in diameter) in the plasma membrane. The particles represented hemichannels (connexin hexamers) because (a) their cross-sectional area could accommodate 24 +/- 3 helices, (b) when their density reached 300-400/microm2, they formed complete channels (dodecamers) in single oocytes, and assembled into plaques, and (c) their appearance in the plasma membrane was associated with a whole-cell current, which was activated at low external Ca2+ concentration ([Ca2+]o), and was blocked by octanol and by intracellular acidification. The Cx50 hemichannel density was directly proportional to the magnitude of the Cx50 Ca2+-sensitive current. Measurements of hemichannel density and the Ca2+-sensitive current in the same oocytes suggested that at physiological [Ca2+]o (1-2 mM), hemichannels rarely open. In the cytoplasm, hemichannels were present in approximately 0.1-microm diameter "coated" and in larger 0.2-0.5-microm diameter vesicles. The smaller coated vesicles contained endogenous plasma membrane proteins of the oocyte intermingled with 5-40 Cx50 hemichannels, and were observed to fuse with the plasma membrane. The larger vesicles, which contained Cx50 hemichannels, gap junction channels, and endogenous membrane proteins, originated from invaginations of the plasma membrane, as their lumen was labeled with the extracellular marker peroxidase. The insertion rate of hemichannels into the plasma membrane (80, 000/s), suggested that an average of 4,000 small coated vesicles were inserted every second. However, insertion of hemichannels occurred at a constant plasma membrane area, indicating that insertion by vesicle exocytosis (60-500 microm2 membranes/s) was balanced by plasma membrane endocytosis. These exocytotic and endocytotic rates suggest that the entire plasma membrane of the oocyte is replaced in approximately 24 h.     

Gastrin-releasing peptides from Xenopus laevis: purification, characterization, and myotropic activity

Two molecular forms of gastrin-releasing peptide (GRP) were isolated from an extract of the intestine of the tetraploid frog Xenopus laevis. The primary structure of GRP-1 (APTSQQHTEQ(10)LSRSNINTRG(20) SHWAVGHLM.NH(2)) differs from that of GRP-2 by a single amino acid substitution (Asn(15)--> Thr(15)). GRP-(20-29) peptide (neuromedin C) was also isolated from the extract. Synthetic GRP-1 produced concentration-dependent contractions of longitudinal smooth muscle strips from Xenopus cardiac stomach (pD(2) = 8.93 +/- 0.32; n = 6). The responses were unaffected by tetrodotoxin, atropine, and methysergide, indicating a direct action of the peptide on smooth muscle cells. GRP-1 elicited concentration-dependent relaxations of precontracted (5 microM carbachol) circular smooth muscle strips from the same region (pD(2) = 8.96 +/- 0.21; n = 8). The responses were significantly (P < 0.05) attenuated (71 +/- 24% decrease in maximum response; n = 6) by indomethacin, indicating mediation, at least in part, by prostanoids. Despite the fact that Xenopus GRP-1 differs from pig GRP at 15 amino acid sites, both peptides are equipotent and equally effective for both contractile and relaxant responses, demonstrating that selective evolutionary pressure has acted to conserve the functional COOH-terminal domain in the peptide. The data suggest a physiologically important role for GRP in the regulation of gastric motility in X. laevis.     

Biosynthesis of thyrotropin releasing hormone in the skin of Xenopus laevis: partial sequence of the precursor deduced from cloned cDNA

Skin of Xenopus laevis contains relatively large quantities of thyrotropin releasing hormone (TRH). Total mRNA isolated from skin was cloned in the plasmid pUC8. Among 1400 cDNA clones, one was found with an insert of 478 nucleotides coding for the amino-terminal part of prepro-TRH. This clone was detected using a mixture of two synthetic undecanucleotides for colony hybridization. The single open reading frame starts with a methionine residue and a stretch of hydrophobic amino acids, as is typical for signal peptides, and terminates at the poly(C) tail without a stop codon. The deduced polypeptide of 123 amino acids contains three copies of the sequences Lys-Arg-Gln-His-Pro-Gly-Lys Arg-Arg and a fourth incomplete copy at the carboxyl end. Typical pro-hormone processing at this sequence would yield pGlu-His-Pro.NH2,i.e. TRH. It is concluded that the cloned part of the mRNA codes for prepro-TRH and that the TRH precursor from skin of X. laevis is a polyprotein containing at least four copies of the end product in its amino acid sequence.     

Sperm incorporation in Xenopus laevis: characterisation of morphological events and the role of microfilaments

Scanning and transmission electron microscopy were used to determine the morphological changes in the egg plasma membrane associated with sperm binding, fusion and incorporation in Xenopus laevis. Sperm incorporation in Xenopus is rapid, occurring within 3-5 min following addition of sperm. Images have been obtained of both early sperm-egg interactions and fertilisation bodies. Additionally, two drugs that specifically alter F-actin dynamics, latrunculin and jasplakinolide, were used to determine whether sperm incorporation is a microfilament-dependent process. Jasplakinolide did not prevent sperm incorporation, cortical granule exocytosis or cortical contraction, suggesting these events can occur without depolymerisation of existing, stabilised filaments. Latrunculin A, which competes with thymosin beta4 in ooplasm for binding actin monomer, did not inhibit cortical granule exocytosis, but blocked cortical contraction in 100% of eggs at a concentration of 5 microM. Although a single penetrating sperm was found on an egg pretreated in latrunculin, fertilisation bodies were never observed. At < 5 microM latrunculin, many eggs did undergo cortical contraction with some exhibiting severe distortions of the plasma membrane and abnormal accumulations of pigment granules. Preincubation of eggs in jasplakinolide before latrunculin mitigated both these effects to some degree. However, eggs incubated in latrunculin either prior to or after insemination never progressed through first cleavage.     

Xenopus laevis FoxE1 is primarily expressed in the developing pituitary and thyroid

The members of the FoxE subfamily of Fox (forkhead) genes are expressed in the developing pituitary, thyroid and lens. Mammalian Foxe1 is expressed primarily in the developing pituitary and thyroid gland, Foxe3 is expressed in the developing lens, while Xenopus FoxE4 is expressed in the developing lens and thyroid. Here we report the identification of Xenopus FoxE1, a gene that is primarily expressed in the developing pituitary and thyroid.     

Outer segment growth and periciliary vesicle turnover in developing photoreceptors of Xenopus laevis

Summary Cytoskeletal alterations in the cytoplasm of chromatolytic neurons of the dorsal root ganglia were studied in chickens after transection of the sciatic nerves. These studies were carried out using cryofixation with a nitrogencooled propane jet. By this method, the morphological complexity of the cytoskeleton in normal perikarya and cell processes can be visualized. The cytoskeleton of the dorsal root ganglion cells (DRG) is composed of an intricate network of microtubules, neurofilaments and microfilaments. The membrane-bounded cell organelles, as well as the cell nucleus and the plasmalemma, are linked to the microtubules and neurofilaments by microfilaments (or crosslinkers). As a result of the transection of the axon, chromatolysis takes place, characterized by dislocation of cell organelles, eccentric position of the nucleus and dispersion of the parallel cisternae of the rough endoplasmic reticulum throughout the cytoplasm. This characteristic phenomenon coincides with a regression of the neurocytoskeletal network. The neurofilaments and microtubules become shorter, and the microfilaments are replaced by strands of globular or granular material. The temporary regression of the microfilaments leads to a dispersion of the cell organelles. During the remodelling of the cytoskeletal structures, proliferation of the neurofilaments in the regenerating neurons may occasionally be observed. These results show that the cytoskeletal structures are responsible not only for the preservation of cell shape, but also for the maintenance of the normal distributional pattern (location and mobility) of the intracellular components.     

Hybridization between the African clawed frogs Xenopus laevis and Xenopus muelleri (Pipidae) increases the multiplicity of antimicrobial peptides in skin secretions of female offspring

Peptidomic analysis was used to compare the distribution of host-defense peptides in norepinephrine-stimulated skin secretions from laboratory-generated female F1 hybrids of the common clawed frog Xenopus laevis (Daudin, 1802) and Mueller's clawed frog Xenopus muelleri (Peters, 1844) with the corresponding distribution in skin secretions from the parent species. A total of 18 peptides were identified in secretions from the hybrid frogs. Eleven peptides (magainin-1, magainin-2, CPF-1, CPF-3, CPF-4, CPF-5, CPF-6, CPF-7, XPF-1, XPF-2, and PGLa) were identified in secretions of both the hybrids and X. laevis. Four peptides (magainin-M1, XPF-M1, CPF-M1, and tigerinin-M1) were previously found in skin secretions of X. muelleri but magainin-M2 and CPF-M2 from X. muelleri were not detected. Three previously undescribed peptides (magainin-LM1, PGLa-LM1, and CPF-LM1) were purified from the secretions of the hybrid frogs that were not detected in secretions from either X. laevis or X. muelleri. Magainin-LM1 differs from magainin-2 from X. laevis by a single amino acid substitution (Gly¹³ → Ala) but PGLa-LM1 and CPF-LM1 differ appreciably in structure from orthologs in the parent species. CPF-LM1 shows potent, broad-spectrum antimicrobial activity and is hemolytic. The data indicate that hybridization increases the multiplicity of skin host-defense peptides in skin secretions. As the female F1 hybrids are fertile, hybridization may represent an adaptive strategy among Xenopus species to increase protection against pathogenic microorganisms in the environment.