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PubMed2003

Polycystic kidney and renal cell carcinoma in Japanese and Chinese toad hybrids.

Frequent development of renal cell carcinomas in hybrids between Japanese toads (Bufo japonicus) and imported Chinese toads (Bufo raddei) was first reported by 2 of our authors in 1987. Such renal tumors of toads had never been observed previously in the laboratory. To confirm the observation and to establish a new animal model system, hybrids between female Japanese and male Chinese toads were newly generated from 3 pairs of parents and pathological changes in their kidneys were examined sequentially over 6 years. In hybrids from 2 of the 3 pairs, bilateral polycystic kidney developed at a high frequency from 3 months after fertilization, this being associated with the emergence of atypical, premalignant-appearing cells in proximal tubules. Papillary lesions developed after 12 months and renal cell carcinomas after 48 months. Such pathological changes were never seen in non-hybrid Chinese or Japanese toads. Electron microscopy showed no evidence of any viral participation. This unique toad model may prove useful for investigation of the underlying mechanisms of genetically determined renal cell carcinogenesis.

Animals↗
PubMed1991

The effects of orchidectomy and gonadotropins on steroidogenesis and oogenesis in Bidder's organs of the toad Bufo woodhousii.

In the family Bufonidae, male toads possess rudimentary ovaries, called Bidder's organs, which are attached to the testes. The mechanisms involved in the inhibition of oogenesis in these structures were investigated in male Bufo woodhousii. Orchidectomized and sham-operated animals were injected with gonadotropins (pregnant mare serum gonadotropin [PMSG] + human chorionic gonadotropin [hCG]) for 26 days and the effects of these hormones on oogenesis and steroidogenic activity (3 beta-hydroxysteroid dehydrogenase [3 beta-HSD] and 17 beta-HSD) in the Bidder's organ were quantified. Bilateral orchidectomy alone resulted in the growth of bidderian oocytes and a shift towards later stages of oogenesis. Gonadotropins enhanced this effect and stimulated the proliferation of new germ cells. In the presence of testes, however, bidderian oogenesis remained inhibited despite high levels of circulating gonadotropins. In both ooplasm and follicular layers of the bidderian oocytes of all toads, 3 beta-HSD and 17 beta-HSD activities were detected by histochemistry. Follicular enzymatic activity increased in orchidectomized toads treated with PMSG + hCG but decreased in sham-operated toads treated with gonadotropins. Testis weights, rudimentary oviduct weights, and plasma steroid levels increased in intact toads injected with hCG + PMSG. Gonadotropins had no effect on plasma steroid levels in orchidectomized toads, however. These results suggest that the testes play a major role in the inhibition of oogenesis in Bidder's organs of B. woodhousii and are a major source of androgens. High circulating levels of gonadotropins do not overcome the inhibitory effects of the testes.

Analysis of Variance↗
PubMed1975

Viability of some metabolic processes in the isolated toad brain adapted to two osmotic environments.

The viability of the isolated toad brain in an aerated Ringer-like medium has been evaluated by the following criteria: 1) amino acid content before and after incubation; 2) accumulation of amino acids in the incubation medium; 3) a comparison of glucose utilization and [U-14C]glucose metabolism with that occurring in vivo; 4) tissue swelling; and 5) tissue lactate contents. On the basis of these criteria, the isolated toad brain, from toads adapted to a fresh-water or a salt-water environment, retains considerable metabolic integrity for at least 2 hr of incubation at 25 degrees C. Specifically, there was no swelling of the tissue, no apparent accumulation of lactate in the tissue, glucose appeared to be utilized at a rate not too different from that calculated for the toad brain in vivo, and the distribution of label from [U-14C]glucose had an overall pattern which resembled that observed in vivo. The tissue levels of amino acids were generally stable in vitro; however, there was a marked decline in the content of aspartate. The accumulation of amino acids in the medium varied considerably from one amino acid to another. Thus, there was very little net efflux of aspartate, GABA, and glutamate from the tissue but considerable net efflux of glutamine. This efflux of amino acids was greater from brains of hyperosmotically adapted toads than from the brains of toads adapted to fresh water by amounts proportional to their initial tissue contents.

Adaptation, Physiological↗
PubMed1993

Comparative study of IGFBP properties in toad and rat sera.

The levels of IGF-I have been simultaneously measured by radioimmunoassay in samples of the toad Bufo arenarum and of normal male Wistar rats. In addition, the different fractions of IGF-I binding proteins (IGFBP) and their binding properties have been identified by ligand blot and Scatchard analysis in the serum of both species. In the toad, we have measured levels of IGF-I (2.78 +/- 0.48 ng/ml) similar to those previously reported in amphibians but far below those found in rats. IGFBP levels were estimated at 129 +/- 23 and 4249 +/- 321 pg/ml in toad and rat serum samples. Two main IGFBP fractions of 30-34 kDa, accompanied by a minor component of 24 kDa and seldom by another of 40 kDa, were identified in toad serum. In rat serum--as already reported--three bands of 40, 30, and 24 kDa were identified, the first being the main component and the last the minor one. The Scatchard analysis of a competitive binding assay showed two types of binding sites in toad serum: one of high affinity-low capacity (Ka1 = 1.6 x 10(10) M-1; R1 = 1.2 x 10(-11) M) and another with low affinity-high capacity (Ka2 = 1.9 x 10(8) M-1; R2 = 1.9 x 10(-10) M). The percentage fraction of these binding sites occupied by IGF-I was 13.5%. The figures for K1 and K2 were lower and those for R1 and R2 were higher in rat than in toad serum. The percentage fraction of occupied rat IGF binding sites was 3.6%.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2001

Angiotensin II elicits water seeking behavior and the water absorption response in the toad Bufo bufo.

Fully hydrated toads, Bufo bufo were acclimated to a simulated terrestrial habitat, with access to shelters and water. To get from the shelters to the water, the toads had to walk across the pan of an Ohaus balance and the body weights were recorded on a computer. Toads were placed inside shelters immediately following injection of human angiotensin II (A II), Thr(8)-saralasin, or Ringer's in the dorsal lymph sac, and their behavior was recorded continuously by video surveillance. The injection doses were 1-100 microg/100 g body weight A II and 100 microg/100 g body weight saralasin dissolved in 0.1 ml Ringer's; control animals received the same volume of Ringer's. The latency from injection to the initiation of water absorption behavior (WR) was significantly shorter in both A-II- and saralasin-injected toads, compared to controls. A-II- and saralasin-injected toads also spent significantly more time in the water than controls. The bladder depots when WR was terminated were significantly larger in A-II- or saralasin-injected toads than in controls. The stimulatory action of Thr(8)-saralasin, an antagonist of A II in mammals, on WR behavior in B. bufo suggests differences in receptor structure and/or receptor distribution between amphibians and mammals.

Angiotensin II↗
PubMed1998

Elevated trace element concentrations in southern toads, Bufo terrestris, exposed to coal combustion waste.

A number of recent studies have linked developmental, physiological, and behavioral abnormalities in amphibians to coal combustion wastes (coal ash). Few studies, however, have determined trace element concentrations in amphibians exposed to coal ash. In the current study we compare total body concentrations of 20 trace elements in adult southern toads, Bufo terrestris, inhabiting coal ash settling basins with toads that were not exposed to the combustion wastes (reference). In addition, we document the accumulation of trace elements in toads transplanted from reference sites to field enclosures in an ash settling basin for 7 and 12 weeks. Arsenic, selenium, and vanadium levels were significantly elevated in toads captured at the ash-contaminated site in comparison to toads from the reference site. All three of these elements were also significantly elevated in toads exposed to the contaminated habitat for only 7 weeks. Our study suggests that adult anurans can bioaccumulate particularly high levels of selenium and may be useful bioindicators in agricultural and coal ash-impacted habitats.

Animals↗
PubMed1990

Insulin-like growth factor-I and insulin-like growth factor-binding protein in the toad, Bufo woodhousei.

Molecular weight characteristics and plasma concentrations of insulin-like growth factor-I (IGF-I) and its binding protein (IGF-BP) were investigated in the toad, Bufo woodhousei. IGF-I and IGF-BP were measured by radioimmunoassay (RIA, Kd = 0.37 +/- 0.04 ng/ml) and charcoal-separated ligand binding assay, respectively, in male toad plasma and adult male human donor plasma using a synthetic human IGF-I standard. Prior to the IGF-I RIA, samples were acid-ethanol extracted. Molecular weight characteristics were determined using size exclusion chromatography. At neutral pH (pH = 7.4), IGF-I immunoreactivity and IGF-BP eluted at molecular weight greater than 66 kDa in both toad and human plasma. Acid chromatography (pH approximately 3) resulted in the separation of IGF-I from its binding protein and consequently a shift of IGF-I immunoreactivity to the low molecular weight fractions (approximately 8 kDa) for both toad and human. IGF-BP activity shifted to molecular weight approximately 50 kDa. Toad plasma IGF-I and IGF-BP activity exhibited differences according to season: IGF-I levels were low in the spring (March = 0.48 +/- 0.11 ng eq/ml), increased progressively to reach a peak in July (5.84 +/- 2.5 ng eq/ml), and decreased to low levels again in the fall (October = 0.60 +/- 0.08, November = 0.45 +/- 0.09 ng eq/ml). Plasma IGF-BP activity demonstrated a similar pattern (March = 17.4 +/- 2.5, July = 35.0 +/- 2.4, November = 12.6 +/- 3.2% specific binding). IGF-I was produced for at least 72 hr when toad liver explants were cultured in serum-free medium, indicating that the liver is a source of IGF-I in anurans.

Animals↗
PubMed1989

The effect of laboratory environment on the morphology of the spleen and the thymus in the yellow-bellied toad, Bombina variegata (L.).

The morphological changes were observed in the spleen and the thymus of the yellow-bellied toad, Bombina variegata, kept under standard laboratory conditions. The mean splenic weights of toads studied soon after capture in July and September were 16.0 mg and 17.7 mg, respectively. In contrast, the mean splenic weights significantly decreased in animals maintained in the laboratory from July till September (to 11.6 mg) and from July till December (to 6.8 mg). In the spleen of toads kept in the laboratory the lymphocyte aggregations were diminished in the white pulp while the amount of connective tissues increased both in the white and in the red pulps. Melano-macrophages were more abundant in the red pulp of toads kept in the laboratory than in freshly collected ones. The thymuses of toads kept in the laboratory were decreased in size and depleted of the majority of their cortical lymphocytes. It is suggested that the morphological changes of the yellow-bellied toad lymphoid organs might be the results of stressful laboratory conditions and lower antigenic stimulation in the laboratory than in nature.

Animals↗
PubMed1991

Comparison of the capsaicin- and amino acid-sensitivity of dorsal root C fibres in the rat and the toad.

1. The C elevation of the compound action potential (CAP) was recorded with suction electrodes from dorsal roots of rats at 25 degrees C and toads (Bufo bufo) at 10 degrees C. The C fibre CAP had a conduction velocity of 0.5 +/- 0.07 SE M per sec (N = 10) and 0.25 +/- 0.04 M per sec (N = 8) in the rat and toad nerves respectively. 2. The depressant effect of applied drugs on the amplitude of the C fibres CAP was measured. Nerves from both species had similar sensitivities to GABA. EC50 5.0 microM +/- 0.5 SEM (N = 3) and 5.5 microM +/- 1.4 (N = 3) for the rat and toad respectively. Maximum depressant effects of GABA produced in rat and toad nerves were 35% +/- 5 SEM and 17% +/- 2.5 respectively. 3. In five out of ten of the rat nerves tested kainate had a clear depressant effect (maximum 36% +/- 4.3 SEM, EC50 6.8 microM +/- 0.9 SEM, N = 3) on the C fibre CAP. Kainate, at concentrations from 100 to 500 microM, had no effect on seven toad nerves. 4. Toad nerves were about 100 times less sensitive, than rat nerves, to capsaicin (ED50 values 430 microM +/- 190 SEM and 0.7 microM +/- 0.2 respectively, N = 4). 5. The similar sensitivity of nerves in both species to GABA and differing sensitivities to kainate and capsaicin suggests that amphibian C fibres specifically lack sensitivity to capsaicin and kainate.

Action Potentials↗
PubMed2004

Tectal responses to potassium loads and subsequent visual stimuli in the toad, Bufo bufo.

Calling male toads were tested behaviourally for their prey catching responses to wormlike stimuli and assigned to groups of non-hungry and hungry depending on their prey catching motivation before being prepared for visual unit, massed unit and slow potential shift (SPS) recording from the optic tectum. Control recordings to visual stimuli were made before recording the effects of application of isotonic solutions containing concentrations of 0-41 mM K(+). Application of solution was followed by presentation of the visual stimulus while the solution still bathed the tectum. The best tectal responses were made to large square visual stimuli in the non-hungry toads, perhaps because recordings were made in the breeding season. Responses of the tectum to solution addition were significant in the concentration range of 7-17 mM K(+). Hungry toads showed an earlier, smaller response than non-hungry (sexually motivated) animals. When the visual stimulus was presented, there were unit and massed unit responses at all bathing solution concentrations, which were larger in non-hungry animals. These experiments revealed that toads motivated to feed respond earlier than non-hungry toads to application of artificial CSF to the tectum, though non-hungry toads responded best to the subsequent visual stimulus.

Animals↗
PubMedDate not supplied

An improved enzyme linked immunosorbent assay for detection of anti-ranavirus antibodies in the serum of the giant toad (Bufo marinus).

An improved ranavirus antibody ELISA (R Ab ELISA) for the specific detection of anti-ranavirus antibodies in toad sera was developed. Sheep anti-epizootic haematopoietic necrosis virus (EHNV) was used as the antigen-capture antibody. EHNV was used as the antigen and sera from field and challenged toads were used to detect the virus. Rabbit anti-toad IgG and IgM were used to detect bound toad antibody. Pre-absorption of toad sera with a monoclonal antibody, raised against the 50 kDa EHNV protein, improved the specificity of the technique. A blocking ELISA, immunofluorescence and immuno-electron microscopy were used to confirm the validity of the ELISA. The assay has potential use in screening sera from Bufo marinus for the presence of antibodies against ranaviruses and to facilitate understanding of the humoral immunological response in toads during virus infection.

Absorption↗
PubMed1998

Gender does not influence neuromuscular properties in dimorphic skeletal muscles of the toad.

The aim of this work was to study gender differences on the physiology of the dimorphic brachial musculature involved in the clasp reflex of the toad (Bufo marinus L.). The neuromuscular transmission, the sensitivity to acetylcholine (ACh) and the cholinesterase activity were compared on the forelimb sternoradialis muscles (SR) from male and female toads. The interosseous muscles of the first finger were used to compare the properties of the nicotinic receptor/ionic channel complex (AChR). All the muscles studied were dimorphic, i.e. significantly smaller in the female than in the male frog in otherwise similar size animals. The SR of either sex contracted to bath application of ACh with similar EC50. In physiological solution the frequency of the miniature end-plate potentials (mepps) was very low (0.1 s-1) and no gender difference was detected. The mepp amplitudes were 0.62 +/- 0.03 and 0.58 +/- 0.03 mV in SR from male and female toads, respectively. To increase exocytosis the muscles were incubated in hypertonic solution (158 mM NaCl). Under this condition mepp frequency was increased by five and seven times and mepp amplitude increased by 1.3 and 1.6 times in SR from male and female toads, respectively. The cholinesterase activity measured by the colorimetric method, did not differ in SR from male and female toads. In muscle fibers dissociated from the dimorphic interosseous muscles of male and female toads, the ionic channel conductance was 43 +/- 5.3 and 44 +/- 4.5 pS, respectively. The mean channel open time was voltage-dependent and not significantly different in preparations from both genders. These observations indicate that neither the ACh-nicotinic receptor interaction, nor the AChR complex kinetics and the nicotinic excitation-contraction coupling or the cholinesterase activity differ in dimorphic muscles from Bufo genders. No gender difference was detected in neuromuscular transmission of the studied muscle. Only a slight increase in mepp frequency and amplitude could be detected when the muscles were incubated in hypertonic solution.

Animals↗
PubMed2000

Expression of polysialic acid, alpha- and beta-cantenins in adult toad testis in hibernation stage and after gonadotrophin--releasing hormone (GnRH) treatment.

The Polysialic Acid (PSA), glycosydic moiety of the Neural Cell Adhesion Molecule (N-CAM), and alpha- and beta-Catenins, which mediate interaction between Cadherins and cytoskeletal proteins, participate in cell adhesion phenomena in numerous organs and tissues. We have performed an immunohistochemical analysis, in hibernating toad testis and in GnRH-reactivated hibernating animals. In hibernating toads we could demonstrate PSA-immunoreactivity (PSA-IR) within the seminiferous tubules, in clusters of primary spermatocytes, spermatids and spermatozoa, in follicular and Sertoli cells. PSA-IR was seen in peritubular, Leydig and efferent duct cells. In GnRH-treated toads PSA-IR persists in primary spermatocyte groups. alpha-Catenin is localized in the basal laminae of seminiferous tubules and in Leydig cells of hibernating toads. This did not change after hormonal treatment. In hibernating toads, beta-Catenin was detected only in Leydig cells and within seminiferous tubules on basal spermatocystes and limiting spermatozoa clusters. In GnRH-treated toads, the beta-Catenin-IR was less intense in Leydig cells and vanished within seminiferous tubules.

Animals↗
PubMed2004

Osmotic properties of the sealed tubular system of toad and rat skeletal muscle.

A method was developed that allows conversion of changes in maximum Ca(2+)-dependent fluorescence of a fixed amount of fluo-3 into volume changes of the fluo-3-containing solution. This method was then applied to investigate by confocal microscopy the osmotic properties of the sealed tubular (t-) system of toad and rat mechanically skinned fibers in which a certain amount of fluo-3 was trapped. When the osmolality of the myoplasmic environment was altered by simple dilution or addition of sucrose within the range 190-638 mosmol kg(-1), the sealed t-system of toad fibers behaved almost like an ideal osmometer, changing its volume inverse proportionally to osmolality. However, increasing the osmolality above 638 to 2,550 mosmol kg(-1) caused hardly any change in t-system volume. In myoplasmic solutions made hypotonic to 128 mosmol kg(-1), a loss of Ca(2+) from the sealed t-system of toad fibers occurred, presumably through either stretch-activated cationic channels or store-operated Ca(2+) channels. In contrast to the behavior of the t-system in toad fibers, the volume of the sealed t-system of rat fibers changed little (by <20%) when the osmolality of the myoplasmic environment changed between 210 and 2,800 mosmol kg(-1). Results were also validated with calcein. Clear differences between rat and toad fibers were also found with respect to the t-system permeability for glycerol. Thus, glycerol equilibrated across the rat t-system within seconds to minutes, but was not equilibrated across the t-system of toad fibers even after 20 min. These results have broad implications for understanding osmotic properties of the t-system and reversible vacuolation in muscle fibers. Furthermore, we observed for the first time in mammalian fibers an orderly lateral shift of the t-system networks whereby t-tubule networks to the left of the Z-line crossover to become t-tubule networks to the right of the Z-line in the adjacent sarcomere (and vice versa). This orderly rearrangement can provide a pathway for longitudinal continuity of the t-system along the fiber axis.

Aniline Compounds↗
PubMed2006

An invasive species induces rapid adaptive change in a native predator: cane toads and black snakes in Australia.

Rapid environmental change due to human activities has increased rates of extinction, but some species may be able to adapt rapidly enough to deal with such changes. Our studies of feeding behaviour and physiological resistance to toxins reveal surprisingly rapid adaptive responses in Australian black snakes (Pseudechis porphyriacus) following the invasion of a lethally toxic prey item, the cane toad (Bufo marinus). Snakes from toad-exposed localities showed increased resistance to toad toxin and a decreased preference for toads as prey. Separate laboratory experiments suggest that these changes are not attributable to learning (we were unable to teach naive snakes to avoid toxic prey) or to acquired resistance (repeated sub-lethal doses did not enhance resistance). These results strongly suggest that black snake behaviour and physiology have evolved in response to the presence of toads, and have done so rapidly. Toads were brought to Australia in 1935, so these evolved responses have occurred in fewer than 23 snake generations.

Adaptation, Physiological↗
PubMed1991

Characterization of a novel receptor in toad retina with dual specificity for insulin and insulin-like growth factor I.

The biochemical properties of insulin receptors from toad retinal membranes were examined in an effort to gain insight into the role this receptor plays in the retina. Competition binding assays revealed that toad retinal membranes contained binding sites that displayed an equal affinity for insulin and insulin-like growth factor I (IGF-I). Affinity labeling of toad retinal membrane proteins with 125I-insulin resulted in the specific labeling of insulin receptor alpha-subunits of approximately 105 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partially reduced (alpha beta-heterodimer) receptors affinity-labeled with 125I-insulin indicated the presence of a disulfide-linked beta-subunit of approximately 95 kDa. Endoglycosidase F digestion of the affinity-labeled alpha-subunits increased their mobility by reducing their apparent mass to approximately 83 kDa. This receptor was not detected by immunoblot analysis with a site-specific antipeptide antibody directed against residues 657-670 of the carboxy terminal of the human insulin receptor alpha-subunit, whereas this antibody did label insulin receptor alpha-subunits from pig, cow, rabbit, and chick retinas. In in vitro autophosphorylation assays insulin stimulated the tyrosine phosphorylation of toad retina insulin receptor beta-subunits. These data indicate that toad retinal insulin receptors have a heterotetrameric structure whose alpha-subunits are smaller than other previously reported neuronal insulin receptors. They further suggest that a single receptor may account for both the insulin and IGF-I binding activities associated with toad retinal membranes.

Animals↗
PubMed1968

The distribution of the T-system along the sarcomeres of frog and toad sartorius muscles.

1. Frog and toad sartorius muscles were soaked in ferritin suspensions and then fixed and prepared for electron microscopy. Ferritin particles were counted in micrographs of regions where fibril striations were in good register, and the number of particles plotted according to position along the sarcomeres.2. The distribution of ferritin particles in both frog and toad muscle sarcomeres could be accounted for by a single peak centred at the Z-line, with a total width of about 0.4 mu.3. The peak in toad fibres was slightly broader than that in frog fibres, and this was due to more frequent branching of the T-system in the toad. In both frog and toad sartorius muscles branching increases the total quantity of T-system by around 30%.4. No peak was found in the ferritin distribution which could account for the peak of albumin distribution observed near the A-I boundary of toad sartorius muscles by Hill (1964).

Albumins↗
PubMed1968

Effect of a saline environment on sodium transport by the toad colon.

1. Colons isolated from saline-adapted or aldosterone-injected toads maintained transmural potential differences with the serosal side positive to the mucosa. The short-circuit currents of colons taken from aldosterone-injected toads could be expressed quantitatively by the net flux of sodium measured isotopically. This did not apply to saline-adapted colons where the net sodium flux was from serosa to mucosa.2. The short-circuit currents of colons taken from aldosterone-injected animals increased as the sodium concentration was raised from 10 to 50 mM, then decreased as the sodium concentration was further increased to 115 mM. Adaptation to saline changed this relationship, the short-circuit current becoming directly dependent on the sodium concentration.3. Faecal sodium was higher than serum sodium in saline-adapted toads. There was little or no change in the level of serum sodium or potassium. The urine of saline-adapted toads also contained high concentrations of sodium.4. The total and ouabain-sensitive ATPase activities of mucosal scrapings taken from saline-adapted colons were about half those found in the aldosterone-injected animal. Ten per cent of the total ATPase activity could be inhibited by ouabain.5. Microsomal fractions of mucosal scrapings taken from saline-adapted toads contained 4 times less Na(+) + K(+)-activated ATPase than did corresponding fractions from aldosterone-injected animals. High concentrations of sodium inhibited the microsomal ATPase activity irrespective of the previous conditions of adaptation.6. Regulation of sodium movements across the toad colon appears to be a complex process with the mucosal cells changing their properties so that they either absorb or secrete sodium ions depending on the physiological state of the animal.

Adaptation, Physiological↗