Showing papers on "Toad published in 2003"

A novel signalling pathway originating in mitochondria modulates rat skeletal muscle membrane excitability.

Abstract: Single skeletal muscle fibres from rat and cane toad were mechanically skinned and stimulated either electrically by initiating action potentials in the sealed transverse (t-) tubular system or by ion substitution causing depolarisation of the t-system to pre-determined levels. Depression of mitochondrial ATP-producing function with three diverse mitochondrial function antagonists (azide: 1-10 mM; oligomycin 1 microg ml-1 and carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP) 1 microM), under conditions in which the cytosolic ATP was maintained high and constant, invariably reduced the excitability of rat fibres but had no obvious effect on the excitability of toad fibres, where mitochondria are less abundant and differently located. The reduction in excitability linked to mitochondria in rat fibres appears to be caused by depolarisation of the sealed t-system membrane. These observations suggest that mitochondria can regulate the functional state of mammalian muscle cells and have important implications for understanding how the balance between ATP utilisation and ATP production is regulated at the cellular level in general and in mammalian skeletal muscle fibres in particular.

Structural and functional aspects of Bufo americanus spermatozoa: Effects of inactivation and reactivation

Abstract: Very little is known about the effects of manipulating toad sperm activity in vitro, and such information is important in the development of a genetic resource bank for bufonid species. The specific objectives of this study were to: 1) identify the optimal inactivation and reactivation solutions for toad spermatozoa colleted in urine; 2) establish the length of time toad spermatozoa can be exposed to an inactivation buffer and still resume motility upon reactivation; 3) evaluate the consequence of inactivation on specific sperm characteristics; and 4) characterize the sperm mitochondria vesicle (MV) and its relationship to motility. Reactivated sperm motility was similar after inactivation in either Simplified Amphibian Ringers (SAR) solution or DeBoer's (DB) solution. Diluting the buffer by 80% with water provided the best method for reactivating sperm. Dilutions with NaCl solutions (10-50 mM) produced inferior results. SAR-inactivated spermatozoa could remain suspended up to 4 hr and still regain 25% of initial motility upon reactivation in water. Compared to the controls, sperm motility was greater (Po0.01) over time for samples treated with SAR, although forward progression was significantly lower. Furthermore, SAR treatment resulted in sperm samples with a greater number of viable, morphologically normal, and intact MVs over time. Electron microscopy and fluorescent staining confirmed that the toad sperm's MV contains a large number of active mitochondria with very few other cytoplasmic structures. Nearly all spermatozoa exhibiting motility had an intact MV, and dissociation of this structure was clearly related to motility loss. In conclusion, toad spermatozoa can be effectively inactivated and reactivated by varying the osmolality of the external solutions and, although sperm forward progression is reduced, all other characteristics are well maintained. Moreover, the increased number of spermatozoa with intact MV after inactivation suggests the process may help preserve this important structure. J. Exp. Zool.

Structural and biochemical characterization of toad liver fatty acid-binding protein.

Abstract: Two paralogous groups of fatty acid-binding proteins (FABPs) have been described in vertebrate liver: liver FABP (L-FABP) type, extensively characterized in mammals, and liver basic FABP (Lb-FABP) found in fish, amphibians, reptiles, and birds. We describe here the toad Lb-FABP complete amino acid sequence, its X-ray structure to 2.5 A resolution, ligand-binding properties, and mechanism of fatty acid transfer to phospholipid membranes. Alignment of the amino acid sequence of toad Lb-FABP with known L-FABPs and Lb-FABPs shows that it is more closely related to the other Lb-FABPs. Toad Lb-FABP conserves the 12 characteristic residues present in all Lb-FABPs and absent in L-FABPs and presents the canonical fold characteristic of all the members of this protein family. Eight out of the 12 conserved residues point to the lipid-binding cavity of the molecule. In contrast, most of the 25 L-FABP conserved residues are in clusters on the surface of the molecule. The helix-turn-helix motif shows both a negative and positive electrostatic potential surface as in rat L-FABP, and in contrast with the other FABP types. The mechanism of anthroyloxy-labeled fatty acids transfer from Lb-FABP to phospholipid membranes occurs by a diffusion-mediated process, as previously shown for L-FABP, but the rate of transfer is 1 order of magnitude faster. Toad Lb-FABP can bind two cis-parinaric acid molecules but only one trans-parinaric acid molecule while L-FABP binds two molecules of both parinaric acid isomers. Although toad Lb-FABP shares with L-FABP a broad ligand-binding specificity, the relative affinity is different.

Effect of lead acetate on the in vitro engulfment and killing capability of toad (Bufo arenarum) neutrophils.

Abstract: Lead is an element of risk for the environment and human health and has harmful effects that may exceed those of other inorganic toxicants. The immune system is one of the targets of lead. Its immunomodulatory actions depend on the level of exposure, and it has been demonstrated that environmental amounts of the metal alter immune function. Very little information is available regarding the effect of the metal on different aspects of the immune system of lower vertebrates, in particular of amphibians. The aim of this study was to investigate the effect of sublethal lead (as acetate) on the function of polymorphonuclear cells of Bufo arenarum. The results revealed that phagocytic and lytic functions of the adherent blood cells collected from sublethal lead-injected toads and incubated with suspensions of Candida pseudotropicalis were affected negatively. The decrease of the phagocytic activity was correlated with increased blood lead levels (P < 0.0001). Additional information referred to the total and differential leukocyte counts was presented; the only difference found was in the number of blast-like cells that resulted augmented in the samples of lead-injected toads. It was concluded that the evaluation of these parameters might be a reliable tool for the biological monitoring of the immune status of amphibians.

Metabolic costs induced by lactate in the toad Bufo marinus: new mechanism behind oxygen debt?

Abstract: The mechanism of an increase in metabolic rate induced by lactate was investigated in the toad Bufo marinus. Oxygen consumption (V˙o 2) was analyzed in fully aerobic animals under hypoxic condition...

Two novel Bv8-like peptides from skin secretions of the toad Bombina maxima

Abstract: Two novel bioactive peptides were purified from skin secretions of the toad Bombina maxima. The partial N-terminal sequences of these two peptides were determined by automated Edman degradation. This allowed the cloning of full-length cDNAs encoding these two peptides from a cDNA library prepared from the toad skin. The deduced complete amino acid sequences indicate that both peptides are composed of 77 amino acids. A fasta search in the databanks revealed that they exhibit 86–91% sequence identity with Bv8, a peptide originally isolated from skin secretions of Bombina variegata. They were thus named as Bv8-like peptide 1 (Bv8-LP1) and Bv8-like peptide 2 (Bv8-LP2), respectively. Sequence differences between Bv8-LP1 and 2 were due to six amino acid substitutions at positions 6, 11, 23, 24, 62 and 63. Bv8-LP1 and 2 differed from Bv8 with eleven and seven amino acid substitutions, respectively. Like Bv8, Bv8-LP1 and 2 possessed contractile activity on isolated guinea pig ileum. Additionally, they stimulated contraction of rabbit aortic rings in a dose-dependent manner at nanomolar concentrations.

Rapid Report: A novel signalling pathway originating in mitochondria modulates rat skeletal muscle membrane excitability

Abstract: Single skeletal muscle fibres from rat and cane toad were mechanically skinned and stimulated either electrically by initiating action potentials in the sealed transverse (t-) tubular system or by ion substitution causing depolarisation of the t-system to pre-determined levels. Depression of mitochondrial ATP-producing function with three diverse mitochondrial function antagonists (azide: 1–10 mm; oligomycin 1 μg ml−1 and carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP) 1 μm), under conditions in which the cytosolic ATP was maintained high and constant, invariably reduced the excitability of rat fibres but had no obvious effect on the excitability of toad fibres, where mitochondria are less abundant and differently located. The reduction in excitability linked to mitochondria in rat fibres appears to be caused by depolarisation of the sealed t-system membrane. These observations suggest that mitochondria can regulate the functional state of mammalian muscle cells and have important implications for understanding how the balance between ATP utilisation and ATP production is regulated at the cellular level in general and in mammalian skeletal muscle fibres in particular.

Characterization of urea transport in Bufo arenarum oocytes.

Abstract: Xenopus laevis oocytes have been extensively used for expression cloning, structure/function relationships, and regulation analysis of transporter proteins. Urea transporters have been expressed in Xenopus oocytes and their properties have been described. In order to establish an alternative system in which urea transporters could be efficiently expressed and studied, we determined the urea transport properties of ovarian oocytes from Bufo arenarum, a toad species common in Argentina. Bufo oocytes presented a high urea permeability of 22.3 × 10−6 cm/s, which was significantly inhibited by the incubation with phloretin. The urea uptake in these oocytes was also inhibited by mercurial reagents, and high-affinity urea analogues. The urea uptake was not sodium dependent. The activation energy was 3.2 Kcal/mol, suggesting that urea movement across membrane oocytes may be through a facilitated urea transporter. In contrast, Bufo oocytes showed a low permeability for mannitol and glycerol. From these results, we propose that one or several specific urea transporters are present in ovarian oocytes from Bufo arenarum. Therefore, these oocytes cannot be used in expression studies of foreign urea transporters. The importance of Bufo urea transporter is not known but could be implicated in osmotic regulation during the laying of eggs in water. J. Exp. Zool. 298A:10–15, 2003. © 2003 Wiley-Liss, Inc.

Natural toad skin for curing AIDS

Abstract: The present invention relates to a kind of AIDS treating Chinese medicine. Toad skin from live toad is produced into toad skin decoction through washnig, drying, crushing, and decoction; and the decoction is then compounded with cell culture liquid into proper concentration. Experiment shows that the toad skin decoction in concentration range of 0.625-1.25 mg/ml and obvious HIV-1 inhibiting effect.