South African Frog Xenopus Laevis Research Articles

The Xenopus Oocyte as an Expression System for Functional Analyses of Fish Aquaporins.

Aquaporins are membrane proteins present in all organisms that selectively transport water and small, uncharged solutes across biological membranes along an osmotic gradient. Recent gene editing technologies in zebrafish (Danio rerio) have started to uncover the physiological functions of the aquaporins in teleosts, but these approaches require methods to establish the effects of specific mutations on channel function. The oocytes of the South African frog Xenopus laevis are widely used for the expression of bacterial, plant, and animal aquaporins, and this heterologous system has contributed to numerous discoveries in aquaporin biology. This chapter focuses on techniques used for oocyte preparation and aquaporin expression and gives an overview of specific methods to determine water and solute permeability of the channels and their intracellular trafficking in oocytes.

Heterologous expression of C. elegans ion channels in Xenopus oocytes

Physiological methods entered the world of C. elegans, a model system used for many years to study development and a plethora of biological processes mainly employing genetic, molecular and anatomical techniques. One of the methods introduced by physiologists is the use of Xenopus oocytes for expression of C. elegans ion channels. Oocytes of the South African frog Xenopus laevis are used widely for the expression of mammalian channels and transporters contributing to numerous discoveries in these fields. They now promise to aid C. elegans researchers in deciphering mechanisms of channels function and regulation with implications for mammalian patho-physiology. Heterologous cRNA can be easily injected into Xenopus oocytes and translated proteins can be studied using several techniques including electrophysiology, immunocytochemistry and protein biochemistry. This chapter will focus on techniques used for oocyte preparation and injection, and will give a brief overview of specific methods. Limitations of the use of Xenopus oocytes will be also discussed.

Environmentally relevant concentrations of ammonium perchlorate inhibit thyroid function and alter sex ratios in developing Xenopus laevis

Embryos and larvae of the South African frog Xenopus laevis were exposed to ammonium perchlorate (AP) or control medium for 70 d. The dosage levels (59 ppb, 14,140 ppb) bracketed a range of perchlorate concentrations measured in surface waters at the Longhorn Army Ammunition Plant (LHAAP) in Karnack, Texas, USA. The experiment also included a 28-d nontreatment recovery period to assess the reversibility of AP effects. There were no significant effects of AP on mortality or hatching success. There were no effects of AP on developmental abnormalities such as bent/asymmetric tails or edema. Ammonium perchlorate inhibited forelimb emergence, the percentage of animals completing tail resorption, and hindlimb development during the 70-d exposure period. Only the upper AP concentration reduced whole-body thyroxine content, whereas both concentrations caused significant hypertrophy of the thyroid follicular epithelium. Both concentrations of AP caused a skewed sex ratio, significantly reducing the percentage of males at metamorphosis. The effects of AP on metamorphosis and thyroid function were reversed during the 28-d nontreatment recovery period. We conclude that AP inhibits thyroid activity and alters gonadal differentiation in developing X. laevis. These effects were observed at concentrations at or below concentrations reported in surface waters contaminated with ammonium perchlorate, suggesting that this contaminant may pose a threat to normal development and growth in natural amphibian populations.

Identification and structural elucidation of lectin-binding oligosaccharides by bioaffinity matrix-assisted laser desorption/ionization Fourier transform mass spectrometry.

Cortical granule lectin (CGL) is released by the egg of the South African toad Xenopus laevis upon fertilization. The lectin binds to oligosaccharides in the extracellular matrix of the egg to form a physical block to prevent additional sperm penetration or polyspermy. To identify the oligosaccharides that bind to CGL, the lectin was immobilized on the surface of a matrix-assisted laser desorption/ionization probe. This bioaffinity probe was used to determine oligosaccharides that bind preferentially to CGL. Structural analyses based on collision-induced dissociation was used to determine that oligosaccharides with the sulfate esters at the nonreducing ends preferentially bind to the lectin.

Effects of gravity on early development

The development of embryonic and larval stages of the South african Toad Xenopus laevis D, was investigated in hyper-g up to 5 g (centrifuge), in simulated 0 g (fast-rotating clinostat), in alternating low g, hyper-g (parabolic flights) and in μg (Spacelab missions D1, D-2). The selected developmental stages are assumed to be very sensitive to enviromental stimuli. The results showed that the developmental reaction processes run normal also in environments different to 1 g and that aberrations in behavior and morphology normalize after return to 1 g. Development, differentiation, and morphology of the gravity perceiving parts of the vestibular system (macula-organs) had not been affected by exposure to different g-levels.

Gone Fishing! Scientists Use Mutant Zebra Fish to Learn How Vertebrate Embryos Develop

M ark C. Fishman has recently netted mutant fish so strange he has trouble believing they even exist. Take the doomed animal whose blood cells literally fell to pieces when he exposed the fish to light. red blood cells became fluorescent and popped. You could watch the cells explode. It was really rather gorgeous, says Fishman, head of cardiovascular research at the Massachusetts General Hospital (MGH) in Boston. The researcher didn't reel in this hapless mutant from the polluted waters of Boston's Charles River. Like scores of other bizarre fish, it was caught in Fishman's laboratory. The mutants are zebra fish, or Danio rerio, and they are the rage among developmental biologists, who study the seemingly miraculous process in which a fertilized egg cell becomes a multicellular adult organism. Using radiation, chemicals, or viruses, these investigators deliberately trigger mutations in zebra fish in hopes of identifying the genetic repertoire employed by a developing vertebrate embryo. The fledgling field of zebra fish research hit a landmark this month with the publication of a special issue of DEVELOPMENT. In more than 3 dozen articles, researchers describe hundreds of mutant fish produced at MGH and at a laboratory in Tiubingen, Germany. It's the largest collection of mutations affecting embryogenesis that exists for any vertebrate, says Wolfgang Driever, who organized the MGH mutant hunt and is now setting up his own zebra fish laboratory in Freiburg, Germany. The journal celebrates its special issue with a bit of whimsy. The upper corners of the right-hand pages hold a series of 230 digitized images chronicling the development of a zebra fish embryo from its two-celled stage. If thumb quickly through the pages, you can actually see cells dividing, says Donald A. Kane of the University of Oregon in Eugene, who worked on the flip book feature. It's an incredibly fun time now to work with zebra fish. It must have been like this with flies many years ago. Kane's remark about flies refers to the extraordinary success developmental biologists have had with Drosophila melanogaster, the common fruit fly Last year, for example, a Nobel prize honored three researchers who in the 1970s studied mutant fruit flies in which embryogenesis has gone awry (SN: 10/14/95, p. 246). While biologists have been startled to learn that many of the genes that guide insect embryogenesis perform similar tasks in vertebrates, there's only so much fruit flies can tell them. Consequently, scientists have longed for a vertebrate whose development is as easy to study. The South African frog Xenopus laevis, whose embryo is quite large and easily manipulated, has been a leading con-

Crystallization and Preliminary Crystallographic Analysis of Recombinant Xenopus laevis Cu, Zn Superoxide Dismutase b

The recombinant Cu,Zn superoxide dismutase from the South African frog Xenopus laevis, expressed in E. coli, has been crystallized in a form suitable for high resolution crystallographic investigations. The crystals grow from polyethylene glycol solutions, at pH 6.0, 28° C, and belong to the orthorhombic space group P2 12 12 1 with unit cell edges a = 73.33, b = 68.86, c = 59.73 Å, one protein dimer (32,000 Mr) per asymmetric unit. Diffraction data have been collected to 3.0 Å resolution, and a molecular replacement solution found for Xenopus laevis superoxide dismutase using the bovine enzyme as search model. The crystallographic R-factor corresponding to this solution is 0.412, in the 15.0-3.0 Å resolution range.

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.

Novel peptide fragments originating from PGLa and the caerulein and xenopsin precursors from Xenopus laevis.

Skin secretions from the South African frog Xenopus laevis have been chromatographed by high performance liquid chromatography (HPLC), fractionated, and analyzed by fast atom bombardment-mass spectrometry (FAB-MS). The HPLC chromatograms showed the secretion to be a complex mixture with over 30 components at similar levels to the four peptides previously isolated from X. laevis skin, i.e. xenopsin, caerulein, thyrotropin-releasing hormone, and PGLa. FAB-MS analysis of the HPLC fractions gave numerous protonated molecular ions ranging from m/z 491 to 2662. Preliminary assignments of these components were made by comparing these experimental molecular weights to those predicted for regions within the xenopsin, caerulein, thyrotropin-releasing hormone, and PGLa precursors. These results suggested that many of these skin secretions were peptides originating from additional processing of the xenopsin, caerulein, and PGLa precursors, primarily involving cleavage at single arginine residues, and a novel cleavage at the NH2-terminal side of single lysines. These assignments were subsequently confirmed by Edman degradation, FAB-MS peptide sequencing, and amino acid analysis. All of these peptides contain one or more lysines and would be expected to have amphiphilic structures. As yet, nothing is known about their activity, although they resemble in composition the mast cell degranulating peptides melittin and the bombolitins. These precursor fragments were also found to have limited sequence homology to bombinin, a hemolytic amphibian peptide isolated from the European Bombina toad.

A mass spectrometric method for the identification of novel peptides in Xenopus laevis skin secretions.

The peptides secreted by the South African frog Xenopus laevis were screened systematically using a strategy based on fabms. Hplc of crude and Sephadex G-10 chromatographed secretions showed that many more peptides were present in these secretions than those previously identified, i.e., xenopsin, caerulein, TRH, and PGLa. Fractions from the hplc were analyzed directly by fabms to determine the molecular weights of these novel peptides. Subsequent analyses, using a combination of fabms, manual Edman degradation, enzymatic digestions, and amino acid analyses identified the partial and sometimes complete sequences of these peptides which had molecular weights ranging from 700-2700. Many peptides with structural features that are often indicative of biological activity, e.g., C-terminal amides and pyroglutamic acid, were readily identified by fabms. In some cases, molecular weight data combined with partial sequence data was sufficient to identify peptides as originating from spacer regions in the precursors to xenopsin, caerulein, and PGLa.

A mass spectrometric assay for novel peptides: Application to Xenopus laevis skin secretions

The peptides secreted by the South African frog Xenopus laevis were screened systematically using a strategy based on fast atom bombardment mass spectrometry (FAB-MS). High performance liquid chromatography (HPLC) of crude and Sephadex G-10 chromatographed secretions showed that many more peptides were present in these secretions than those previously identified, i.e., xenopsin, caerulein. TRH and PGLa. Fractions from the HPLC were analyzed directly by FAB-MS to determine the molecular weights of these novel peptides. Subsequent analyses, using a combination of FAB-MS, manual Edman degradation, enzymatic digestions and amino acid analyses, identified the partial and sometimes complete sequences of these peptides which had molecular weights ranging from 700–2,700. Many peptides with structural features that are often indicative of biological activity, e.g., C-terminal amides and pyroglutamic acid, were readily identified by FAB-MS. In some cases, molecular weight data combined with partial sequence data was sufficient to identify peptides as originating from PGLa and the spacer regions in the precursors to xenopsin and caerulein.

Some bio-electric parameters of early Xenopus embryos

ABSTRACT Membrane potential and resistance were measured in eggs, cleavage stages and blastulae of the South African toad Xenopus laevis, using intracellular microelectrodes. The membrane potential increased from – 6·5 ±2 mV in eggs to –57 ±8· 0 mV at the mid-blastula stage. The input resistance of fertile eggs ranged from 0·5 MΩ to 5 ·0 MΩ corresponding to a specific resistance of 20–200 kΩ cm2. During the first two or three division cycles the input resistance usually decreased by a factor of 2– 10 and then subsequently rose during the blastula stages from a mean value of 600 ± 100 kΩ at stage 5 to 2·0 ±0·5 MΩ at stage 8. At all developmental stages examined, point polarization of a surface cell in the embryo by rectangular current pulses of 0·5–6 × 10−8 A produced voltage deflexions in other surface cells. This was seen even when several (7–8) cell junctions intervened between the current passing and voltage recording microelectrodes at distances of more than 1 mm. These measurements suggest that the junctional resistance is low compared with that at the surface, though the geometrical arrangement of cells is not favourable for calculation of absolute values of membrane resistance. Current spread between cells occurred apparently less easily during mid-blastula stages than at earlier stages in development, perhaps indicating an increase in junctional resistance during development. A comparison has been drawn between the present measurements and similar ones made in another amphibian, Triturus.

The South African frog Xenopus laevis as a test object in hormone analyses

Acta Medica ScandinavicaVolume 130, Issue S206 p. 512-515 The South African frog Xenopus laevis as a test object in hormone analyses Haqvin Malmros, Haqvin MalmrosSearch for more papers by this author Haqvin Malmros, Haqvin MalmrosSearch for more papers by this author First published: January/December 1948 https://doi.org/10.1111/j.0954-6820.1948.tb12095.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Volume130, IssueS206January/December 1948Pages 512-515 RelatedInformation