Isolation and primary structure of urotensin II from the brain of a tetrapod, the frog Rana ridibunda.

A peptide related to urotensin II has been isolated in pure form from an extract of the brain of the European green frog, Rana ridibunda. The primary structure of the peptide was established as Ala-Gly-Asn-Leu-Ser-Glu-Cys-Phe-Trp-Lys-Tyr-Cys-Val and this sequence was confirmed by chemical synthesis. Frog urotensin II contains an additional amino acid residue compared with fish urotensin II peptides but the structure of the cyclic region of the molecule has been fully conserved. The data show that urotensin II is not confined to the caudal neurosecretory system of fish but is present in the central nervous system of a tetrapod.     

Isolation of peptide hormones from the pancreas of the bullfrog (Rana catesbeiana). Amino acid sequences of pancreatic polypeptide, oxyntomodulin, and two glucagon-like peptides

Insulin, pancreatic polypeptide, glucagon, oxyntomodulin, and two distinct glucagon-like peptides were isolated from acidic ethanol extracts of bullfrog pancreas by gel filtration followed by high pressure liquid chromatography. The amino acid sequences of pancreatic polypeptide, oxyntomodulin, and both glucagon-like peptides were determined. Frog pancreatic polypeptide contains 36 amino acid residues and has a COOH-terminal phenylalaninamide. It is more homologous with human pancreatic polypeptide (61%) than other characterized members of this family of peptides. Frog glucagon has an amino acid composition identical to the NH2-terminal 29 residues of the larger, more abundant oxyntomodulin and was not sequenced. The finding of a single form of glucagon and oxyntomodulin, but two glucagon-like peptides in frog pancreas extract is similar to that found or deduced for mammals.     

The amphibian globin gene repertoire as revealed by the Xenopus genome

The draft genome sequence of the Western clawed frog Xenopus (Silurana) tropicalis facilitates the identification, expression analysis and phylogenetic classification of the amphibian globin gene repertoire. Frog and mammalian neuroglobin display about 67% protein sequence identity, with the expected predominant expression in frog brain and eye. Frog and mammalian cytoglobins share about 69% of their amino acids, but the frog protein lacks the mammalian-type extension at the C-terminus. Like in mammals, X. tropicalis cytoglobin is expressed in many organs including neural tissue. Neuroglobin and cytoglobin genomic regions are syntenically conserved in all vertebrate classes. Frog and fish globin X show only 57% amino acid identity, but gene synteny analysis confirms orthology. The expression pattern of X. laevis globin X differs from that in fish, with a prominent expression in the eye and weak expression in most other examined tissues. Globin X is possibly present as two paralogous copies in X. tropicalis, with one copy showing transition stages of non-functionalization. The amphibian genome contains a previously unknown globin type (tentatively named 'globin Y') which is expressed in a broad range of tissues and is distantly related to the cytoglobin lineage. The globin Y gene is linked to a cluster of larval and adult hemoglobin alpha and beta genes which contains substantially more paralogous hemoglobin gene copies than previously published. Database and gene synteny analyses confirm the absence of a myoglobin gene in X. tropicalis.     

The Frog Prince: a reconstructed transposon from Rana pipiens with high transpositional activity in vertebrate cells

Members of the Tc1/mariner superfamily of transposable elements isolated from vertebrates are transpositionally inactive due to the accumulation of mutations in their transposase genes. A novel open reading frame-trapping method was used to isolate uninterrupted transposase coding regions from the genome of the frog species Rana pipiens. The isolated clones were ~90% identical to a predicted transposase gene sequence from Xenopus laevis, but contained an unpredicted, ~180 bp region encoding the N-terminus of the putative transposase. None of these native genes was found to be active. Therefore, a consensus sequence of the transposase gene was derived. This engineered transposase and the transposon inverted repeats together constitute the components of a novel transposon system that we named Frog Prince (FP). FP has only ~50% sequence similarity to Sleeping Beauty (SB), and catalyzes efficient cut-and-paste transposition in fish, amphibian and mammalian cell lines. We demonstrate high-efficiency gene trapping in human cells using FP transposition. FP is the most efficient DNA-based transposon from vertebrates described to date, and shows ~70% higher activity in zebrafish cells than SB. Frog Prince can greatly extend our possibilities for genetic analyses in vertebrates.     

Habitat-mediated impact of alien mink predation on common frog densities in the outer archipelago of the Baltic Sea

Alien predators have been recognised as one possible cause for amphibian declines around the world, but little is known of habitat-mediated predation impacts especially on adult amphibians. We studied common frog Rana temporaria under American mink Mustela vison predation in the outer archipelago of the Baltic Sea, south-western Finland. Using egg batches as an index of breeding frog female numbers we compared frog numbers and densities between a large, long-term mink-removal area and a comparable control area. Frog numbers in the removal area were at least 2.7-fold higher than those in the control area. In the presence of mink, frog densities increased with the amount of vegetation cover on the islands, indicating that mink predation affected frog densities especially on less-vegetated islands. An opposite trend appeared to be true for frogs in the mink-removal area, where other predators like snakes could induce a decline of frog densities on more vegetated islands. Shrub or grass vegetation seems to provide frogs shelter against alien mink predation. Our result highlights the importance of landscape-level habitat management as a conservation tool for amphibian populations.     

Somatostatin modulation of neurally mediated pepsinogen secretion from frog esophageal mucosa

Frog esophageal mucosa contains peptic glands which are innervated by cholinergic neurons. When incubated in a medium containing 1.5 mM CaCl₂, pepsinogen release from esophageal mucosa was increased by a high potassium concentration (55 mM KCl), 1,1-dimethyl-4-phenylpiperazinium (DMPP) or bethanechol. Whereas the response to bethanechol remained little changed, the response to high KCl concentrations or DMPP was abolished in the absence of Ca²⁺. The stimulatory effects of high KCl concentrations and DMPP were also eliminated by the presence of atropine or somatostatin. Furthermore, pepsinogen release in response to bethanechol was dose-dependently inhibited by somatostatin. Frog esophagus was found to contain somatostatin-like immunoreactivity, with a higher density at the end adjacent to the stomach. Chromatography of mucosa extract on Sephadex G-50 revealed a single peak of somatostatin-like immunoreactivity that coeluted with somatostatin-14. Immunohistochemical staining of the mucosa with peroxidase antiperoxidase technique demonstrated the presence of two varieties of somatostatin-like immunoreactivity-containing cells, one individually dispersed within the intercalated septa and the other in groups within the interlobular septa of the peptic glands. These results seem to indicate that somatostatin or somatostatin-like immunoreactivity may play a modulatory role in neurally mediated pepsinogen secretion in the frog esophagus.     

The effect of ethanol on ion transport in frog skin

Frog skin has been used as a model epithelial sodium-transporting system to study the effect of ethanol on ion transport. Treatment of the outside of frog skin with ethanol decreased the net sodium transport due to inhibition of ²²Na⁺ influx. Ethanol did not alter sodium outflux when bathing the outside of the skin. The inhibition was in proportion to the concentration of ethanol, 0.25 M resulting in 50% inhibition. The chloride permeability of the skin was increased several-fold when the skin was exposed to ethanol in either bathing solution. With 0.4 M ethanol in the inner bathing solution, all the unidirectional fluxes of Na⁺ and Cl^? were increased. The movement of Cl^? was evaluated by comparison of Cl^? flux with urea flux, since urea is thought to move passively across frog skin via an extracellular (shunt) pathway. Chloride flux was increased to a greater extent than urea flux. These experiments indicate that ethanol affects chloride permeability beyond an increase in extracellular ion flow and independent of its effect on Na⁺ transport.     

Hepatic ornithine decarboxylase from the frog, Rana negromaculata: dietary induction, purification and some properties

1. In the liver of the frog, Rana negromaculata, the activity of ornithine decarboxylase (ODC) was induced by dietary stimuli and was rapidly lost upon intraperitoneal injection of cycloheximide or putrescine. 2. Frog liver ODC, purified by DEAE-Cellulofine and immunoaffinity column chromatographies, was used in a comparative study with mouse kidney ODC, also purified by the same method. 3. The purified frog ODC showed three bands on SDS-polyacrylamide gel electrophoretic analysis, as confirmed by [3H]alpha-difluoromethylornithine binding. 4. Frog ODC was found to be similar to mouse enzyme in some properties, for example molecular weight, immunoreactivity and inhibition by rat antizyme, except for a slightly higher Km value for ornithine.     

Seasonal variation and an “outbreak” of frog predation by tamarins

We report temporal variation and an “outbreak” of frog predation by moustached tamarins, Saguinus mystax, in north-eastern Peruvian Amazonia. Frog predation rates were generally very low, but strongly increased in October 2015. Other high rates, identified by outlier analyses, were also observed in September–November of other years. Over all study years, predation rates in this 3-month period were significantly higher than those in the remainder of the year, suggesting a seasonal pattern of frog predation by tamarins. Reduced fruit availability or increased frog abundance or a combination of both may be responsible for both the seasonal pattern and the specific “outbreak” of frog predation.     

The molecular weight of rhodopsin and the nature of the rhodopsin-digitonin complex

The sedimentation behavior of aqueous solutions of digitonin and of cattle rhodopsin in digitonin has been examined in the ultracentrifuge. In confirmation of earlier work, digitonin was found to sediment as a micelle (D-1) with an s₂₀ of about 6.35 Svedberg units, and containing at least 60 molecules. The rhodopsin solutions sediment as a stoichiometric complex of rhodopsin with digitonin (RD-1) with an s₂₀ of about 9.77 Svedberg units. The s₂₀ of the RD-1 micelle is constant between pH 6.3 and 9.6, and in the presence of excess digitonin. RD-1 travels as a single boundary also in the electrophoresis apparatus at pH 8.5, and on filter paper at pH 8.0. The molecular weight of the RD-1 micelle lies between 260,000 and 290,000. Of this, only about 40,000 gm. are due to rhodopsin; the rest is digitonin (180 to 200 moles). Comparison of the relative concentrations of RD-1 and retinene in solutions of rhodopsin-digitonin shows that RD-1 contains only one retinene equivalent. It can therefore contain only one molecule of rhodopsin with a molecular weight of about 40,000. Cattle rhodopsin therefore contains only one chromophore consisting of a single molecule of retinene. It is likely that frog rhodopsin has a similar molecular weight and also contains only one chromophore per molecule. The molar extinction coefficient of rhodopsin is therefore identical with the extinction coefficient per mole of retinene (40,600 cm.² per mole) and the E(1 per cent, 1 cm., 500 mµ) has a value of about 10. Rhodopsin constitutes about 14 per cent of the dry weight, and 3.7 per cent of the wet weight of cattle outer limbs. This corresponds to about 4.2 x 10⁶ molecules of rhodopsin per outer limb. The rhodopsin content of frog outer limbs is considerably higher: about 35 per cent of the dry weight, and 10 per cent of the wet weight, corresponding to about 2.1 x 10⁹ molecules per outer limb. Thus the frog outer limb contains about five hundred times as much rhodopsin as the cattle outer limb. But the relative volumes of these structures are such that the ratio of concentrations is only about 2.5 to 1 on a weight basis. Rhodopsin accounts for at least one-fifth of the total protein of the cattle outer limb; for the frog, this value must be higher. The extinction (K₅₀₀) along its axis is about 0.037 cm.² for the cattle outer limb, and about 0.50 cm.² for the frog outer limb.     

Identification of new atrial natriuretic peptides in frog heart

It has been observed that mammalian atrial natriuretic peptide (ANP)-like immunoreactivity is found in frog heart, but to date the natriuretic factors have not yet been identified. Isolation from bull-frog heart extract was performed mainly by immunoaffinity chromatography on a column linked with anti-hANP IgG. From the low molecular weight fraction, 24- and 21-amino acid peptides were purified to homogeneity. Both peptides were found to elicit diuretic-natriuretic as well as vasorelaxant activity, and were named "frog ANP-24" and "frog ANP-21", respectively. Complete amino acid sequences of the peptides were established by microsequencing and confirmed by syntheses. Frog ANP-21 was identified as an N-terminally three amino acid deleted form of frog ANP-24. Remarkable sequence homology was observed between frog ANP and mammalian ANP, especially in the regions flanked by two half-cystine residues.     

Luteinizing hormone-releasing factor (LRF)-like immunoreactivity in rat pancreatic islet cells.

Luteinizing hormone-releasing factor (LRF)-like immunoreactive material was demonstrated by the three-layer immunoperoxidase method in formalin-fixed tissue sections of the rat pancreas. Anti-LRF antiserum was prepared in rabbits by immunizing with synthetic LRF coupled to bovine serum albumin (BSA). The immunoreactive site of LRF reacting with antiserum resided between residues Tyr⁵ and Gly¹⁰-NH₂. A positive staining reaction was observed in the islet cells with the use of anti-LRF antiserum after solid phase immunoadsorption with BSA, whereas no staining was observed when adjacent control sections were prepared with anti-LRF antiserum after immunoadsorption with an LRF-BSA conjugate, or with rabbit anti-oxytocin antiserum. LRF-like immunoreactive material was isolated from the rat pancreata by methanol extraction. This material coeluted with synthetic and hypothalamic LRF in cation exchange chromatography on carboxymethyl cellulose, and dilutions of it gave an inhibition curve parallel to that of synthetic LRF in radioimmunoassay. The concentration of LRF-like material in the rat pancreas is 1.1 pg/mg wet weight. These results suggest that LRF or a closely LRF-related peptide is shared by the central nervous system and the gastrointestinal tract.     

THE PRESENCE OF CYSTEINE IN FROG MYELIN BASIC PROTEIN

—Frog myelin basic protein, when subjected to ion-exchange chromatography at alkaline pH, underwent conversion to a higher molecular weight form. Treatment of the latter with 2-mercaptoethanol regenerated the monomeric basic protein. Amino acid analysis of the monomer and the higher molecular weight species after performic acid oxidation demonstrated the presence of approximately 1 mol of cysteic acid per mol of protein of molecular weight 19,700. Treatment of the monomer with the mild oxidizing agent azodicarboxylic acid bis dimethyl amide resulted in its partial conversion to the higher molecular weight form. These studies demonstrate that the frog myelin basic protein, unlike those of all other species hitherto examined, contains a single cysteinyl residue in its polypeptide chain.     

Cell to Cell Adhesion Systems in Xenopus laevis, the South African Clawed Frog I. Detection of Ca2+ Dependent and Independent Adhesion Systems in Adult and Embryonic Cells

The reaggregation kinetics of frog ( Xenopus laevis ) cells prepared using several different dissociation procedures were monitored. Two distinct modes of cell adhesion were revealed, one mediated by Ca²⁺ dependent cell-cell adhesion system (CDS) and the other mediated by Ca²⁺ independent one (CIDS).
CDS was detected in frog embryonic (two-cell stage to neurula) cells and in cells of epithelial cell lines (A6 and A8), while CIDS was detected only in A6 cells. Frog CDS was resistant to 0.01% tryptic digestion in the presence of 1 mM Ca²⁺ and CIDS was resistant to dilute (0.0001%) tryptic digestion. Cells with both mechanisms were prepared by dissociation with 1 mM EDTA and both mechanisms were absent in cells dissociated with 0.01% trypsin and 1 mM EDTA. Frog CDS and CIDS functioned in a temperature dependent and independent manner, respectively. Properties of frog CDS and CIDS revealed in this study were the same as those of mammalian or avian CDS and CIDS, respectively. Frog cells with CDS cross-adhered to mammalian epithelial (F9) cells but not to fibroblastic (V79) cells Ca²⁺ dependently. Monoclonal antibody ECCD-1 raised against mouse epithelial CDS blocked aggregation of frog A8 cells.
These results suggest the similarity between frog CDS and mammalian epithelial type CDS.     

Characterization of the cDNA encoding proopiomelanocortin in the frog Rana ridibunda

In the amphibian pars intermedia, secretion of proopiomelanocortin (POMC)-derived peptides is controlled by multiple factors including classical neurotransmitters and neuropeptides. To pursue questions concerning the regulation of POMC gene expression in Rana ridibunda, we have isolated and characterized a full-length cDNA for frog POMC. A cDNA clone isolated from a frog pituitary library contains an open-reading frame of 780-bp that predicts a 260 amino acid POMC protein. The structure of frog POMC demonstrates considerable amino acid sequence similarity with POMC from other species. In particular, the sequence of alpha-melanotropin (alpha-MSH) is identical in frog and all mammalian species studied so far, while adrenocorticotropin (ACTH) and beta-endorphin exhibit 79% and 84% homology with their human counterpart. Frog POMC contains only one potential asparagine-linked N-glycosylation signal (Asn-Ser-Thr) within the gamma-MSH domain. The alpha-MSH sequence is C-terminally flanked by the Gly-Lys-Lys amidation signal while the joining peptide is not amidate.     

Electrophoretic analysis of liver and testis histones of the frog Rana pipiens

Histones were extracted from frog livers and testes and analyzed by electrophoresis on long polyacrylamide gels and on sodium dodecyl sulfate (SDS)-containing polyacrylamide gels. Frog histones were found to be similar to those of calf thymus except that frog histone fraction F2A2 showed a marked dependence on the temperature at which the long gels were run, and frog histone fraction F3 could be separated from frog F2B on SDS-containing gels. Comparisons between frog liver and frog testis histones indicated that the testis contains as its major F1 component a fast migrating species not found in liver. Testis histones also showed less microheterogeneity of fractions F3 and F2A1 than liver histones. These were the only differences observed between liver and testis histones, even when testis histones were prepared from sperm suspensions that were rich in cells in the late stages of spermiogenesis. Thus it seems that, in Rana, the electrophoretic properties of the basic proteins of sperm differ from those of somatic cells only in the nature of histone F1 and in the degree of microheterogeneity of fractions F2A1 and F3.     

Frog lysozyme. V. Isolation and some physical and immunochemical properties of lysozyme isozymes of the leopard frog, Rana pipiens.

Frog Lysozyme has been purified by sequential application of acid extraction, salt fractionation, CM-cellulose chromatography, heat treatment, and gel filtration. Eight isozymes of purified lysozyme were found to be stable during prolonged storage. Isozymes were separated by preparative polyacrylamide gel electrophoresis, Ninety percent of the lytic activity of frog ovarian egg was represented by forms 7 and 8, the most highly charged isozymes. Seventy-eight percent of frog liver lysozyme activity was that of form 4. Forms 7 and 8 differed from form 4 by being larger (apparent molecular weight of 18,000 vs. 16,000), by remaining active in more acidic environment, and by exhibiting a dependency upon NaCl for activity. Antiserum prepared against frog form 4 did not react with frog forms 7 and 8 and antiserum to chicken egg-white lysozyme did not react with any frog lysozymes. All frog lysozymes showed identical reversible binding to deaminated chitin. Apparent size differences and lack of immunological cross-reactivity suggest that at least some of the isozymes are non-allelic.     

Population Recovery following Decline in an Endangered Stream-Breeding Frog (Mixophyes fleayi) from Subtropical Australia

Amphibians have undergone dramatic declines and extinctions worldwide. Prominent among these have been the stream-breeding frogs in the rainforests of eastern Australia. The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has been postulated as the primary cause of these declines. We conducted a capture-mark-recapture study over a 7-year period on the endangered Fleay’s barred frog (Mixophyes fleayi) at two independent streams (30 km apart) in order to assess the stability of these populations. This species had undergone a severe decline across its narrow geographic range. Mark-recapture modelling showed that the number of individuals increased 3–10 fold along stream transects over this period. Frog detection probabilities were frequently above 50% but declined as the populations increased. Adult survival was important to overall population persistence in light of low recruitment events, suggesting that longevity may be a key factor in this recovery. One male and female were present in the capture record for >6 years. This study provides an unambiguous example of population recovery in the presence of Bd.