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PubMed1989

Altitudinal distribution and blood values in the toad, Bufo spinulosus Wiegmann.

1. Red blood cell (RBC) count, RBC size, hematocrit, cell and blood hemoglobin concentrations and plasma total solid concentration were measured in 16 lowland (from near sea level up to 2700 m) and 18 highland (3200 up to close to 4500 m) adult toads (Bufo spinulosus). 2. Lowland toads showed higher hematocrit values than highland toads, but their blood hemoglobin concentration and plasma solid concentration were not significantly different. 3. Highland toads had smaller RBC size, higher corpuscular hemoglobin concentration, a trend toward larger RBC count and a considerably smaller body size. These features may contribute to their successful life at high altitude.

Adaptation, Physiological↗
PubMed1994

Melatonin desensitizing effects on the in vitro responses to MCH, alpha-MSH, isoproterenol and melatonin in pigment cells of a fish (S. marmoratus), a toad (B. ictericus), a frog (R. pipiens), and a lizard (A. carolinensis), exposed to varying photoperiodic regimens.

Melatonin is a weak dose-independent lightening agonist in fish skin, a moderate dose-dependent lightening agonist in toad skin and a potent lightening agent in frog and lizard skins (reversing in a dose-dependent manner the darkening caused by alpha-melanocyte-stimulating hormone). In frog skins, previous exposure to melatonin reduced further lightening actions of the indoleamine, and in toad skins, increasing concentrations of melatonin elicited decreasing lightening responses, suggesting an autodesensitizing action of the hormone. Various concentrations of melatonin diminished the responses to the lightening agonist melanin-concentrating hormone (MCH) in fish skins and to the darkening agonists alpha-MSH in toad, frog and lizard skins and isoproterenol in frog skins. In vitro inhibitory actions of melatonin are mimicked in the absence of the hormone in skin preparations from toads kept in continuous darkness for 48 hr. The lipophylic nature of the indoleamine associated with the results herein described suggests intracellular actions of melatonin on vertebrate pigment cells.

Animals↗
PubMed1980

Effect of vasopressin on cyclic AMP-dependent protein kinase in toad urinary bladder.

The effect of vasopressin on the toad urinary bladder has been shown to be mediated by cyclic AMP. It has been assumed that, as demonstrated for other systems, this involves activation of cyclic AMP-dependent protein kinase. In order to test this hypothesis we investigated the effect of vasopressin on cyclic AMP-dependent protein kinases in epithelial cells of toad bladders. About 80% of protein kinase activity and cyclic AMP-binding capacity was found to be in the cytosol. DEAE-cellulose chromatography showed a pattern of 15--20% type I and 80--85% type II cyclic AMP-dependent protein kinase. Cytosolic kinase was activated 3--4-fold by cyclic AMP with half-maximal activation at 5 . 10(-8) M. Similarly, half-maximal binding of cyclic AMP occurred at 7 . 10(-8) M. Incubation of toad bladders in Ringer's solution containing 0.1 mM 3-isobutyl-1-methylxanthine, prior to homogenization and assay, showed stable cyclic AMP-binding capacity and protein kinase ratio --cyclic AMP/+cyclic AMP. Exposure of bladders to 10 mU/ml of vasopressin for 10 min caused intracellular activation of protein kinase and decrease in cyclic AMP-binding capacity that were maintained for at least 30 min. Incubation of bladders with increasing concentrations of vasopressin (0.5--100 mU/ml) resulted in a discrepancy between a progressive increase in cyclic AMP levels and a levelling off at 10 mU/ml of vasopressin for the changes in protein kinase ratio and cyclic AMP-binding capacity. The increase in kinase ratio was due to higher activity in the absence of exogenous cyclic AMP and was fully inhibitable by a specific protein kinase inhibitor. Using Sephadex G-25-CM50 column chromatography for separation of holoenzyme and free catalytic subunit we demonstrated that the activation of protein kinase in the vasopressin-treated bladders is due to intracellular dissociation of the kinase. These results show that the effect of vasopressin on the toad bladder involves activation of a cytosolic cyclic AMP-dependent protein kinase. The time course and the dose-response curve of the kinase activation closely parallel vasopressin's effect on osmotic water flow.

Animals↗
PubMed1978

Partial characterization of pepsins and gastricsins and their zymogens from human and toad gastric mucosae.

1. Three zymogens have been isolated from human gastric mucosae and two from the stomachs of the toad Caudiverbera caudiverbera. 2. Human zymogens I and III were immunologically related and cross-reacted with antisera prepared against porcine pepsinogen. The third, (II), showed no cross-reactivity. 3. Human zymogens I and III and toad zymogen ZII gave rise to two human pepsins and to a pepsin-like enzyme, respectively. 4. Human zymogen II (gastricsinogen) and toad zymogen ZI gave rise to human gastricsin and to a gastricsin-like enzyme respectively. 5. The toad enzymes showed much greater stability at neutral and alkaline pH values than the human enzymes.

Animals↗
PubMed1984

The effect of calcium ions on the responses to motoneurons to substance P and eledoisin-related peptide in the toad and rat spinal cord.

The effects of changes in the external calcium ion concentration [Ca2+]0 on segmental reflexes and the responses of motoneurons to bath applied agonists have been studied in the toad and rat spinal cords in vitro. Reducing [Ca2+]0 enhanced polysynaptic reflexes in the toad, with maximal discharges occurring at 0.3 mM. Monosynaptic reflexes in the rat were reduced by lowering [Ca2+]0. Responses of toad motoneurons to substance P and eledoisin-related peptide were enhanced by lowering [Ca2+]0, maximum responses occurring at 0.3 mM. Lowering [Ca2+]0 also enhanced responses to L-glutamate but the effect was smaller and less consistent. This effect of Ca2+ was abolished by the addition of tetrodotoxin to the bathing solution. Toad motoneuron responses to gamma-aminobutyrate and glycine were not affected by alterations in [Ca2+]0. Rat motoneuron responses to substance P and eledoisin-related peptide were also enhanced by reductions in [Ca2+]0 but the effect was more pronounced in younger (less than 5 days post partum) than older (5-10 days post partum) animals. These results are consistent with the idea that motoneuron responses to peptides in normal solutions result from activation of receptors on both motoneurons and interneurons: enhancement of the responses by lowering [Ca2+]0 results from the potentiation of the transynaptic component.

Animals↗
PubMed2006

Temperature and humidity alter prolactin receptor expression in the skin of toad (Bufo bankorensis and Bufo melanostictus).

Bufo bankorensis and Bufo melanostictus, the only two species of Bufonidae genus in Taiwan, live in habitats that differ in altitude and humidity. This study tested the hypothesis that prolactin receptor (PRLR) expression responds to environmental change. Western blot analysis showed that the PRLR protein was widely distributed in brain, lung, liver, kidney, dorsal skin and ventral skin of toads. The level PRLR protein was elevated in the dorsal skin of the two toad species treated with dry or wet conditions for 14 days. The increase in PRLR of dorsal skin in B. bankorensis was higher than that in B. melanostictus. This experimental result suggests that B. bankorensis secretes more mucus to reduce water evaporation from its thinner cuticle than B. melanostictus. The expression of PRLR protein was increased in the lung of B. bankorensis and decreased in the lung of B. melanostictus. Moreover, PRLR protein levels were increased in the kidneys in the two species toad, likely due to reduction in water lost through lung and urine. The two toad species were subjected to varying temperatures (25 degrees C, 15 degrees C and 10 degrees C) for 14 days. The lowest PRLR protein expression was observed at 10 degrees C. Comparison of the decreasing trend in PRLR protein levels demonstrated that the variation in B. bankorensis was significantly higher than that in B. melanostictus. Comparisons of variation in PRLR protein expression in the two species under different environments suggest that B. bankorensis is more adaptable to different environments than B. melanostictus.

Adaptation, Physiological↗
PubMed1998

A deep brain photoreceptive molecule in the toad hypothalamus.

We have isolated a cDNA clone encoding a deep brain photoreceptive molecule from the hypothalamic cDNA library of the toad, Bufo japonicus. The deduced amino acid sequence showed the highest similarity to that of pinopsin (75-76%) among vertebrate retinal opsins, indicating the expression of toad pinopsin in the deep brain. Antibodies raised against the C-terminal tail of toad pinopsin stained cell bodies and the knob-like structures of the cerebrospinal fluid-contacting neurons in the anterior preoptic nucleus. This region is known to play an important role in breeding behavior, suggesting that toad pinopsin acts as a photosensor for the photoperiodic gonadal response.

Amino Acid Sequence↗
PubMed1984

A global model of the neural mechanisms responsible for visuomotor coordination in toads.

A model of how the nervous system of toads processes visual information to control motor behaviour is proposed. The problem of visuomotor coordination in toads is studied through the integration of two different approaches: a top-down approach through schema theory developed in the studies of cognitive psychology, artificial intelligence and brain theory; and a bottom-up approach through the integration of physiological, anatomical, ethological, and neural modelling. The model proposes that visual information is processed in a parallel distributed way through different brain layers whose interaction defines the proper motor response for that specific situation. It is postulated that visual processing of information is organized into main schemata, which set the goal to be attained and, depending on the specific circumstances of the animal, activates different brain layers; the main schemata may use other schemata to solve a specific subproblem to reach the schemata, and a programme of schema co-ordination. With this model we have simulated how toads plan their route to reach a prey or the route to go away form a predator, depending on the state of their three-dimensional world. The model postulates specific hypotheses that could be tested experimentally on the processing of information in the toad's nervous system.

Animals↗
PubMed1997

A model of Weber and noise gain control in the retina of the toad Bufo marinus.

We present several variations of a model of gain control in the retina of the toad Bufo marinus, and use them to fit the threshold-vs-intensity data of an actual toad ganglion cell [Donner et al. (1990). Journal of General Physiology, 95, 733-753]. Our models are based on a proposal by Donner et al. that the gain (neural spike per photon ratio) of toad ganglion cells is set by a sequence of two retinal gain control stages. The first stage consists of a Weber gain control mechanism at the level of the red rods. The second is a more proximal "noise gain" stage, which multiplies the (incremental) input signal by a factor that is inversely proportional to the standard deviation of the random ganglion cell input and, under conditions that produce the de Vries-Rose threshold law, is also proportional to the standard deviation of the photon fluctuations within the ganglion cell receptive field. We demonstrate that noise gain control arises naturally from modeling ganglion cell spike generation with either of two common types of spike generation models: integrate-and-fire models or threshold accommodation models. We simulate the process of spike generation in both types of models and show that either model can account for the basic overall shape of the toad t.v.i. curve. However, although integrate-and-fire models appropriately generate noise gain control, they cannot quantitatively fit the threshold data with realistic retinal parameters. Integrate-and-fire models also fail to account for the observed relationship between the generator potential of the ganglion cell and its spiking probability. A threshold accommodation model with realistic retinal parameters, on the other hand, can account for both the threshold data and the generator potential-spike probability relationship. When a Weber gain stage is added to the model at the photoreceptor level, the resulting two-stage gain control model is shown to account quantitatively for the ganglion cell t.v.i. curve of Bufo marinus over the full range of background levels studied by Donner et al.

Adaptation, Ocular↗
PubMed2000

Baroreceptor control of heart rate in the awake toad: peripheral autonomic effectors and arterial baroreceptor areas.

Systemic injection of sodium nitroprusside (30 microg/kg, i.v.) in the awake Bufo paracnemis toad induced a fall in arterial blood pressure and tachycardia. This tachycardia, but not the hypotension, was significantly reduced in toads with bilateral electrolytic lesion of the caudal and commissural regions of the solitary tract nucleus and in animals with transection of the spinal cord, 2 mm below the obex. This indicates that the tachycardia is reflex, depends on the integrity of the solitary tract nucleus and is due to descending spinal autonomic activation. Pretreatment with propranolol (4 mg/kg, i.v.) significantly reduced the tachycardia but did not block it completely, showing the importance of beta-adrenoceptors in its genesis. The reflex increase in heart rate induced by nitroprusside was not statistically significant in animals with bilateral section of the laryngeal nerve, whose baroreceptor fibers originate from the pulmocutaneous artery or in animals in which the bilateral section of the laryngeal nerve was performed together with section of the glossopharyngeal nerves, which incorporate fibers originating from the carotid labyrinth. The reduction of the reflex tachycardia was significant in toads with aortic arch denervation alone or combined with section of the laryngeal nerves or in animals with complete denervation of the three baroreceptors areas. These results suggest that the region of the aortic arch, when submitted to unloading, is the most important baroreceptor zone for cardiac compensation in toads.

Animals↗
PubMedDate not supplied

An enzyme-linked immunosorbent assay for measuring anti-sheep red blood cells antibodies in lead-exposed toads.

INTRODUCTION: Immune function assays to screen immunotoxic effects of xenobiotics has recently become of major interest. In the framework of our studies, we standardized methods to quantify the humoral response of an amphibian species (Bufo arenarum, Amphibia, Anura) exposed to sublethal lead (as acetate). METHODS: The levels of agglutinins to heterologous red blood cells (RBC) were measured in serum from adult B. arenarum. Since agglutinin titers were very low, a noncompetitive enzyme-linked immunosorbent assay (ELISA) method was carried out. As toad serum showed marked nonspecific binding, we developed a new ELISA on microtiter plates for the quantitative determination of the heterophile antibodies. The method was based on that described by Hirvonen et al. [Vox Sang. 69 (1995) 341], employing sheep red blood cells (SRBC) sensitized with amphibian antibodies that were transferred to microplates; later the measurement of bound immunoglobulins was performed. Different variables such as the amount of antigen, blocking agents, and other experimental conditions (fixing solution and commercial plates) were studied. Toads (n=22) received a weekly subcutaneous injection of 50 mg/kg lead (acetate) for 6 weeks, and the control ones (n=26) were injected with Na acetate at the same time. RESULTS: The anti-sheep RBC antibodies titers of adult toads were obtained with the improved ELISA method, being the absorbance range 0.12 to 1.58 AU (1/200 diluted serum). Titers from lead-exposed toads were also determined, being the final titers (expressed as (-)x +/-S.E.M.) higher (0.79+/-0.06 AU), than those of Day 0 (0.57+/-0.06) (P<.01). DISCUSSION: It was concluded that the ELISA technique we developed was useful for measuring the humoral immune response in this animal model and that in these preliminary studies, lead showed an immunostimulating action on the humoral immune system.

Animals↗
PubMed1999

An electrophoretic study of myosin heavy chain expression in skeletal muscles of the toad Bufo marinus.

In this study we developed an SDS-PAGE protocol which for the first time separates effectively all myosin heavy chain (MHC) isoforms expected to be expressed in iliofibularis (IF), pyriformis (PYR), cruralis (CRU) and sartorius (SAR) muscles of the toad Bufo marinus on the basis of previously reported fibre type composition. The main feature of the method is the use of alanine instead of glycine both in the separating gel and in the running buffer. The correlation between the MHC isoform composition of IF, SAR and PYR muscles determined in this study and the previously reported fibre type composition of IF and SAR muscles in the toad and of PYR muscle in the frog was used to tentatively identify the MHC isoforms expressed by twitch fibre types 1, 2 and 3 and by tonic fibres. The alanine-SDS electrophoretic method was employed to examine changes in the MHC composition of IF, PYR, CRU and SAR muscles with the ontogenetic growth of the toad from post-natal life (body weight < 1 g) to late adulthood (body weight 200-450 g). The developmental changes in the MHC isoform composition of the toad IF muscle observed in this study are in very good agreement with those in the fibre type composition of the developing IF muscle reported in the literature.

Animals↗
PubMed1984

Steroid-induced protein synthesis in giant-toad (Bufo marinus) urinary bladders. Correlation with natriferic activity.

We have identified a group of proteins (Mr approximately 70 000-80 000; pI approximately 5.5-6.0) in giant-toad (Bufo marinus) urinary bladders whose synthesis appears to be related to aldosterone-stimulated Na+ transport. Spironolactone, a specific mineralocorticoid antagonist in renal epithelia, inhibits the synthesis of these proteins as well as the natriferic effect of the hormone. Since a variety of other steroids (some of which are traditionally considered to be glucocorticoids) also stimulate Na+ transport in toad urinary bladders, we examined whether their natriferic activity was expressed in a fashion similar to that of aldosterone. Short-circuit current was used to measure Na+ transport, and epithelial-cell protein synthesis was detected with high-resolution two-dimensional polyacrylamide-gel electrophoresis and autoradiography. At a concentration of approximately 100 nM, dexamethasone, corticosterone and aldosterone were equinatriferic. Dexamethasone and aldosterone had identical dose-response curves, maximal and half-maximal activity being evident at concentrations of approximately 100 nM and 10 nM respectively. In contrast, at a concentration of approximately 10 nM, corticosterone had no effect on Na+ transport. The natriferic activities of these three steroids correlate with their known affinities for the putative mineralocorticoid receptor in toad urinary bladders. Natriferic concentrations of dexamethasone and corticosterone (140 nM) induced the synthesis of proteins with characteristics identical with those induced by aldosterone. Spironolactone, at an antagonist/agonist ratio of 2000:1, inhibited steroid-induced Na+ transport and the synthesis of these proteins. Thus it appears that all natriferic steroids share a common mechanism of action in toad urinary bladders. Natriferic activity can be correlated not only with relative steroid-receptor affinity but also with the induction of a specific group of epithelial-cell proteins.

Aldosterone↗
PubMed1993

Isolation of highly purified, functional endosomes from toad urinary bladder.

Endosomes are difficult to isolate as they share size and density properties with much more abundant cellular organelles such as mitochondria. In cultured cell lines the tandem use of charge-dependent isolation techniques and differential centrifugation is necessary to isolate endosomes. Endosomal populations of the toad urinary bladder are of special interest because they are thought to contain a water channel. Understanding of the molecular structure of the water channel has been constrained, as there is currently no practical method to isolate functional water-channel-containing vesicles. This study reports the tandem use of charge-dependent techniques and centrifugation to isolate populations of endosomes from the toad urinary bladder. To purify water-channel-containing vesicles aqueous two-phase partition was utilized to fractionate a preparation partially purified by differential centrifugation. Populations of endosomes were analysed by small-particle flow cytometry techniques. A 5-fold enrichment in endosomes, achieved with aqueous two-phase partition, allowed us to identify two populations of endosomes of diverse size in a toad bladder endosomal fraction. Preenrichment also improved the efficiency of flow cytometry sorting, allowing isolation of the two endosomal populations in sufficient quantities for secondary analysis. A population of larger endosomes had very high water permeability, indicating the presence of water channels. The two populations had different SDS/PAGE fingerprints. Electron micrographs of the flow-sorted material shows a uniform population of membrane vesicles devoid of mitochondria and other identifiable cellular organelles. Hence, aqueous two-phase partition and flow cytometry allow identification of two populations of endosomes in the toad urinary bladder which have diverse structural and functional properties. Isolation of functional water-channel-containing vesicles allows co-localization of water-channel function with candidate water-channel proteins.

Animals↗
PubMed2000

The genetics of amphibian declines: population substructure and molecular differentiation in the yosemite toad, Bufo canorus (Anura, bufonidae) based on single-strand conformation polymorphism analysis (SSCP) and mitochondrial DNA sequence data.

We present a comprehensive survey of genetic variation across the range of the narrowly distributed endemic Yosemite toad Bufo canorus, a declining amphibian restricted to the Sierra Nevada of California. Based on 322 bp of mitochondrial cytochrome b sequence data, we found limited support for the monophyly of B. canorus and its closely related congener B. exsul to the exclusion of the widespread western toad B. boreas. However, B. exsul was always phylogenetically nested within B. canorus, suggesting that the latter may not be monophyletic. SSCP (single-strand conformation polymorphism) analysis of 372 individual B. canorus from 28 localities in Yosemite and Kings Canyon National Parks revealed no shared haplotypes among these two regions and lead us to interpret these two parks as distinct management units for B. canorus. Within Yosemite, we found significant genetic substructure both at the level of major drainages and among breeding ponds. Kings Canyon samples show a different pattern, with substantial variation among breeding sites, but no substructure among drainages. Across the range of B. canorus as well as among Yosemite ponds, we found an isolation-by-distance pattern suggestive of a stepping stone model of migration. However, in Kings Canyon we found no hint of such a pattern, suggesting that movement patterns of toads may be quite different in these nearby parklands. Our data imply that management for B. canorus should focus at the individual pond level, and effective management may necessitate reintroductions if local extirpations occur. A brief review of other pond-breeding anurans suggests that highly structured populations are often the case, and thus that our results for B. canorus may be general for other species of frogs and toads.

Animals↗
PubMed2001

Nux vomica 30 prepared with and without succession shows antialcoholic effect on toads and distinctive molecular association.

Adult toads, Bufo melanostictus, were administered Nux vomica (Nux v) 30 prepared with and without succussion on the tongue. The drug was mixed with sterile distilled water at the rate 0.05ml/ml water and given orally 0.05ml/individual. The control consisted of blank ethanol solution. Seeds of Strychnos nuxvomica were ground and extracted with 90% ethanol in the laboratory. Nux v 30 was prepared by successive dilution and succussion in 30 steps, Nux v 30 u was prepared by successive dilution only. Four hours after treatment, toads were given 25% ethanol i.p. at 8g/kg body weight. The duration of ethanol induced sleep time was recorded for each toad. Both Nux v 30 and Nux v 30 u significantly reduced ethanol induced sleep time in toads as compared to their respective controls. Electronic, infra red and nuclear magnetic resonance spectra of Nux v 30, Nux v 30 u and their diluent medium (90% ethanol) show marked differences from each other. These dilutions and ethanol 30 and ethanol 30 u show marked differences from each other with respect to spin-lattice relaxation time (T1) and chemical shift. The difference has been attributed to the variation in intra and inter-molecular association of ethanol and water.

Administration, Oral↗
PubMed2006

Developmental plasticity mirrors differences among taxa in spadefoot toads linking plasticity and diversity.

Developmental plasticity is found in most organisms, but its role in evolution remains controversial. Environmentally induced phenotypic differences may be translated into adaptive divergence among lineages experiencing different environmental conditions through genetic accommodation. To examine this evolutionary mechanism, we studied the relationship between plasticity in larval development, postmetamorphic morphology, and morphological diversity in spadefoot toads, a group of closely related species that are highly divergent in the larval period and body shape and are distributed throughout temperate areas of both the New and the Old World. Previous studies showed that accelerated metamorphosis is adaptive for desert-dwelling spadefoot toads. We show that even under common garden conditions, spadefoot toad species show divergent reaction norms for the larval period. In addition, experimentally induced changes in the larval period caused correlated morphological changes in postmetamorphic individuals such that long larval periods resulted in relatively longer hindlimbs and snouts. A comparative analysis of morphological variation across spadefoot toad species also revealed a positive correlation between the larval period and limb and snout lengths, mirroring the effects of within-species plasticity at a higher taxonomic level. Indeed, after approximately 110 Ma of independent evolution, differences in the larval period explain 57% of the variance in relative limb length and 33% of snout length across species. Thus, morphological diversity across these species appears to have evolved as a correlated response to selection for a reduced larval period in desert-dwelling species, possibly diverging from ancestral plasticity through genetic accommodation.

Animals↗
PubMed1988

The induction of neoplastic lesions by aflatoxin-B1 in the Egyptian toad (Bufo regularis).

The carcinogenic activity of aflatoxin-B1, the metabolic product of the mold Aspergillus flavus (a commonly occurring contaminant of groundnuts and other foodstuffs), was tested using the Egyptian toad (Bufo regularis). Injecting the toads with aflatoxin-B1 at a dose level of 0.01 mg/50 g body wt in 1 ml corn oil once a week for 15 weeks induced hepatocellular carcinomas in 19% of the experimental toads. Four toads developed tumors in the kidney due to metastases from the primary hepatocellular carcinomas.

Aflatoxin B1↗