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PubMed1998

Mechanism of action of honey bee (Apis mellifera L.) venom on different types of muscles.

1. The effect of crude honeybee (Apis mellifera) venom on the skeletal, smooth as well as cardiac muscles were studied in this investigation. 2. Perfusion of gastrocnemius-sciatic nerve preparation of frogs with 1 microgram/ml venom solution has weakened the mechanical contraction of the muscle without recovery. Blocking of nicotinic receptors with 3 micrograms/ml flaxedil before bee venom application sustained normal contraction of gastrocnemius muscle. 3. The electrical activity of duodenum rabbits was recorded before and after the application of 1 microgram/ml venom solution. The venom has depressed the amplitude of the muscle contraction after 15 min pretreatment with atropine nearly abolished the depressor effect of the venom on smooth muscle. 4. In concentrations from 0.5-2 micrograms/ml, bee venom caused decrease of heart rate of isolated perfused toad heart. This bradycardia was accompanied by elongation in the P-R interval. A gradual and progressive increase in the R-wave amplitude reflected a positive inotropism of the venom. Application of 5 micrograms/ml verapamil, a calcium channels blocking agent, abolished the noticed effect of the venom. 5. Marked electrocardiographic changes were produced within minutes of the venom application on the isolated perfused hearts, like marked injury current (elevation or depression of the S-T segment), atrioventricular conduction disturbances and sinus arrhythmias. Atropine and nicotine could decrease the toxic effect of the venom on the myocardium. 6. Results of the present work lead to the suggestion that bee venom is mediated through the peripheral cholinergic neurotransmitter system. General neurotoxicity of an inhibitory nature involving the autonomic as well as neuromuscular system are established as a result of the venom, meanwhile a direct effect on the myocardium membrane stabilization has been suggested.

Animals↗
PubMed1995

Effect of neuropeptide-Y on tectal field potentials in the toad.

Field potentials (FP) recorded from the surface of toad's optic tectum (OT) to electrical stimulation of the contralateral optic nerve (ON) show initial positive deflections P*, followed by negative wave N and positive wave P; the former result from axonal inputs, the latter two resemble excitatory and inhibitory postsynaptic processes, respectively. Electrostimulation of the pretectum ipsilaterally to the recorded OT--preceeding ON-stimulation--strongly attenuates the N wave, suggesting pretectotectal inhibitory influences. The N wave of the tectal FP evoked by ON-stimulation is reduced, too, after application of the neuropeptide-Y (NPY) to the tectal surface. Previous authors have shown that frog's pretectum contains NPY immunoreactive pretectotectal projecting cells.

Animals↗
PubMed2005

Antimicrobial activity of the bufadienolides marinobufagin and telocinobufagin isolated as major components from skin secretion of the toad Bufo rubescens.

The increase in the emergence of antibiotic-resistant microorganisms and difficult to treat infections caused by these pathogens stimulate research aiming the identification of novel antimicrobials. Skin secretion of amphibian contains a large number of biologically active compounds, including compounds that performance defense mechanisms against microorganisms. In the present work, two antimicrobial bufadienolides, telocinobufagin (402.1609 Da) and marinobufagin (400.1515 Da), were isolated from skin secretions of the Brazilian toad Bufo rubescens. The specimens were collected in Brasilia (Distrito Federal, Brazil), the skin secretions extracted by electric stimulation, and submitted to purification by RP-HPLC. The molecular structure and mass determination were done by (1)H and (13)C NMR and mass spectrometry data, respectively. The antimicrobial activity was performed by liquid growth inhibition against Staphylococcus aureus and Escherichia coli. The minimum inhibitory concentrations of telocinobufagin and marinobufagin were, respectively, 64.0 and 16.0 microg/mL for E. coli and both 128 microg/mL for S. aureus. Besides the antimicrobial activity both bufadienolides promoted an increase of the contraction force in isolated frog ventricle strips.

Amphibian Venoms↗
PubMed2003

Phylogenetic relationships of Pelobatoidea re-examined using mtDNA.

Pelobatoidea is a clade of ancient anurans with obscure relationships to the remaining clades of frogs. We used partial sequences of two mitochondrial genes (cytochrome b and 16S RNA) from all Pelobatoidea subclades, including all species of Pelobatidae and Pelodytidae and four outgroup taxa (Xenopus, Ascaphus, Discoglossus, and Rana), to propose a phylogenetic hypothesis for relationships within Pelobatoidea. Maximum likelihood and Bayesian analyses support the monophyly of Pelobatoidea, but our hypothesis of internal relationships differs substantially from all previous hypotheses. Megophryidae is sister to Pelobates, and this clade is sister to Pelodytes. The most basal clade within Pelobatoidea is formed by Scaphiopus and Spea. The family Pelobatidae, as previously defined is not monophyletic, and it is split into Eurasian spadefoot toads Pelobates which retain the name Pelobatidae and North American spadefoot toads Scaphiopus and Spea which comprise the revived taxon Scaphiopodidae. Our analysis uncovers the existence of morphologically cryptic taxa within previously recognized species of the genus Spea and reveals marked genetic differentiation within Iberian Pelodytes. We discuss biogeographic implications and the evolution of fossoriality in the light of the new phylogenetic hypothesis.

Adaptation, Biological↗
PubMed1995

Developmental changes in melanin-concentrating hormone in Rana temporaria.

Melanin-concentrating hormone (MCH) is a vertebrate neuropeptide produced in hypothalamic neurons. In bony fish, such as trout, MCH acts as a neurohypophysial hormone which, once released into the circulation, acts on pigmented skin cells with the result that the fish turns pale to camouflage itself against a light colored background. In other animals the role of MCH is not clearly established but it appears to be a neuromodulator/transmitter within the central nervous system rather than a hormone. The present study examines MCH function in the grass frog, Rana temporaria. Using immunocytochemistry the location and morphology of irMCH neurons were followed, from tadpole to adult frog. In adult R. temporaria a group of MCH neurons appeared to comprise small and large-celled populations located in the ventral and dorsal infundibular regions, respectively. A group of MCH neurons in the preoptic area is proposed, although the perikarya were rarely immunostainable. Immunoreactive fibers were seen in various areas of the brain, including the olfactory lobes, optic tecta, habenular nucleus, and spinal cord. Immunoreactive MCH cells were only visible in midmetamorphic climax stages, and cellular morphology suggested low secretory activity until the animal first emerged onto land at which time nuclear size and granulation increased significantly. No such increase was observed in equivalent stages of the South African clawed toad, Xenopus laevis, an animal which is fully aquatic throughout its life in contrast to R. temporaria which is terrestrial.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Detection of N-acetylated forms of beta-endorphin and nonacetylated alpha-MSH in the intermediate pituitary of the toad, Bufo marinus.

Steady-state analysis of the acid extracts of the intermediate pituitary of the toad, Bufo marinus, revealed the presence of multiple forms of beta-endorphin and alpha-MSH. Approximately 98% of the immunoreactive beta-endorphin was N-acetylated. The major form of N-acetylated beta-endorphin, which represented 81.5% of the total beta-endorphin recovered from this tissue, had an apparent molecular weight of 1.2 kDa and a net charge of +1 at pH 2.75. Approximately 98% of the immunoreactive alpha-MSH present in the Bufo intermediate pituitary had reverse phase HPLC properties similar to the nonacetylated form of alpha-MSH, ACTH(1-13)amide. These observations are in agreement with studies on the intermediate pituitary of the frog, Xenopus laevis, which have shown that the N-acetylation of alpha-MSH in this species is a cosecretory processing event, whereas the N-acetylation of beta-endorphin is a posttranslational processing event (2, 5, 15). These observations indicate that the N-acetylation of beta-endorphin and alpha-MSH occurs at distinct subcellular sites in intermediate pituitary cells of anuran amphibians. The Bufo intermediate pituitary will serve as a good model system for studying these novel N-acetyltransferase reactions.

Acetylation↗
PubMed1980

In vitro evidence for T-B lymphocyte collaboration in the clawed toad, Xenopus.

Requirements for an in vitro secondary antibody response to the soluble antigens dinitrophenylated keyhole limpet hemocyanin and dinitrophenylated flow gamma-globulin are described for the clawed toad, Xenopus. Priming of both hapten and carrier-specific cells is required in order to obtain good responses to the hapten. The carrier-reactive ("helper") cells do not adhere to nylon wool, are X ray-resistant and surface Ig-negative. The cell adherence and X ray sensitivity characteristics of these Xenopus "T" cells and their ability to generate a mixed lymphocyte response, allogeneic cytotoxic and helper activities, are identical to those of thymus-derived (T) cells in higher vertebrates. The hapten-reactive, antibody-secreting cell precursors are nylon-adherent, X ray-sensitive and surface Ig-positive. In the absence of other distinguishing serological markers for T cells in frogs, and considering the thymus dependency of the helper activity described here, the evidence presented is interpreted as demonstrating the requirement for antigen-specific T-B cell cooperation in response to soluble protein antigens in vitro for Xenopus.

Animals↗
PubMed2001

Natriuretic peptide receptors in the central vasculature of the toad, Bufo marinus.

Natriuretic peptide receptors in the central vasculature of the toad, Bufo marinus, were characterized using autoradiographical, molecular, and physiological techniques. Specific 125I-rat ANP binding sites were present in the carotid and pulmonary arteries, the lateral aorta, the pre- and post-cava, and the jugular vein, and generally occurred in each layer of the blood vessel. The 125I-rat ANP binding was partially displaced by the specific natriuretic peptide receptor C ligand, C-ANF, which indicates the presence of two types of natriuretic peptide receptors in the blood vessels. This was confirmed by a RT-PCR study, which demonstrated that guanylyl cyclase receptor (NPR-GC) and NPR-C mRNAs are expressed in arteries and veins. An in vitro guanylyl cyclase assay showed that frog ANP stimulated the production of cGMP in arterial membrane fractions. Physiological recordings from isolated segments of the carotid and pulmonary arteries and the lateral aorta, which had been pre-constricted with arginine vasotocin, showed that rat ANP, frog ANP and porcine CNP relaxed the vascular smooth muscle with relatively similar potency. Together, the data show that the central vasculature contains two types of natriuretic peptide receptors (NPR-C and NPR-GC) and that the vasculature is a target for ANP and CNP.

Animals↗
PubMed1983

Exchange diffusion, electrodiffusion and rectification in the chloride transport pathway of frog skin.

Measurements of chloride flux ratios across frog skin at different clamping voltages showed that chloride transport at clamping voltages from 0 mV to and beyond the spontaneous potential is probably electrodiffusion. At reversed potentials a significant fraction of chloride transport could be described formally as exchange diffusion. Chloride conductance was found to be highly voltage dependent, being largest at hyperpolarizing clamping voltages. The transition from the less conducting state to the more conducting one was studied by recording the time course of the current after a step change in clamping voltage from 0 mV to hyperpolarizing voltages. The shape of the curve is sigmoidal, and the relative rate of change of current increases with increasing hyperpolarization. It is proposed that the change in conductance is governed by the same mechanism as in the toad skin, namely a change in chloride permeability due to voltage gating of chloride channels. The time course of transepithelial conductance after addition of amiloride to the outside solution indicates that a fraction of the decrease in conductance is due to closure of chloride channels caused by the change in intracellular potential due to the inhibition of the sodium channels.

Animals↗
PubMed1994

Different potential of cellular and viral activators of transcription revealed in oocytes and early embryos of Xenopus laevis.

Many protein domains for transcriptional activation also function when fused to a heterologous DNA binding domain. In mammalian/HeLa cells, we have previously characterized the activation domains of several transcription factors using GAL4 fusion proteins. Here we have tested their transcriptional activity in oocytes and developing embryos of the clawed toad Xenopus laevis. We find that the "acidic" C-terminal domain of the herpesvirus VP16 (= Vmw65) activator, which is active from yeast to man, is also very active in the two Xenopus systems. The constitutive nature of this viral domain may have evolved to be refractory to cellular defense mechanisms. By contrast, activation domains from cellular eukaryotic transcription factors (TFE3, ITF2, MTF-1) are differentially active in oocytes and early embryos. This indicates that their activity can be regulated by protein modification and/or availability of specific coactivators. We have also compared VP16 induced enhancement of transcription from remote and promoter-proximal positions. In both oocytes and late blastula embryos, activation from a promoter-proximal position was more than 50 fold, while only a moderate stimulation (3-8 fold) was observed from remote positions. This may mean that frog oocyte and early embryos are not yet fully geared for gene control by remote enhancers, i.e. respond predominantly to close-by regulatory sequences. The fact that cellular enhancers are naturally located at various distances from the responsive promoters may thus be exploited by multicellular organisms for differential gene control at early and late stages of development.

Animals↗
PubMed1993

Poorly selective cation channels in apical membranes of epithelia.

The apical membrane of frog skin contains two types of pathways which allow the passage of several monovalent cations in the absence of external Ca2+. Differences between the two pathways concern their open-close kinetics, selectivity, and the affinity for several blocking agents. Type S channels open and close relatively slowly, whereas type F channels display fast open-close kinetics. Both channel types allow the passage of Na+, K+, and Rb+ currents which are blocked by divalent cations and La3+ added to the extracellular side. Type F channels are permeable for Cs+ which is, however, excluded from type S channels. Shifts in open-close kinetics induced by Mg2+ occur at concentrations below 5 microM for type F channels, whereas more than a tenfold higher dose is required for the type S pathway. UO2(2+) concentrations up to 100 microM only occlude type S channels while 100 microM tetracaine selectively blocks type F channels. Apical membranes of toad urinary bladder, cultured amphibian renal epithelia (A6), and toad colon contain only type F channels. In toad bladder and A6 cells volume expansion strongly activates this pathway. Macroscopic currents carried by Ba2+ and Ca2+ could be recorded after activation of toad bladders with oxytocin and treatment of the apical surface with nanomolar concentrations of Ag+, which seems to interact with a site located at the channel interior.

Amiloride↗
PubMed1993

Ca(2+)-blockable, poorly selective cation channels in the apical membrane of amphibian epithelia. Tetracaine blocks the UO2(2+)-insensitive pathway.

We examined the effect of the local anesthetic tetracaine on the Ca(2+)-blockable, poorly selective cation channels in the isolated skin of Rana temporaria and the urinary bladder of Bufo marinus using noise analysis and microelectrode impalements. Experiments with frog skin demonstrated that mucosal concentrations of the compound up to 100 microM did not affect the Na+ current through type S channels (slowly fluctuating, UO2(2+)-blockable channels) and the associated noise. On the other hand, 20 microM mucosal tetracaine already suffices to inhibit approximately 50% of the current carried by Cs+ and Na+ through channel type F (fast fluctuating, UO2(2+)-insensitive channel) and So of the associated Lorentzian component. With 100 microM of the inhibitor the current and So values were reduced by at least 70-80%. The time course of the response to serosal tetracaine was markedly slower and the effects on the current and So were smaller. Possible effects on the basolateral K+ conductance were excluded on the basis of the lack of response of transepithelial K+ movements to 100 microM tetracaine. UO2(2+) and tetracaine together blocked the poorly selective cation pathways almost completely. Moreover, both agents retain their inhibitory effect in the presence of the other. In toad urinary bladder, the Ca(2+)-blockable channel is also tetracaine blockable. The concentration required for half-maximal inhibition is approximately 100 microM in SO4(2-) and approximately 20 microM in Cl-. The data with tetracaine complement those obtained with UO2(2+) and support the idea that the Ca(2+)-blockable current proceeds through two distinct classes of cation channels. Using tetracaine and UO2(2+) as channel-specific compounds, we demonstrated with microelectrode measurements that both channel types are located in the granulosum cells.

Animals↗
PubMed1987

The distribution of motoneurons supplying hind limb muscles in the clawed toad, Xenopus laevis.

The distribution of motoneurons in the lumbar spinal cord (spinal segments 8-10) of the clawed toad, Xenopus laevis, was studied with the horseradish peroxidase technique. In a total of 13 different hind limb muscles this tracer was applied in a slow-release gel. Motoneurons innervating a particular hind limb muscle were clustered in longitudinally arranged motor pools. Motor pools of different muscles did show considerable overlap both in the rostrocaudal and transverse plane. But, the various motor pools clearly show a somatotopic organization of motoneurons even in such a condensed lumbar spinal cord as in Xenopus laevis. Motoneurons innervating more distally positioned muscles are generally found in more caudal segments, while proximal muscles (with the exception of the m. adductor magnus) are supplied by motoneurons more or less throughout the lumbar enlargement. Flexor muscles usually are innervated by motoneurons situated ventrolaterally in the ventral horn, extensor muscles by dorsomedially found motoneurons. This pattern is particularly apparent for proximal (thigh) muscles, less so for more distal (shank and foot) muscles. The present data are in keeping with those obtained with the retrograde cell degeneration technique in ranid frogs and are consistent with observations in other tetrapods, although a more clear separation of motor pools is evident in "higher" vertebrates such as birds and mammals.

Animals↗
PubMed1998

Neural substrate for motor control of feeding in amphibians.

Descending pathways to premotor/motor centers and their cell groups of origin were studied by means of retrograde biocytin tracing experiments in the frog Discoglossus pictus and the plethodontid salamander Plethodon jordani, which differ remarkably in the structure and function of their feeding apparatus and their feeding strategy. Labeled neurons were found in 30 major cell groups located in the telencephalon, diencephalon, synencephalon, mesencephalon and rhombencephalon. The number and distribution of nuclei are very similar in both species. Furthermore, the descending pathways of these groups of neurons take the same courses inside the medulla oblongata. Axons of most nuclei descend either in the ventromedial or ventrolateral medulla oblongata, and it is concluded that the spatial arrangement of pathways is identical in the species studied. Bilateral electrical stimulation of the optic tectum of the plethodontid salamander Hydromantes italicus elicited strong discharges of short latencies in the hypoglossal nerve. In most hypoglossal motor neurons, excitatory postsynaptic potentials (EPSPs) of short latencies followed paired shocks applied at intervals as short as 3 ms, but showed temporal and spatial facilitation, suggesting that the EPSPs include mono- as well as polysynaptic components. In the ventral white matter, orthodromic single units were found that are candidates for excitatory reticular interneurons. These properties of tectal descending pathways in salamanders strongly differ from those found in toads. Differences in feeding behavior and its control by the premotor/motor networks between the species investigated do not appear to result from anatomically altered input or from a different organization of descending pathways to these premotor/motor centers, but rather from differences in local properties of reticular premotor networks as well as from different effects of neuromodulatory systems.

Animals↗
PubMed1988

Apical Na+ permeability of frog skin during serosal Cl- replacement.

Gluconate substitution for serosal Cl- reduces the transepithelial short-circuit current (Isc) and depolarizes short-circuited frog skins. These effects could result either from inhibition of basolateral K+ conductance, or from two actions to inhibit both apical Na+ permeability (PapNa) and basolateral pump activity. We have addressed this question by studying whole-and split-thickness frog skins. Intracellular Na+ concentration (CcNa) and PapNa have been monitored by measuring the current-voltage relationship for apical Na+ entry. This analysis was conducted by applying trains of voltage pulses, with pulse durations of 16 to 32 msec. Estimates of PapNa and CcNa were not detectably dependent on pulse duration over the range 16 to 80 msec. Serosal Cl- replacement uniformly depolarized short-circuited tissues. The depolarization was associated with inhibition of Isc across each split skin, but only occasionally across the whole-thickness preparations. This difference may reflect the better ionic exchange between the bulk medium and the extracellular fluid in contact with the basolateral membranes, following removal of the underlying dermis in the split-skin preparations. PapNa was either unchanged or increased, and CcNa either unchanged or reduced after the anionic replacement. These data are incompatible with the concept that serosal Cl- replacement inhibits PapNa and Na,K-pump activity. Gluconate substitution likely reduces cell volume, triggering inhibition of the basolateral K+ channels, consistent with the data and conclusions of S.A. Lewis, A.G. Butt, M.J. Bowler, J.P. Leader and A.D.C. Macknight (J. Membrane Biol. 83:119-137, 1985) for toad bladder. The resulting depolarization reduces the electrical force favoring apical Na+ entry. The volume-conductance coupling serves to conserve volume by reducing K+ solute loss. Its molecular basis remains to be identified.

Animals↗
PubMed1988

Morphological classification of retinal ganglion cells in adult Xenopus laevis.

Retrograde transport of horseradish peroxidase (HRP) was used to characterise the soma and dendritic arborization of retinal ganglion cells in adult Xenopus laevis toad. HRP was administered to the cut end of the optic nerve and the morphological characteristics of HRP-filled ganglion cells were analysed in retinal wholemount preparations using computer assisted morphometry. Ganglion cells were classified according to their soma size, dendritic branching pattern, dendritic field and the number of shaft dendrites. Ganglion cells were divided into 3 major classes on the basis of soma sizes and extent of dendritic field: large (soma size, mean 258.04 micron 2 +/- 52.03 SD; dendritic field size 0.104 mm2 +/- 0.23), medium size (126.7 micron 2 +/- 37.01; 0.041 mm2 +/- 0.013) and small (87.3 micron 2 +/- 22.69; 0.0061 mm2 +/- 0.0035). A more detailed analysis allowed 12 morphologically distinct subgroups to be identified (Types I-XII). Quantitative studies showed that large cells comprise about 1%, medium size about 8-9% and the small cells over 90% of total ganglion cell population. The number of large and medium size ganglion cells corresponded well with the number of myelinated optic fibres and the number of small neurons with the number of unmyelinated optic fibres in the optic nerve. Large ganglion cells were correlated with Class 4 and 5, medium size ganglion cells with Class 3 and small ganglion cells with Class 1 and 2 functionally characterized ganglion cells in the frog retina (Maturana et al. 1960). The retinal distribution of large ganglion cells appear to suggest certain similarities to mammalian alpha type ganglion cells.

Animals↗
PubMed1986

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.

Amino Acid Sequence↗