Showing papers on "Toad published in 1998"

Purification and characterization of insulin, glucagon, and two glucagon-like peptides with insulin-releasing activity from the pancreas of the toad, Bufo marinus.

Abstract: Insulin and four peptides derived from the posttranslational processing of proglucagon have been isolated in pure form from the pancreas of the cane toad, Bufo marinus. Although Bufo insulin contains 9 amino acid substitutions, compared with human insulin, all those residues that are considered to be involved in receptor-binding and in dimer and hexamer formation have been conserved. Bufo insulin was, however, more potent (4-fold) than human insulin in inhibiting the binding of [I-125-Tyr-A14] insulin to the soluble full-length recombinant human insulin receptor, which is probably a consequence of the substitution (Thr --> Kis) at position A-8. Bufo glucagon was isolated in two molecular forms: glucagon-29 shows only one amino acid substitution (Thr29 --> Ser), compared with human glucagon; and glucagon-36 comprises glucagon-29, extended from its C-terminus by Lys-Arg-Ser-Gly-Gly-Met-Ser. The human proglucagon gene contains one copy of glucagon-like peptide (GLP)-1, a potent insulin secretogogue, and one copy of GLP-2 that is devoid of insulin-releasing activity. In contrast, two proglucagon-derived peptides with 32- and 37-amino acid residues (GLP-32 and GLP-37), displaying greater structural similarity to human GLP-1 than to GLP-2, were isolated from Bufo pancreas. Both peptides produced concentration-dependent increases in insulin release from glucose-responsive rat insulinoma-derived BRIN-BD11 cells. The threshold concentrations producing a significant (P < 0.001) effect were 10(-8) M (GLP-32) and 10(-9) M (GLP-37), and the maximum increase in the rate of insulin release produced by 10(-6) M concentrations of both peptides was approximately 5-fold.

Isolation and characterization of novel endogenous digitalis-like factors in the ovary of the giant toad, Bufo marinus.

Abstract: We have previously described the structures of four novel unconjugated bufadienolides in the ovary of the toad, Bufo marinus. In this study, we report the separation and characterization of three novel bufadienolide conjugates. These compounds were purified by HPLC, and their structures were determined to be 11alpha, 19-dihydroxytelocinobufagin-3-(12-hydroxydodecanoic acid) ester, 11alpha,19-dihydroxytelocinobufagin-3-(14-hydroxy-7-tetra decenoic acid) ester, and 11alpha, 19-dihydroxytelocinobufagin-3-(14-hydroxytetradecanoic acid) ester on the basis of NMR and MS data. Numerous dicarboxylic acid esters of bufadienolides have previously been described, but the three bufadienolide conjugates described in this report differ from previously described esters in that they contain hydroxylated monocarboxylic acids. The function of these three conjugates is not known but they are, like bufotoxins, potent inhibitors of Na+, K+-ATPase and may play a developmental role in the differentiation of toad oocytes.

Effect of nifedipine on depolarization-induced force responses in skinned skeletal muscle fibres of rat and toad

Abstract: The effect of the dihydropyridine, nifedipine, on excitation-contraction coupling was compared in toad and rat skeletal muscle, using the mechanically skinned fibre technique, in order to understand better the apparently disparate results of previous studies and to examine recent proposals on the importance of certain intracellular factors in determining the efficacy of dihydropyridines. In twitch fibres from the iliofibularis muscle of the toad, 10 microM nifedipine completely inhibited depolarization-induced force responses within 30 s, without interfering with direct activation of the Ca(2+)-release channels by caffeine application or reduction of myoplasmic [Mg2+]. At low concentrations of nifedipine, inhibition was considerably augmented by repeated depolarizations, with half-maximal inhibition occurring at < 0.1 microM nifedipine. In contrast, in rat extensor digitorum longus (EDL) fibres 1 microM nifedipine had virtually no effect on depolarization-induced force responses, and 10 microM nifedipine caused only approximately 25% reduction in the responses, even upon repeated depolarizations. In rat fibres, 10 microM nifedipine shifted the steady-state force inactivation curve to more negative potentials by < 11 mV, whereas in toad fibres the potent inhibitory effect of nifedipine indicated a much larger shift. The inhibitory effect of nifedipine in rat fibres was little, if at all, increased by the absence of Ca2+ in the transverse tubular (t-) system, provided that the Ca2+ was replaced with sufficient Mg2+. The presence of the reducing agents dithiothreitol (10 mM) or glutathione (10 mM) in the solution bathing a toad skinned fibre did not reduce the inhibitory effect of nifedipine, suggesting that the potency of nifedipine in toad skinned fibres was not due to the washout of intracellular reducing agents. The results are considered in terms of a model that can account for the markedly different effects of nifedipine on the two putative functions of the dihydropyridine receptor, as both t-system calcium channel and a voltage-sensor controlling Ca2+ release.

Effect of chloride on Ca2+ release from the sarcoplasmic reticulum of mechanically skinned skeletal muscle fibres.

Abstract: The effect of intracellular Cl– on Ca2+ release in mechanically skinned fibres of rat extensor digitorum longus (EDL) and toad iliofibularis muscles was examined under physiological conditions of myoplasmic [Mg2+] and [ATP] and sarcoplasmic reticulum (SR) Ca2+ loading. Both in rat and toad fibres, the presence of 20 mM Cl–in the myoplasm increased Ca2+ leakage from the SR at pCa (i.e. –log10 [Ca2+]) 6.7, but not at pCa 8. Ca2+ uptake was not significantly affected by the presence of Cl–. This Ca2+-dependent effect of Cl– on Ca2+ leakage was most likely due to a direct action on the ryanodine receptor/Ca2+ release channel, and could influence channel sensitivity and the resting [Ca2+] in muscle fibres in vivo. In contrast to this effect, acute addition of 20 mM Cl– to the myoplasm caused a 40–50% reduction in Ca2+ release in response to a low caffeine concentration both in toad and rat fibres. One possible explanation for this latter effect is that the addition of Cl– induces a potential across the SR (lumen negative) which might reduce Ca2+ release via several different mechanisms.

The lung of the common toad, Bufo arenarum (Anura: Bufonidae). A light and electron microscopy study.

Abstract: The present study describes the lung morphology of the common toad, Bufo arenarum, as observed by light and electron microscopy. The lung wall consists of four layers: mesothelium, dense connective tissue with thin elastic fibers, loose connective tissue containing smooth muscle fibers and an internal respiratory epithelium. The lung presents three types of folds defined by their epithelia. First order folds are coated by ciliated epithelium containing numerous goblet cells. Second order folds present the same type of epithelium but devoid of goblet cells, while third order folds are only lined by respiratory epithelium. The respiratory surface of the lung is lined by a single cell type, the pneumocyte, which presents characteristics of both type I and type II alveolar cells of higher vertebrates. The pneumocytes are prismatic in shape and possess attenuated cytoplasmatic processes which spread over the pulmonary capillaries to form the outer layer of the air-blood barrier. These cells present microvilli in the apical extreme and contain different types of cytoplasmic bodies: electron dense, multivesicular and lamellar.

Developmental studies for identification of the inhibitory center of melanotropes in the toad, Bufo japonicus

Abstract: Two series of experiments were performed to identify the inhibitory center of the melanotropes in the intermediate lobe of hypophysis of the toad, Bufo japonicus. First, developmental changes in the distribution of dopaminergic neurons were examined from hatching stage to postmetamorphosis using an antiserum against dopamine synthase (tyrosine hydroxylase, TH). In the postmetamorphic toads, TH-positive cell bodies were localized in three clusters. One was the preoptic recess organ (PRO) in the prechiasmatic area, the other two were the paraventricular organ (PVO) and infundibular nucleus (IN) in the postchiasmatic area. Each of them exhibited different ontogenetic changes. During larval development, TH-positive cell bodies were first detected in the PVO and IN at a premetamorphic stage. The number of immunoreactive cells increased rapidly in both loci as metamorphosis proceeded, although the two nuclei showed different growth profiles. By contrast, in the PRO, a very small number of immunoreactive cells were observed before the onset of the prometamorphic period. Although the number of immunoreactive neurons increased as metamorphosis progressed, early neurons were confined to the caudal area of the PRO (cPRO), the rostral area of the PRO (rPRO) being devoid of TH-positive cells. Immunoreactive TH neurons appeared in the rPRO for the first time at the end of metamorphic climax. This timing coincided well with the development of TH-positive nerve endings in the pars intermedia (PI) and median eminence. In the second series of experiments, the embryonic primordium of the PRO was surgically extirpated from open neurulae to examine the effects of PRO-ectomy. In 75% of the operated animals, background adaptation was not observed, their dermal melanophores remained permanently dispersed even on the white background. Dopaminergic neurons in the rPRO and the immunoreactive nerve endings in the PI and median eminence were scarcely observed in these animals. It was concluded that the present data strongly support the hypothesis that rPRO is the center of white-background adaptation.

Tachykinin receptors in the small intestine of the cane toad (bufo marinus): a radioligand binding and functional study

Abstract: In this study, we have used radioligand binding and functional techniques to investigate tachykinin receptors in the small intestine of the cane toad Bufo marinus. The radioligand [125I]Bolton-Hunter [Sar9,Met(O2)11]substance P (selective at mammalian NK-1 receptors) showed no specific binding. Specific binding of [125I]Bolton-Hunter substance P ([125I]BHSP) was saturable, of high affinity (Kd 0.3 nM) and was inhibited by SP (IC50 0.64 nM) > ranakinin scyliorhinin I $ [Sar9]-SP $ neurokinin B > SP(7–11) App[NH]p, indicating a G-protein coupled receptor. The order of potency of tachykinins and analogues in contracting the isolated lower small intestine was carassin (EC50 1.4 nM) > eledoisin SP(6–11) > scyliorhinin II $ neuropeptide γ > neurokinin B [Sar9]SP > SP(7–11). In both studies, the selective mammalian NK-1, NK-2 and NK-3 receptor agonists [Sar9,Met(O2)11]SP, [Lys5,MeLeu9,Nle10]NKA(4–10) and senktide were weak or ineffective. There was a strong positive correlation between the pD2 and pIC50 values for mammalian tachykinins and analogues (r=0.907), but not for the non-mammalian tachykinins, which were all full agonists but variable binding competitors. [Sar9,Met(O2)11]-SP(pD2 5.7) was approximately 25-fold less potent as an agonist than [Sar9]SP, which was itself 25-fold weaker than SP. Responses to SP were significantly reduced (n = 8, P<0.001) by the antagonist [D-Arg1,D-Trp7,9,Leu11]-SP (spantide; 1 μM). Highly selective NK-1 receptor antagonists including CP 99994 and GR 82334 (both 1 μM) were ineffective in both functional and binding studies. Tetrodotoxin (1 μM) did not inhibit contractile responses to SP, NKA and senktide. In summary, this study has shown the presence of one or more tachykinin receptor in the toad intestine. The binding site recognised by [125I]BHSP prefers SP and ranakinin. This toad “NK-1-like receptor” differs from the mammalian NK-1 receptor in having a low affinity for all mammalian NK-1 selective ligands, including antagonists. For some non-mammalian peptides, their high potency as contractile agonists relative to their poor binding affinity suggests the existence of other tachykinin receptors in the toad small intestine.

Lack of Inhibitory Control of Melanophore-Stimulating Hormone Secretion in the Larval Toad, Bufo japonicus

Abstract: In general, both larval and adult amphibians are known to change their skin color depending on the background color. However, in the Japanese toad Bufo japonicus, the larvae were found to be devoid of the ability of responding to the background color. Melanin granules in the dermal melanophores were in a state of constant dispersion independently of the color of the background to which they were subjected. Pharmacological and histochemical studies revealed that in the toad tadpoles, melanophore-stimulating hormone is being released without inhibition, presumably because innervation of catecholaminergic fibers in the intermediate lobe is incomplete until they finish metamorphosis. Biological significance of the dark color of Bufo tadpoles is discussed in conjunction with another characteristic that they have a tendency to aggregate to form a dense black mass.

Opposite effects of cooling on twitch contractions of skeletal muscle isolated from tropical toads (Leptodactylidae) and northern frogs (Ranidae)

Abstract: Cooling increases the twitch force of frog skeletal muscle (Rana temporaria; Rana pipiens), but decreases the twitch force of tropical toad muscle (Leptodactylus insularis). Action potentials and intramembranous charge movement in frog and toad fibers were slowed identically by cooling. Cooling increased the integral of twitch Ca2+ detected by aequorin in frog fibers (1.4-fold), while also decreasing the peak and slowing the rate of decay. Conversely, cooling decreased the integral (0.6-fold) and the peak of twitch Ca2+ in toad fibers, without affecting the rate of decay. The difference in entire Ca2+ transients may account for cold-induced twitch potentiation in frogs and twitch paralysis in toads. In sustained contractions of toad fibers, cooling markedly decreased maximum force caused by: (i) tetanic stimulation, (ii) two-microelectrode voltage clamp steps, (iii) high [K+], or (iv) caffeine. Maximum force in sustained contractions was decreased moderately by cooling frog fibers. Rapid rewarming and simultaneous removal of high [K+] or caffeine during a sustained contraction, caused toad muscle force to rise towards the value corresponding to the warm temperature. This did not occur after removing high [K+] or caffeine from toad fibers kept in the cold. Transmission electron micrographs showed no relevant structural differences. Parvalbumins are thought to promote relaxation of frog muscle in the cold. The unique parvalbumin isoforms in toad muscle apparently lack this property.

Anthropogenic mortality of the common toad (Bufo bufo) in Scotland

Abstract: oration in older Arabian horses has been suggested to be due to the destruction of melanocytes through an immunopathologic or cytotoxic process (Lemer and Cage 1974). Disturbances in melanin metabolism associated with greying act to stimulate formation of new melanoblasts, or to stimulate their activity, resulting in focal areas of overproduction in the dermis with the perineum, genitalia and tail being at high risk for development of melanocytic tumours (Madewell and Theilen 1987, Pulley and Stannard 1990). Although on the basis of the current classification of melanocytic tumours (Pulley and Stannard 1990) the neoplasm in the skin was defined as dermal melanoma, a recent study (Valentine 1995) has included a new type, dermal melanomatosis, which occurs in grey horses, over 15 years of age, and most frequently involves the central tail, perineum and extemal genitalia. The study indicated that dermal melanomas occurring in younger animals, have a better prognosis and generally appear as single masses in various locations. Apart from the differential clinical features, dermalmelanoma and dermal melanomatosis are histologically indistinguishable. Malignant melanomas can metastasise via lymph vessels and blood, although regional lymph nodes are commonly the first site affected. The wide distribution of melanomas in this case would indicate that metastases had occurred through both routes. Three other cases of lameness caused by melanomas in horse have been described. In two reports, posterior paresis developed secondary to spinal cord compression (Traver and others 1977, Schott and others 1990) and in one report pelvic limb lameness resulted primarily from infiltration of neoplastic cells into sacral and ischiatic nerves (Kirker-Head and others 1985). Except for lymphosarcomas and neurofibromas in cattle, all tumours involving the nervous system are rare in large animals. Most tumours that cause compression of the spinal cord originate from surrounding structures such as the bone, cartilage, fibrous tissue and blood vessels of the vertebrae and, less commonly, from the haematopoietic elements of bone and tissue outside the vertebral column, including muscle, fat and paraganglia (Prata and Carrillo 1985, Madewell and Theilen 1987). Tumour metastases from sites elsewhere in the body may also be found in the vertebrae, epidural space, meninges and, rarely, the parenchyma of the spinal cord. Secondary tumours occur due to haematogenous or lymphatic spread of tumour emboli and include hemangiosarcoma, lymphosarcoma, mammary, pulmonary and prostatic adenocarcinoma and malignant melanoma (Prata and Carrillo 1985). The clinicopathological findings in the present case were those of a transverse myelopathy (LeCouteur 1986) due to lumbar spinal stenosis or cauda equina compression. Mechanical deformation is likely to be the major factor in the pathogenesis of the spinal cord lesions; however, the circulation disturbance may also have played an important role. This case illustrates that melanoma may be encountered causing signs of transverse myelopathy in Arabian horses due to metastases of dermal melanomas.

Effects of sodium chloride and sodium gluconate concentration on hydration behavior and water transport in the red-spotted toad, Bufo punctatus

Abstract: Effects of Sodium Chloride and Sodium Gluconate Concentration on Hydration Behavior and Water Transport in the Red spotted Toad, Bufo punctatus by Polly Ann Sullivan Dr. Karin Hoff and Dr. Stan Hillyard, Examination Committee Co-chairs Professors of Biology University of Nevada, Las Vegas Toads hydrate from standing water or moist substrates by osmotic absorbtion across the ventral skin. On land toads adopt a distictive posture called the water absorption response (WR) to maximize contact with the substrate and facilitate water absorption. Experiments using hydration behavior on moist substrates and rehdration rate in standing water or salt solutions in dehydrated Red-spotted toads (fiufo punctatus) were used to demonstrate that: 1) toads can distinguish among NaCl concentrations, 2) NaCl facilitates water uptake across the skin, and 3) transport of the chloride anion facilitates sodium uptake and affects sodium detection by the toad. The results of these studies also strongly suggest that general osmotic mechanisms as well as epithelial sodium channels function in chemosensation across the amphibian skin and that the ventral skin and the feet may have different roles in chemosensation.