Bufo Vulgaris Research Articles

Quantitative structure–toxicity relationship study of lethal concentration to tadpole (Bufo vulgaris formosus) for organophosphorous pesticides

In the present study more than 1000 structural parameters of 41 organophosphorus pesticides (OPs) were calculated using the software ChemOffice 8.03 and Dragon 2.1. Then, with multivariate linear regression and best subset regression analyses, different equations were derived to calculate the lethal toxicity, LC50, for these 41 organophosphorous pesticides found in tadpoles (Bufo vulgaris formosus). An equation was developed for all selected OPs, especially those with relatively low toxicity levels (LC50>4.5mM) that accounted for 89.09% of the variability in the toxic effect. The equation indicated that the main contributions to OPs toxicity with tadpoles were the electrostatic contribution qH+ (maximum net positive H atomic charge), spatial autocorrelation (MATS7m) and hydrophobicity (lgKow), with the two former being the most important parameters. For OPs with high toxicity, however, different structural parameters were introduced. The following equation was developed with LC50<4.5mM. These equations implied that with different levels of toxicity there could have different mechanisms in the tadpole. Furthermore, the results showed that molecular structural parameters had a particular value in modeling chemical reactivity within a homologous series of compounds.

Effects of pH, aluminium, and soft water on larvae of the amphibians Bufo bufo and Triturus vulgaris

Larval Bufo bufo (L., 1758) and Triturus vulgaris (L., 1758) were exposed to soft water (0.5 mg·L–1 Ca2+) experimentally acidified to pH 3.9 to 5.9 and total aluminium concentrations of &lt;10, 150, and 300 µg·L–1. Below pH 4.5 both species experienced increased mortality. The LC50 (168 h) for &lt;10 and 150 µg·L–1 Al was pH 4.3 and 4.1 for B. bufo and 4.2 and 4.1 for T. vulgaris. However, Al3+ increased the survival of both species, which may be due to the contribution of Al3+ to the ionic strength. No B. bufo larvae died at pH &gt;4.5, whereas T. vulgaris at higher Al concentrations suffered relatively high mortality at pH 5.1–5.9, where Al occurs mainly as Al(OH)2+ and Al(OH)2+. Unlike external gills (T. vulgaris), internal gills (B. bufo) have their own internal environment and are probably better protected against the presence of these toxic Al species in the water. These Al species thus seem to be toxic to T. vulgaris larvae but not to B. bufo. Chloride was seen to be important for survival in water of low ionic strength, since the survival of T. vulgaris larvae, particularly at low Al concentration, increased at pH levels down to pH 4.3 when the water was acidified with HCl.

Bile salts of the toad, Bufo marinus: characterization of a new unsaturated higher bile acid, 3 alpha,7 alpha,12 alpha,26-tetrahydroxy-5 beta-cholest-23-en-27-oic acid.

The bile salts present in gallbladder bile of the toad, Bufo marinus, were found to consist of a mixture of bile alcohol sulfates and unconjugated bile acids. The major bile alcohol was 5 beta-bufol; 5 alpha- and 5 beta-cholestane-3 alpha,7 alpha,12 alpha, 26-tetrols occurred as the minor bile alcohols. Bile acids of Bufo marinus were cholic acid, allocholic acid, 3 alpha,7 alpha,12 alpha-trihydroxy-5 alpha- and 5 beta-cholestan-26-oic acids, 3 alpha,7 alpha,12 alpha-trihydroxy-5 alpha- and 5 beta-cholest-23-en-26-oic acids, 3 alpha,7 alpha,12 alpha, 26-tetrahydroxy-5 beta-cholestan-27-oic acid, and a C27 bile acid which has not been previously described. By chromatographic behavior, mass spectral data, and identification of the products of catalytic hydrogenation and ozonolysis, the structure of the new higher bile acid was elucidated as 3 alpha,7 alpha,12 alpha,26-tetrahydroxy-5 beta-cholest-23-en-27-oic acid. The bile salt pattern of Bufo marinus closely resembles that of Bufo vulgaris formosus, except for the absence of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-22-ene-24-carboxylic acid, the major bile acid of the latter toad.

Changes in intercellular contacts, motility, and adhesiveness of the presumptive mesodermal cells of Bufo vulgaris embryos before and during gastrulation

AbstractThe morphology and adhesiveness of the presumptive mesodermal cells (PMCs) of Bufo vulgaris embryos were examined before and during gastrulation. Before the late‐blastula stage, the PMCs were in close contact with one another and were polyhedral in shape. Intercellular contacts became loose and cells were round in shape at the late‐blastula stage. At the initial stage of gastrulation, the PMCs began to form hyaline blebs. The blebbing cells appeared to crowd in one region (the bleb‐forming region [BFR]), located in the deep marginal mesoderm: The blebbing cells decreased in number and changed into filopodia‐ and lamellipodia‐forming cells with the start of the migration of PMCs at the early‐gastrula stage.Cells in the BFR and in the presumptive BFR were isolated, and adhesiveness of cells to fibronectin was examined. The results showed that the BFR cells begin to adhere to fibronectin at the early‐gastrula stage when the PMCs start to migrate along the inner surface of the blastocoelic wall.A series of changes (i.e., loosening of intercellular contacts, the formation of hyaline blebs, and the development of the ability to adhere to fibronectin) is thus evident in the PMCs before and during gastrulation. These serial changes are discussed within the context of the way in which PMCs start to migrate at the early‐gastrula stage.

Chemo- and baroreceptor innervation of the aortic trunk of the toad Bufo vulgaris

In the toad Bufo vulgaris, afferent impulses were recorded from the fine branch of the vagus which terminates in the aortic trunk. Spontaneous single unit discharge was random. When the aorta was artificially perfused, units fired in response to flow change, hypoxia and hypoxia-hypercapnia of the perfusion fluid, and administration of NaCN or Ach. A few baroreceptor fibers fired synchronously with the blood pressure fluctuation. In anesthetized toads with other reflexogenic areas denervated, the intravenous administration of NaCN caused an increase in breathing and a slight elevation of blood pressure. When the nerve concerned was cut, these responses were abolished. Electrical stimulation of the nerve's central cut end brought about the same type of response as that to NaCN. Special cells closely resembling the glomus cells in the carotid labyrinth were found in the aortic wall. In amphibia, there is an aortic nerve corresponding to that of mammals, and the aorta is one of receptive areas predominantly contributing to respiratory control.

Contribution of intracellular stored calcium to contractile activation in contractures of stomach circular muscle of Bufo vulgaris formosus.

Contractile responses of stomach circular muscle of Bufo to high-K, to acetylcholine (ACh) in normal Ringer or in high-K solution, and to calcium in Ca-free high-K solution showed a phasic contraction which relaxed completely in 30-45 sec. K-induced contracture was abolished in Ca-free solution containing 1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) within 20 sec, while ACh-induced contracture was not abolished and 10-25% of control tension was kept up to 40 sec. This response increased to 40-50% when all extracellular Na was replaced with tris(hydroxymethyl)-aminomethane (Tris). K-induced contracture was inhibited completely by 1 mM La. ACh-induced contracture in the muscle depolarized by high-K solution was dependent on the depolarization time, 0-10, 60-70, and nearly 100% of control after 1, 3, and 10 min depolarization, respectively. These ACh-induced contractures were not inhibited by 1 mM EGTA or La. All contractures mentioned above were markedly inhibited by 5 mM procaine. These results suggest that activation of both contractures induced by high-K and ACh were, at least partly, dependent on the Ca at the intracellular Ca storage sites. Ca-induced contracture was dependent on depolarization time as was ACh-induced contracture, when the muscle was depolarized by Ca-free high-K solution without pre-treatment with Ca-free Ringer solution. These results suggest that activation of Ca-contracture is also dependent on intracellular stored Ca.

The minor bile acids of the toad, Bufo vulgaris formosus.

Bile from the toad, Bufo vulgaris formosus, was found to contain a number of minor bile acids along with two major bile acids, 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholest-22-ene-24-carboxylic acid and 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholest-23-en-26-oic acid. The following minor bile acids were identified by combined gas-liquid chromatography-mass spectrometry: cholic acid, allocholic acid, 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestan-26-oic acid, 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 alpha-cholestan-26-oic acid, 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 alpha-cholest-22-ene-24-carboxylic acid, 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 alpha-cholest-23-en-26-oic acid, varanic acid, and 3 alpha, 7 alpha, 12 alpha, 24-tetrahydroxy-24-methyl-5 beta-cholestan-26-oic acid. The fact that the toad bile contains not only cholic acid but also 3 alpha, 7 alpha, 12 alpha-trihydroxy- and 3 alpha, 7 alpha, 12 alpha,24-tetrahydroxy-5 beta-cholestan-26-oic acids, which have been recognized as biosynthetic intermediates of cholic acid in mammals, suggests that the toad is capable of synthesizing cholic acid by the same pathway as that for the biosynthesis of the C24 bile acid in mammals.

The glomus cell of the carotid labyrinth of Xenopus laevis.

The ultrastructure of the glomus cells of the carotid labyrinth was investigated in the anuran, Xenopus laevis. These cells show many catecholamine containing granules. About 50 cells in groups of 3-5 are located near the sinusoids. Morphologically, their organelles are similar to those previously reported in Bufo vulgaris. Striking features are (1) intimate appostion of the glomus cell to smooth muscle (g-s connection), (2) gap junctions between adjacent glomus cells, (3) exocytotic figures. Based on these findings a possible function of the glomus cell is discussed.

A reflexogenic area for controlling the blood pressure in toad (Bufo vulgaris formosa).

In the toad, Bufo vulgaris, the pulmocutaneous artery, which is innervated by a fine branch of the laryngeal nerve, was shown to be a reflexogenic area affecting the systemic blood pressure. Distension of the vascularly isolated pulmocutaneous artery evoked falls in systemic blood pressure. Impulses in bursts, synchronous with the systolic rise of the blood pressure, were recorded from the peripheral cut end of the laryngeal nerve when all branches, save the one innervating the pulmocutaneous artery, had been divided. Differing from mammalian baroreceptors, those in toads occurred with a marked latency in response to rises in blood pressure. A hyperbolic relation was obtained between the pressure and the latency. Existence of these baroreceptors in pulmocutaneous artery was confirmed in preparations in vitro. Punctate stimulation was used to localize the baroreceptive site.

Studies on cardiotonic steroids from the skin of Japanese toad.

The isolation and characterization of the cardiotonic steroid constituents in the skin of the Japanese toad, Bufo vulgaris formosus BOULENGER, has been carried out. Three new types of bufotoxins in which the succinoyl, adipoyl, and pimeloyl groups are displaced for the suberoyl residue of the so-calledbufotoxin, separated and identified. In addition, the occurrence of bufogenin 3-sulfates and analogous conjugates of cardenolide named cardenobufotoxin was also demonstrated. The ethanolic extract of the skin obtained from 1800 toads was separated by chromatography on Amberlite XAD-2 resin, silica gel, and Sephadex LH-20, followed by highperformance liquid chromatography to provide new cardiotonic steroid conjugates. The structures of these substances were elucidated to be resibufogenin 3-succinoylarginine ester (Id), gamabufotalitoxin homologs (IIg, IIk, IIn, IIp), bufalitoxin homologs (IIIc, IIIe, IIIg, IIIi), cinobufotoxin homologs (IVc, IVe, IVg, IVl), arenobufotoxin (Vd), cinobufotalitoxin (VIb), cardenobufotoxins (VIIId, VIIIf), bufogenin 3-sulfates (IIq, IIIj, Ve, VIId), and sarmentogenin 3-sulfate (VIIIg) by degradative and synthetic means.

Glycopeptides from the egg jelly coat of the toad Bufo vulgaris

1. 1. Glycopeptides isolated by exhaustive pronase digestion from the egg jelly coat of toad, Bufo vulgaris, were separated by DEAE-ccellulose chromatography into two fractions. These fractions each gave a single band when they were subjected to electrophoresis on cellulose acetate strips. The glycopeptides behaved as a kind of “macroglycopeptide” on gel chromatography. 2. 2. The glycopeptide fractions contained as sugar components galactosamine, glucosamine, galactose, fucose and N-acetylneuaminic acid. The amino acid composition was characterized by the presence of threonine and serine, which accounted for approximately 70% of the total amino acids. 3. 3. Large amounts of galactosamine, threonine and serine were destroyed by treatment with alkali, indicating that the carbohydrate—peptide linkage in the glycopeptides is mostly an O-glycosidic bond between N-acetylgalactosamine and the hydroxy groups of threonine and serine.

EFFECTS OF MEDIA OF DIFFERENT IONIC COMPOSITION ON THE ACTIVATION POTENTIAL OF ANURAN EGG CELLS.

Activation potentials of the eggs of two anuran species, Bufo vulgaris formosus and Rana rugosa, were measured in media of different ionic composition, and the mechanism of their generation is discussed. The same trends of ionic effects upon the activation potential were consistently obtained in both species. The membrane potential of the unactivated eggs was negative with respect to tap water, Ringer's solution, and the media described below except isotonic KCl and KNO3 , in which the potentials were nearly zero or sometimes slightly positive. Upon activation induced by pricking in tap water, isotonic Na2 SO4 , or 42 mM or 63 mM buffered sodium phosphate solution, depolarization followed by reversal of the membrane potential took place, associated with a decrease in the effective resistance. Then the potential gradually decreased and returned to the initial value. On the other hand, in Ringer's solution, or in isotonic NaBr, NaNO3 , KCl or KNO3 , the activation potential occurred in a hyperpolarizing direction, associated with a decrease in the effective resistance. These results suggest that the ooplasmic membrane of the anuran egg surface during activation is selectively permeable to NO3 - and Br- as well as Cl- , but not to SO4 - and phosphate ions, or alternatively, that the activation potential is due to this selective permeability and to the concentration gradients of these monovalent anions across the ooplasmic membrane.

The Nutritional Efficiency of Cannibalism and an Artificial Feed for the Growth of Tadpoles of Japanese Toad

Tadpoles of Japanese toad (Bufo vulgaris) were cultured by an artificial feed and by forced cannibalism from the egg until metamorphosis finished. The best growth was found in the artificial feed group, however, the highest feed efficiency was observed in the cannibalism group. The tadpoles were metamorphosed by the culture with amino acid mixture feed but no body weight increase was seen in this case. The nutritional disturbance hindered the metamorphosis. The relative amino acid composition was constant throughout all the stages of the tadpole growth. The relative amino acid composition of the toad was diversed according to the species. Several unidentified ninhydrine-positive substances were detected on the column chromatograms of amino acid analysis of tadpoles and their adult forms.

Relationships between disaccharide hydrolysis and sugar transport in amphibian small intestine.

1. A study is described of the relationships which exist between disaccharide hydrolysis and glucose transport in the small intestine of Rana pipiens and Bufo vulgaris. The experiments were undertaken on the intestine perfused in vitro through the vascular system and with fluid circulating through the intestinal lumen. For this system it was found that, with [U-(14)C]glucose in the intestinal lumen, the apparent specific activity of the glucose appearing in the vascular effluent was not significantly different from that in the lumen.2. Changes of ionic composition of vascular and luminal fluids, and the presence of phloridzin or strophanthin, had little effect upon the maltase activity in situ in R. pipiens, although this activity was somewhat reduced when the sodium of the intestinal lumen was replaced by lithium. In contrast, in all cases a marked reduction was found in the rate of glucose translocation in the vascular effluent.3. With Tris substituted for the luminal sodium, there was evidence of a competitive inhibition of the maltase activity in situ by the buffer cation. At the same time, the rate of glucose translocation into the vascular effluent was but little affected and there was an apparent increase in the efficiency with which the cellular systems responsible for the translocation were able to capture the glucose liberated.4. It was found that competition for transepithelial translocation occurred between the glucose initially present in the intestinal lumen, and glucose derived from either maltose or trehalose. There was no evidence for competition for hydrolysis between maltose and trehalose, yet the glucose units derived from these two disaccharides competed with each other for translocation.5. The significance is discussed of the finding that it is possible to dissociate the processes of disaccharide hydrolysis from those underlying the translocation of hexose units into the vascular effluent. It is suggested that monosaccharide units released by the hydrolysis of disaccharide molecules have access to a pool of glucose which is equally accessible to glucose free in the intestinal lumen. It is also suggested that the rate of transport of glucose from the pool into the vascular effluent (i.e. glucose translocation) is determined by the concentration of glucose within the pool. From consideration of the properties of a model operating upon these principles, it is possible to predict the relative contribution of disaccharides and monosaccharides in the intestinal lumen to the glucose appearing in the vascular effluent. The experimentally determined contributions of the two sources are very similar to those predicted from the model. The implications of possible sites for the postulated pool are discussed.

Effect of urea derivatives and analgetics on the permeability to water and electrical properties of the urinary bladder of the toad.

Four urea derivatives and six analgetics examined here, when added to the serosal medium bathing the urinary bladder of the toad, Bufo vulgaris japo-nicus, increased the permeability to water despite the presence or the absence of vasopressin and this effect was completely inhibited by 10-55M epinephrine. Oxyphenbutazone on the serosal side inhibited the, response to cyclic 3', 5'-AMP, while in contrast phenylmethylurea, acetaminophen and aminopyrine slightly enhanced this response. Potential difference, short-circuit current and conductance across the bladder were depressed by these compounds. In brief the mode of action of these compounds seems to be similar to that of chlorpropamide, but theophylline-like action could not be excluded. A common chemical structure was also discussed.

Histochemical demonstration of catecholamines in the toad sympathetic ganglia.

With the aid of histochemical procedures developed by Falck and Hillarp, the lumber sympathetic ganglia and the toxic gland of the lower limb of the toad (Bufo vulgaris japonica) were examined.1. Both SB and SC systems were considered to be adrenergic or catecholamine containing ones.2. The toxic gland of the lower limb was supposed to be innervated by adrenergic nerve fibres originated from sB neurons in the sympathetic ganglia.3. These nerve fibres which were supposed to innervate the toxic gland were assumed to innervate the smooth muscle-like cells surrounding the secretory cells of the toxic gland.4. The function of chromaffin cells in the sympathetic ganglia was briefly discussed.

Effect of sulfonylureas on the permeability to water and electrical properties of the urinary bladder of the toad.

Chlorpropamide induced an increase in the permeability to water of the urinary bladder of Bufo vulgaris japonicus only when added to the serosal medium even in the absence of vasopressin and this effect was inhibited by epinephrine, glutathione and 10-22M calcium. Sulfonylureas (chlorpropamide, tolbutamide, carbutamide and acetohexamide) potentiated the vasopressin induced effect on the osmotic flow of water. In contrast chlorpropamide added to the serosal medium obviously inhibited the effect of exogenous cyclic 3', 5'-AMP on the permeability to water in the presence of epinephrine and also markedly depressed the D.C. conductance of the bladder. One possible explanation for the mode of action of chlorpropamide was presented.

Hindlimb development and malformations of toes in the larvae reared at high temperature in the toad, Bufo vulgaris formosus

Hindlimb development and malformations of toes in the larvae reared at high temperature in the toad, Bufo vulgaris formosus

Disaccharide absorption by amphibian small intestine in vitro.

1. An account is given of the absorption of disaccharides by the small intestine of Rana temporaria, R. pipiens and Bufo vulgaris perfused in vitro through the vascular system. Maltase and trehalase activity are found in the intestine of all three species; very small amounts of sucrase are present in the intestine of R. pipiens but there is no evidence for the presence of lactase in any of the animals studied.2. During maltose absorption free glucose appears in the vascular effluent and in the intestinal lumen. Only very small quantities of disaccharide are found in the vascular effluent. The concentration of free glucose in the intestinal lumen during maltose absorption is not high enough to account for the rates of glucose transport observed. The rate of appearance of glucose in the vascular effluent is determined by the concentration of disaccharide in the luminal fluid, and hexose, free in solution in the lumen, is not an obligatory intermediate in the process of disaccharide absorption.3. For R. pipiens more than 90% of the maltase activity in the system is present in the intestinal wall and the rate of maltose hydrolysis by maltase, free in the intestinal lumen, is found to be inadequate to account for the rates of appearance of glucose observed to occur in the lumen and in the vascular effluent. It is not possible to wash away maltase activity from the intestinal wall.4. The kinetic properties of maltase and trehalase acting in situ are of the Michaelis-Menten type; the apparent K(m) is 2 mM for maltase, and 3 mM for trehalase.5. The relationship which exists between the rate of absorption of glucose and the concentration in the luminal fluid of either disaccharide or free glucose is of the Michaelis-Menten type. Expressed in molar units, the apparent K(m) for the glucose transport is about one fifth that of the disaccharidase. The maximum rate of glucose transport observed is less than the maximum rate of disaccharide hydrolysis. In R. pipiens equimolar concentrations in the intestinal lumen of the monomer free glucose, or of the dimer, maltose, yield approximately equal rates of transport of the free hexose.6. It is concluded that in the amphibian, either intestine disaccharide hydrolysis and glucose transport are functions of separate subcellular systems which spatially are very closely related, or that the hydrolysis and transport are different facets of the activity of a common system.

Immunological investigations of embryonic haemoglobin of Bufo vulgaris.

An antigenic difference could be established between embryonic haemoglobin, fetal haemoglobin and adult haemoglobin of Bufo vulgaris. The distribution of embryonic haemoglobin in the surface layer of the yolk platelets was demonstrated.