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PubMed2004

Membrane translocation mechanism of the antimicrobial peptide buforin 2.

The antimicrobial peptide magainin 2 isolated from the skin of the African clawed frog Xenopus laevis crosses lipid bilayers by transiently forming a peptide-lipid supramolecular complex pore inducing membrane permeabilization and flip-flop of membrane lipids [Matsuzaki, K., Murase, O., Fujii, N., and Miyajima, K. (1996) Biochemistry 35, 11361-11368]. In contrast, the antimicrobial peptide buforin 2 discovered in the stomach tissue of the Asian toad Bufo bufo gargarizans efficiently crosses lipid bilayers without inducing severe membrane permeabilization or lipid flip-flop, and the Pro(11) residue plays a key role in this unique property [Kobayashi, S, Takeshima, K., Park, C. B., Kim, S. C., and Matsuzaki, K. (2000) Biochemistry 39, 8648-8654]. To elucidate the translocation mechanism, the secondary structure and the orientation of the peptide in lipid bilayers as well as the effects of the peptide concentration, the lipid composition, and the cis-trans isomerization of the Pro peptide bond on translocation efficiency were investigated. The translocation efficiencies of F10W-buforin 2 (BF2), P11A-BF2, and F5W-magainin 2 (MG2) across egg yolk L-alpha-phosphatidyl-DL-glycerol (EYPG)/egg yolk L-alpha-phosphatidylcholine (1/1) bilayers were dependent supralinearly on the peptide concentration, suggesting that the translocation mechanisms of these peptides are similar. The incorporation of the negative curvature-inducing lipid egg yolk L-alpha -phosphatidylethanolamine completely suppressed the translocation of BF2, indicating the induction of the positive curvature by BF2 on the membrane is related to the translocation process, similarly to MG2. In pure EYPG, where the repulsion between polycationic BF2 molecules is reduced, membrane permeabilization and coupling lipid flip-flop were clearly observed. Structural studies by use of Fourier transform infrared-polarized attenuated total reflection spectroscopy indicated that BF2 assumed distorted helical structures in EYPG/EYPC bilayers. A BF2 analogue with an alpha-methylproline, which fixed the peptide bond to the trans configuration, translocated similarly to the parent peptide, suggesting the cis-trans isomerization of the Pro peptide bond is not involved in the translocation process. These results indicate that BF2 crosses lipid bilayers via a mechanism similar to that of MG2. The presence of Pro(11) distorts the helix, concentrating basic amino acid residues in a limited amphipathic region, thus destabilizing the pore by enhanced electrostatic repulsion, enabling efficient translocation.

Amino Acid Sequence↗
PubMed2001

Volume changes of individual melanosomes measured by scanning force microscopy.

Black pigment cells, melanophores, e.g. located in the epidermis and dermis of frogs, are large flat cells having intracellular black pigment granules, called melanosomes. Due to a large size, high optical contrast, and quick response to drugs, melanophores are attractive as biosensors as well as for model studies of intracellular processes; e.g. organelle transport and G-protein coupled receptors. The geometry of melanosomes from African clawed toad, Xenopus laevis, has been measured using scanning force microscopy (SFM). Three-dimensional images from SFM were used to measure height, width, and length of the melanosomes (100 from aggregated cells and 100 from dispersed cells). The volumes of melanosomes isolated from aggregated and dispersed melanophores were significantly different (P < 0.05, n=200). The average ellipsoidal volume was 0.14+/-0.01 (aggregated) and 0.17+/-0.01 microm3 (dispersed), a difference of 18%. The average major diameter was 810+/-20 and 880+/-20 nm for aggregated and dispersed melanosomes, respectively. To our knowledge, this is the first time SFM has been used to study melanosomes. This may provide an alternative non-destructive technique that may be particularly suitable for studying morphological aspects of various melanin granules.

Animals↗
PubMed2002

Hans H. Ussing--scientific work: contemporary significance and perspectives.

As a zoologist, Hans H. Ussing began his scientific career by studying the marine plankton fauna in East Greenland. This brought him in contact with August Krogh at the time George de Hevesy, Niels Bohr and Krogh planned the application of artificial radioactive isotopes for studying the dynamic state of the living organism. Following his studies of protein turnover of body tissues with deuterium-labeled amino acids, Ussing initiated a new era of studies of transport across epithelial membranes. Theoretical difficulties in the interpretation of tracer fluxes resulted in novel concepts such as exchange diffusion, unidirectional fluxes, flux-ratio equation, and solvent drag. Combining methods of biophysics with radioactive isotope technology, Ussing introduced and defined the phrases 'short-circuit current', 'active transport pathway' and 'shunt pathway', and with frog skin as experimental model, he unambiguously proved active transport of sodium ions. Conceived in his electric circuit analogue of frog skin, Ussing associated transepithelial ion fluxes with the hitherto puzzling 'bioelectric potentials'. The two-membrane hypothesis of frog skin initiated the study of epithelial transport at the cellular level and raised new questions about cellular mechanisms of actions of hormones and drugs. His theoretical treatment of osmotic water fluxes versus fluxes of deuterium labeled water resulted in the discovery of epithelial water channels. His discovery of paracellular transport in frog skin bridged studies of high and low resistance epithelia and generalized the description of epithelial transport. He devoted the last decade of his scientific life to solute-coupled water transport. He introduced the sodium recirculation theory of isotonic transport, and in an experimental study, he obtained the evidence for recirculation of sodium ions in toad small intestine. In penetrating analyses of essential aspects of epithelial membrane transport, Ussing provided insights of general applicability and powerful analytical methods for the study of intestine, kidney, respiratory epithelia, and exocrine glands-of equal importance to biology and medicine.

Animals↗
PubMed1977

Cobalamin binding proteins in stomach and serum from various animal species data for B12 binding capacities and molecular sizes of the binding proteins.

1. The unsaturated cobalamin-binding capacity of stomach mucosa and serum from 37 animals and the size of the binders have been measured. 2. The binding capacity in stomach mucosa was from 1 to 600 nmole kg-1 wet wt, the highest values occurring in pig, guinea pig, porpoise and earthworm. In serum it varied from 0.4 to 270 nmole 1(-1), the highest values occurring in duck, grass snake, toad and spiny dogfish. 3. The size in terms of Stokes radius of the cobalamin binders in stomach mucosa varied from 1 nm in hagfish (Mr approximately 14,000) to 7 nm in lamprey (Mr approximately 210,000). In serum it was from 1.4 nm (Mr approximately 17,000) to 7.4 nm (Mr approximately 240,000) both in frog.

Animals↗
PubMed2007

Myosin VI and VIIa distribution among inner ear epithelia in diverse fishes.

Unconventional myosins are critical motor proteins in the vertebrate inner ear. Mutations in any one of at least six different myosins can lead to human hereditary deafness, but the precise functions of these proteins in the ear are unknown. This study uses a comparative approach to better understand the role of myosins VI and VIIa in vertebrate ears by examining protein distribution for these two myosins in the ears of evolutionarily diverse fishes and the aquatic clawed toad Xenopus laevis. Both myosins are expressed in the inner ears of all species examined in this study. Myo7a localizes to hair cells, particularly the actin-rich hair bundle, in all species studied. Myo6 also localizes to hair cells, but its distribution differs between species and end organs. Myo6 is found in hair bundles of most fish and frog epithelia examined here but not in anterior and posterior utricular hair bundles of American shad. These results show that myo7a distribution is highly conserved in diverse vertebrates and suggest functional conservation as well. The finding of myo6 in fish and Xenopus hair bundles, however, suggests a novel role for this protein in anamniotic hair cells. The lack of myo6 in specific American shad utricular hair bundles indicates a unique quality of these cells among fishes, perhaps relating to ultrasound detection capability that is found in this species.

Actins↗
PubMed2006

Social transmission of novel foraging behavior in bats: frog calls and their referents.

The fringe-lipped bat, Trachops cirrhosus, uses prey-emitted acoustic cues (frog calls) to assess prey palatability . Previous experiments show that wild T. cirrhosus brought into the laboratory are flexible in their ability to reverse the associations they form between prey cues and prey quality . Here we asked how this flexibility can be achieved in nature. We quantified the rate at which bats learned to associate the calls of a poisonous toad species with palatable prey by placing bats in three groups: (a) social learning, in which a bat inexperienced with the novel association was allowed to observe an experienced bat; (b) social facilitation, in which two inexperienced bats were presented with the experimental task together; and (c) trial-and-error, in which a single inexperienced bat was presented with the experimental task alone. In the social-learning group, bats rapidly acquired the novel association in an average of 5.3 trials. In the social-facilitation and trial-and-error groups, most bats did not approach the call of the poisonous species after 100 trials. Thus, once acquired, novel associations between prey cue and prey quality could spread rapidly through the bat population by cultural transmission. This is the first case to document predator social learning of an acoustic prey cue.

Animals↗
PubMed2001

Caffeine-induced depolarization in amphibian skeletal muscle fibres: role of Na+/Ca2+ exchange and K+ release.

Caffeine (4 mM) produces a depolarization of about 10 mV in frog muscle fibres (Leptodactylus ocellatus). The aim of this work was to study the mechanisms of this effect. An approximately threefold rise in membrane resistance [Cl--free (SO(4)2-) medium] substantially increased, and both Na+-free medium and Ni2+ (5 mM) reduced, the caffeine-induced depolarization. In voltage-clamped (-60 mV) short fibres from lumbricalis muscle of the toad (Buffo arenarum), caffeine generated an inward current of 4.13 +/- 0.48 microA cm(-2). This caffeine-induced current was reduced by 60% in Na+-free medium, 44% in the presence of 5 mM amiloride and 48% by 5 mM Ni2+, suggesting that the activation of the Na+-Ca2+ exchanger in its forward mode may play a role in the observed electrical effects of the drug. Caffeine also produced a marked release of K+. Net K+ efflux increased from 3.5 +/- 0.2 (control) to 22.1 +/- 2.3 pmol s(-1) cm(-2) (caffeine). It is shown that in the presence of the drug, [K+] in the lumen of the T tubules may well increase to levels which could produce, in part, both the observed depolarization and the caffeine-induced current under voltage clamp conditions. The caffeine-induced K+ efflux was not reduced by 5 mM Ni2+. At a holding potential of 30 mV the caffeine-induced current was reversed (outward) and roughly halved by 5 mM Ni2+. The Ni2+-sensitive fraction of the caffeine-induced current, assumed to represent the Na+-Ca2+ exchanger current, had an estimated reversal potential close to 12 mV ([Na+]o = 115 mM; [Ca2+]o = 1 mM). In conclusion, the depolarizing effect of caffeine described here would be produced by two mechanisms: (a) an inward current generated by the activation of the Na+-Ca2+ exchanger in its forward mode, and (b) the rise of the external [K+] in restricted spaces like the T tubules.

Amiloride↗
PubMed1997

Mucormycosis in a free-ranging green tree frog from Australia.

Mucor amphibiorum is reported for the first time from a free-ranging native amphibian, a green tree frog (Litoria caerulea) from Queensland, Australia. The nasal cavity was largely replaced by granulomatous inflammatory tissue, and most internal organs had nodular granulomas. Typical mother and daughter sphaerules of M. amphibiorum occurred in these nodules which were due to granulomatous inflammation as well as areas of more active mixed inflammation with necrosis. Tissue homogenate from the spleen was inoculated into two cane toads (Bufo marinus), and one toad became infected with M. amphibiorum.

Animals↗
PubMed1996

Analysis of isolates within species of anuran trypanosomes using random amplified polymorphic DNA.

A total of 20 decamer primers were used to generate random applied polymorphic DNA (RAPD) markers from 5 isolates of Trypanosoma fallisi, 3 isolates of T. ranarum, 2 isolates of T. rotatorium, and 2 isolates of T. rotatorium-like trypanosomes in addition to 2 species from the American Type Culture Collection, T. chattoni (ATCC 50294) and Trypanosoma sp. (ATCC 50295). A slight polymorphism was observed among the four isolates of T. fallisi obtained form American toads, Bufo americanus, collected in Algonquin Park, Ontario, Canada, and an isolate obtained from the same species of host collected in Marquette, Michigan, United States, and produced similarity coefficients ranging from 80.7% to 96.9%. Pronounced polymorphism was recorded among the three isolates of T. ranarum from bullfrogs, Rana catesbeiana, collected in Ontario, Canada, and in Maryland, United States, and from a Northern leopard frog, R. pipiens, collected in Minnesota (USA). The similarity coefficients ranged from 54.7% to 59.5%, suggesting that alleles of these isolates were conserved over a wide geographic range. The high degree of polymorphism observed in two isolates of T. rotatorium from a bullfrog collected in Ontario and two isolates of a T. rotatorium-like parasite from the green frog R. clamitans, collected in Louisiana (USA) suggests that they are different species. These results reflect the high similarity among isolates from the same geographic location and the pronounced polymorphism apparent among isolates from distant geographic locations.

Animals↗
PubMed1992

Somitogenesis in the marsupial frog Gastrotheca riobambae.

This paper reports the first description of somitogenesis in a non-aquatic-developing amphibian, the Andean marsupial frog, Gastrotheca riobambae. This frog develops from an embryonic disk located on top of a large yolky egg, bearing some resemblance to the embryo of the chick. Besides the histological characterization of somite formation, we quantified cell number in the developing somites of Gastrotheca, Xenopus laevis and the chick. Gastrotheca was found to have a mode of somitogenesis which has previously been encountered in the aquatic-developing toad Bombina. Somitic cell number was found to be an order of magnitude higher than that of Xenopus, and approximately double that of the chick. We discuss the possible relation between mode of somitogenesis, somitic cell number, speed of development and egg size.

Animals↗
PubMed1991

Study of frog (Rana esculenta) proopiomelanocortin processing in the intermediate pituitary. Identification of alpha-melanotropin, beta-melanotropin, Lys-gamma-melanotropin, and corticotropin-like intermediate lobe peptide.

The proteolytic processing of frog (Rana esculenta) proopiomelanocortin in melanotropic cells of the intermediate pituitary gland has been examined through purification of the mature fragments by reverse-phase high-pressure liquid chromatography and microsequencing of isolated peptides. alpha-Melanotropin, beta-melanotropin, Lys-gamma-melanotropin, corticotropin-like intermediate lobe peptide, and hinge peptide have been isolated and chemically characterized. The results show a high preservation in the processing sites of frog proopiomelanotropin when compared to bovine counterparts. They reveal also a great conservation of the processing enzyme equipment of melanotropic cells in tetrapods species. Identification of Lys-gamma-melanotropin suggests the occurrence of an endopeptidase able to cleave between two basic residues. On the other hand alpha-melanotropin does not appear to be N-acetylated, as previously found in the clawed-toad Xenopus laevis, and this feature might distinguish amphibian from mammalian proopiomelanocortin processing.

Adrenocorticotropic Hormone↗
PubMed1999

Behavioral, molecular and integrative mechanisms of amphibian osmoregulation.

Amphibian water balance has been studied at many levels of biological order. Terrestrial species must react to environmental cues that relate to water availability while some arboreal species have cutaneous skin secretions that can reduce evaporative water loss. The Indian tree frog. Polypedates maculatus, uses cutaneous secretions and wiping behavior to lower evaporation but also relies on moist microclimates to endure prolonged survival away from water. The related species, P. leucomystax, inhabits wetter forest habitats. Preliminary studies with this species are unable to demonstrate the expression of wiping behavior, indicating that arid habitats may be a powerful selective force for this behavior. Laboratory experiments on rehydrating toads in the genus Bufo indicate that animals are able to detect changes in barometric pressure and humidity that might result in the availability of water under field situations. Experiments with Bufonid species and with spadefoot toads, Scaphiopus couchi, show that the peptide hormone, angiotensin II, stimulates cutaneous drinking in a similar manner seen for oral drinking by other vertebrate classes. Amphibian tissues have long been used as a model for the study of basic physiological principles of epithelial ion and water transport. Recent progress with tissue cultures has provided information on the molecular structure of ion and water channels that can be applied to obtain a better understanding, at the molecular level, of ion and water balance strategies used by the wide variety of amphibian species. Terrestrial amphibians are more tolerant of dehydration than are other vertebrates and are able to store dilute urine in their urinary bladder. Toads appear to be able to detect the presence of water in their bladders in addition to the availability of water in their environment. Dehydrated toads are able to rehydrate very rapidly by the coordination of behavioral and physiological mechanisms to enhance cutaneous water absorption. The integration of behavior with cutaneous water gain, renal handling of ions and water and the role of the lymphatic system in overall water balance involves complex interactions between neural and hormonal factors. Experiments are summarized that describe the contribution of individual factors however much more information is needed before the nature of these interactions are fully understood.

Adaptation, Physiological↗
PubMed2002

Glutathione transferase isoenzymes from frog (Xenopus laevis) liver and embryo.

The expression of glutathione transferase isoenzymes has been investigated in embryo and adult liver of the frog Xenopus laevis. By analysing the GST isoenzymes recovered from GSH-affinity chromatography in terms of electrophoretic mobility, HPLC elution profile, immunological reactivity, N-terminal amino acid sequence and mass spectrometry molecular mass no significant difference in the GST subunit composition between embryos and liver was found. In both tissues the same three subunits, showing similarity to mu, alpha and sigma class GSTs, are present. These results, together with those previously reported for toad (Bufo bufo), strongly support the notion that the transition from an aquatic environment to a terrestrial atmosphere containing high oxygen concentration has accompanied specific GST gene expression.

Amino Acid Sequence↗
PubMed1993

Differential processing of provasotocin: relative increase of hydrin 2 (vasotocinyl-Gly) in amphibians able to adapt to an arid environment.

Hydrin 2 (vasotocinyl-Gly) is an intermediate in pro-vasotocin processing found, along with vasotocin, only in the neurohypophysis of anuran amphibians. It increases cutaneous water permeability in the frog and is likely involved in neuroendocrine control of osmoregulation. The relative amounts of vasotocin and hydrin 2 stored in neurohypophysis have been measured on the one hand in amphibian species known not to adapt in dry areas, on the other hand in two species, Bufo regularis (Africa) and Bufo viridis (Near-East) able to survive in an arid environment. In the first group, the proportions of the two peptides are approximately equal whereas in the two toads the molar ratio hydrin 2 to vasotocin reaches 2. The ratio does not appear to vary significantly when these toads are either submitted to dehydration or placed in saline solutions. Predominance of hydrin 2 suggests an adaptive decrease of the activity of the alpha-amidating enzymatic system involved in the conversion of vasotocinyl-Gly into mature amidated vasotocin.

Acclimatization↗
PubMed2005

Functional roles for the compartmentalization of the subcutaneous lymphatic sacs in anuran amphibians.

Compliance of the subcutaneous lymph sacs of the hindlimbs increases from distal to proximal, as does limb segment mass (and presumably rate of lymph formation), for the semiaquatic bullfrog Rana catesbeiana and the cane toad Bufo marinus but not the aquatic clawed toad Xenopus laevis. Subcutaneous lymph-sac compliances vary interspecifically. The distal-to-proximal increase in lymph-sac compliance and estimates of lymph formation rate in the various hindlimb segments indicate that partitioning of hindlimb subcutaneous lymphatic sacs establishes a differential decrease in the intra-lymph-sac pressure for R. catesbeiana and B. marinus. These pressure differentials constitute a "compliance pump" that drives distal-to-proximal intersac lymph flow. The compliance pump alone explains lymphatic return for the aquatic frog X. laevis but does not explain how lymph would reach the dorsally located lymph hearts for terrestrial anurans, so we hypothesize that skeletal muscle pumps return lymph from the femoral and pubic lymph sacs to the lymph heart. This is a fundamentally different role of the subcutaneous lymph-sac system than has been previously proposed. We suggest that the more proximal subcutaneous lymph sacs are important for fluid storage because they have a relatively high compliance.

Analysis of Variance↗
PubMed1988

Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells.

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.

Animals↗
PubMed2000

Murine Bv8 gene maps near a synteny breakpoint of mouse chromosome 6 and human 3p21.

The genomic structure of the murine Bv8 gene was determined in 129/SvJ mouse, and the chromosomal localization was identified. Bv8 has first been characterized from skin secretion of the yellow-bellied toad, Bombina variegata. When injected into rat brain, this polypetide causes hyperalgesia. The murine Bv8 gene was shown to consist of four exons and was localized on chromosome 6 between the microsatellite markers D6Mit66 and D6Mit36 near the gene mem1, whereas the human counterpart was assigned to the non-syntenic region 3p21.1. Furthermore, the primary Bv8 transcript appeared to be alternatively spliced. The first variant contained all four exons yielding a product with a stretch highly enriched in basic amino acids in its central part. This domain is absent in the peptides from frog as well as in a splice variant expressed in mouse testis. A third variant gives rise to a truncated polypeptide.

Alternative Splicing↗
PubMed1983

Membrane potential plays a dual role for chloride transport across toad skin.

The Cl- -current through toad skin epithelium depends on the potential in a way consistent with a potential-controlled Cl- permeability. Computer analysis of the Koefoed-Johnsen Ussing two-membrane model provided with constant membrane permeabilities indicates that the voltage- and time-dependent currents are not caused by a trivial Goldmand-type rectification and ion redistributions following transepithelial potential pertubations. Extended with a dynamic Cl- permeability in the apical membrane according to a Hodgkin-Huxley kinetic scheme, the model predicts voltage clamp data which closely resemble experimental observations. This extension of the classic frog skin model implies that the Cl- permeability is activated by a voltage change caused by the inward Na+ current through the apical membrane.

Animals↗