Purification, characterization, and spasmogenic activity of neurotensin from the toad Bufo marinus

Neurotensin (NT) was isolated from an extract of the intestine of the cane toad, Bufo marinus and its primary structure established as: pGlu-Ala-Ile-Val-Ser-Lys-Ala-Arg-Arg-Pro-Tyr-Ile-Leu. This amino acid sequence shows five substitutions (Leu2 --> Ala, Tyr3 --> Ile, Glu4 --> Val, Asn5 --> Ser, and Pro7 --> Ala) compared with bovine NT. Synthetic Bufo NT (pD2 = 8.05 +/- 0.28) was equipotent and equally effective as bovine NT (pD2 = 8.24 +/- 0.38) in producing spasmogenic contraction of isolated segments of toad small intestine. However, the maximum response produced by Bufo NT was only 35 +/- 2% of that produced by substance P. The potencies, but not the maximum responses, to Bufo and bovine NT were significantly (p < 0.05) attenuated by pre-treatment with atropine but neither parameter was significantly diminished by tetrodotoxin and indomethacin. The data suggest that the action of NT involves interaction with receptors on toad intestinal smooth muscle that recognize the C-terminal region of NT (residues 8-13) that has been fully conserved during evolution of tetrapods. Contractile activity is mediated, at least in part, by release of acetylcholine.     

Digitalis-like and vasoconstrictor effects of endogenous digoxin-like factor(s) from the venom of Bufo marinus toad

Digitalis glycoside-like properties of the Bufo marinus toad crude venom and one of its constituents, bufalin, were studied in various assay systems. In concentrations 0.3-30 micrograms/ml crude venom increased the contractility of isolated electrically driven rat atria, constricted rat aortic rings, inhibited ouabain-sensitive Na+,K(+)-ATPase in rat erythrocytes and the Na+,K(+)-pump in rat aorta, and cross-reacted with antidigoxin antibody from the dissociation enhanced lanthanide fluoroimmunoassay (DELFIA). These effects were unaffected by adrenoceptor blockers and the 5-HT antagonist, deseril, but were blocked by antidigoxin antibody. Bufalin (10-30 microM) increased myocardial contractility and inhibited Na+,K(+)-ATPase in rat erythrocytes similarly to crude Bufo marinus venom. In rat aorta bufalin showed weak and delayed vasoconstrictor activity which was antagonized by 2 microM phentolamine, and had a biphasic effect on the Na+,K(+)-pump; 0.5-1.0 microM bufalin stimulated the pump, while higher concentrations inhibited its activity. Although the effects of bufalin were blocked by antidigoxin antibody, bufalin showed very low digoxin-like immunoreactivity in the DELFIA. These observations suggest that, in addition to bufalin, Bufo marinus venom contains at least one more digitalis-like steroid with significant intrinsic vasoconstrictor activity which, unlike bufalin, constricts the blood vessels acting directly via inhibition of the sodium pump in the vascular smooth muscle membrane.     

Metabolism and cardiovascular effects of leukotrienes in the marine toad Bufo marinus

Interspersed repeated DNA sequences are characteristic features of both prokaryotic and eukaryotic genomes. REP sequences are defined as conserved repetitive extragenic palindromic sequences and are found in Escherichia coli, Salmonella typhimurium and other closely related enteric bacteria. These REP sequences may participate in the folding of the bacterial chromosome. In this work we describe a unique class of 28 conserved complex REP clusters, about 100bp long, in which two inverted REPs are separated by a singular integration host factor (IHF) recognition sequence. We term these sequences RIP (for repetitive IHF-binding palindromic) elements and demonstrate that IHF binds to them specifically. It is estimated that there are about 70 RIP elements in E. coli. Our analysis shows that the RIP elements are evenly distributed around the bacterial chromosome. The possible function of the RIP element is discussed.     

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.     

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.     

Morphology and distribution of Müller cells in the retina of the toad Bufo marinus

We have previously shown that an antibody against neuron-specific enolase (NSE) selectively labels Müller cells (MCs) in the anuran retina (Wilhelm et al. 1992). In the present study the light- and electron-microscopic morphology of MCs and their distribution were described in the retina of the toad, Bufo marinus, using the above antibody. The somata of MCs were located in the proximal part of the inner nuclear layer and were interconnected with each other by their processes. The MCs were uniformly distributed across the retina with an average density of 1500 cells/mm2. Processes of MCs encircled the somata of photoreceptor cells isolating them from each other by glial sheath, except for those of the double cones. Some of the photoreceptor pedicles remained free of glial sheath. Electron-microscopic observations confirmed that MC processes provide an extensive scaffolding across the neural retina. At the outer border of the ganglion cell layer these processes formed a non-continuous sheath. The MC processes traversed through the ganglion cell layer and spread beneath it between the neuronal somata and the underlying optic axons. These processes formed a continuous inner limiting membrane separating the optic fibre layer from the vitreous tissue. Neither astrocytic nor oligodendrocytic elements were found in the optic fibre layer. The significance of the uniform MC distribution and the functional implications of the observed pattern of MC scaffolding are discussed.     

Müller cells in the retina of the cane toad, Bufo marinus, express neuropeptide Y-like immunoreactivity

We have used light- and electron-microscopic immunohistochemistry to identify the presence of immunoreactivity to neuropeptide Y (NPY) within Müller cells in the retina of the cane toad, Bufo marinus. Müller cells containing NPY-like immunoreactivity (NPY-LI) were identified at the light-microscopic level by the coexistence with immunoreactivity to glial fibrillary acidic protein (GFAP) and at the ultrastructural level by their characteristic relationship to neuron cell bodies and processes. At the light-microscopic level, those cells which contained both NPY-LI and GFAP-LI usually had small cell bodies in the inner nuclear layer, while those cells which contained only NPY-LI were identified as large and small amacrine cells. The radially oriented primary processes in the inner plexiform layer and the vitreal end feet of GFAP-LI Müller cells also expressed NPY-LI. At the ultrastructural level, thin lamellar processes of Müller cells with NPY-LI enclosed some amacrine cell bodies in the inner nuclear layer and amacrine cell dendrites in the inner plexiform layer. These observations suggest that NPY-LI is localized in Müller cells in addition to two types of amacrine cells previously identified in the Bufo retina. This study provides the first evidence that glial elements in the vertebrate retina express NPY-LI.     

The role of pulmocutaneous baroreceptors in the control of lymphatic heart rate in the toad Bufo marinus

We examined the effect of NO on collagen-induced whole blood aggregation and platelet activation in whole blood by using impedance aggregometry and flow cytometry. For the extracellular generation of NO, we chose sodium nitroprusside dihydrate (SNP), and as intracellular generators of NO, L-arginine and isosorbide dinitrate (ISDN). The latter two significantly inhibited whole blood aggregation, whereas SNP had no such effect. The inhibitory effect of ISDN was diminished by addition of methylene blue (MB) or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl 3-oxide (carboxyl-PTIO), and the inhibitory effect of L-arginine was diminished by addition of N(G)-monomethyl-L-arginine monoacetate (L-NMMA). Although the addition of ISDN increased the cyclic guanosine monophosphate (cGMP) level in whole blood and in the suspension of platelets and white blood cells (PLTs + WBCs), no increase was found in platelet-rich plasma (PRP). The P-selectin expression on the platelet surface in whole blood was reduced by ISDN and L-arginine. These findings suggest that the intracellular generation of NO inhibits whole blood aggregation, and this mechanism may play an important role in its antithrombotic effect in whole blood.     

Purification of endogenous digitalis-like factors from normal human urine

We detected two candidates for endogenous digitalis-like factors in human urine based on the inhibition of 3H-ouabain binding to human erythrocytes. Two ouabain-displacing compound(ODC)s were consistently eluted off the C18 reverse phase HPLC column with 18% and 31% acetonitrile. The more-polar ODC-1 was ubiquitously found in mammals, markedly increased after acute and chronic salt loading in humans, and was thought to be a natriuretic factor with vasoactive property. ODC-1 mostly resembled ouabain in biological, physicochemical, and chromatographic properties and may correspond to ouabainlike compound purified by other investigators. The less-polar ODC-2 was indistinguishable from digoxin in proton nuclear magnetic resonance(NMR) and fast atom bombardment(FAB) mass spectrum.     

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

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 approximately neurokinin A (NKA) > or = SP(5-11) > or = neuropeptide gamma > or = scyliorhinin II > scyliorhinin I > or = [Sar9]-SP > or = neurokinin B approximately physalaemin approximately carassin > SP(7-11) approximately eledoisin > or = SP(4-11) approximately SP(6-11). Binding was also inhibited by Gpp[NH]p > or = GTPgammaS > 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 approximately SP > or = physalaemin > or = ranakinin > SP(6-11) > scyliorhinin II > or = neuropeptide gamma > neurokinin B approximately NKA approximately scyliorhinin I > or = SP(4-11) > or = SP(5-11) > [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,Me-Leu9,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 microM). Highly selective NK-1 receptor antagonists including CP 99994 and GR 82334 (both 1 microM) were ineffective in both functional and binding studies. Tetrodotoxin (1 microM) 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.     

Ammonia transport by the urinary bladder of Bufo marinus

Among cyanobacteria, the heterocystous, N2-fixing Anabaena variabilis and the unicellular Anacystis nidulans have recently been shown to possess an NAD+-dependent, bidirectional hydrogenase. A 5.0 kb DNA segment of the A. nidulans genome is now identified to harbor the structural genes hoxUYH coding for three subunits of the bidirectional hydrogenase. The gene arrangement in A. nidulans and in A. variabilis is remarkably dissimilar. In A. nidulans, but not in A. variabilis, the four accessory genes hoxW, hypA, hypB and hypF could be identified downstream of hoxH. An insertional homozygous mutant in hoxH from A. nidulans was completely inactive in performing Na2S204-dependent H2 evolution but could utilize the gas with almost 50% of the activity of the wild type. These findings with the first defined hydrogenase mutant in any photosynthetic, 02-evolving microorganism indicate that the unicellular cyanobacterium A. nidulans possesses both an uptake and a bidirectional hydrogenase. The physiological role(s) of the two hydrogenases in unicellular non-N2-fixing cyanobacteria is not yet understood.     

Acid-base status in the toad Bufo viridis in vivo

The acid-base status in the toad Bufo viridis was assessed on blood samples taken through a chronical cannula from unrestrained animals. Blood gases and the pH were very constant in animals which were kept either under control conditions (free access to tap water) or immersed in shallow water. pH was 7.646 +/- 0.032 (mean +/- SEM) in 14 control toads at 26 degrees C. PCO2 was 13.1 +/- 1.2 mm Hg and [HCO3-] was 17.0 +/- 0.7 mmol/l at the same temperature. pH and PCO2 were independent of the hematocrit and the haemoglobin in the blood. Diamox induced a characteristic metabolic acidosis which is apprently the result of the inhibition of carbonic anhydrase both in the kidney and in the skin. The results are discussed in relation to the role of the kidney and the skin in regulating the acid-base balance in the toad.     

Phylogeography of Bufo marinus from its natural and introduced ranges

The marine toad, Bufo marinus, has a broad natural distribution extending from the south-west of the USA to southern Peru and the central Amazon. It was introduced to several localities in the Caribbean and Pacific Oceans to control sugar cane pests. We sequenced 468 bp of mitochondrial DNA (mtDNA) containing the ND3 gene, and flanking tRNA genes from toads spanning the broad natural and introduced ranges. Consistent with the known history of introductions and expected effects of serial bottlenecks, mtDNA within introduced populations in Hawaii and Australia was uniform and most closely related to samples from eastern Venezuela and French Guiana. However, mtDNA nucleotide diversity in the geographic region spanning the source areas is also relative low (0.18-0.46%) and the absence of variation in the introduced populations precludes quantitative assessment of the reduction in genetic diversity. Unexpectedly, there was a large phylogeographic break (5.4% sequence divergence) within the natural range separating populations east and west of the Venezuelan Andes. We hypothesize that the two major lineages of B. marinus were isolated by the uplift of the eastern Andean cordillera which was completed approximately 2.7 Ma. Another species of the marinus group, B. paracnemis, had mtDNA paraphyletic, with marinus, being nested within the eastern lineage. Thus, at least one speciation event within the marinus group postdates the split within marinus. These findings suggest that the taxonomy of B. marinus should be re-evaluated and that the search for pathogens to control Australian populations should be conducted in populations from both lineages in the natural range.     

Isolation, characterization and synthesis of a novel paradaxin isoform

Recently our laboratory found that tolerance to morphine-induced antinociception could be completely reversed with intracerebroventricular (i.c.v.) administration of a protein kinase A inhibitor, whereas intrathecal (i.t.) administration of the inhibitor produced hyperalgesia in morphine-tolerant mice. In the experiments described here, we sought to characterize further the role of phosphorylation events in supraspinal versus spinal opioid-mediated pain pathways and how such events might be involved in the development of antinociceptive tolerance. Two phosphatase inhibitors were administered centrally to determine whether they affected morphine-induced antinociception in naive or chronically morphine-treated mice. By the i.c.v. route, okadaic acid enhanced morphine-induced antinociception in tolerant mice and produced toxicity by the i.t. route. The calcineurin inhibitor ascomycin had no effect on antinociception following acute or chronic morphine treatment. These results suggest that increased activity of protein phosphatase types 1 and/or 2A in the brain may contribute to the development of morphine tolerance.     

The effects of melanocortin peptides and corticosterone on habituation in the great plains toad, Bufo cognatus

Four patients with long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency are presented. Clinical onset in the form of acute encephalopathy occurred between the ages of 9 months and 3 years. The clinical course included recurrent metabolic crises in 4 patients, cardiac involvement and retinopathy in 3, and myopathy in 2. None had signs of peripheral neuropathy. Three patients died and one is currently well. Hypoketotic hypoglycemia with C6-C14 3-hydroxy-dicarboxylic aciduria during metabolic crises associated with decreased plasma carnitine levels was the main biochemical finding. Enzymologic studies disclosed long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency in all patients. Homozygosity for a G to C mutation at position 1528 in the encoding region of the enzyme was found in 2 patients. Histologic and electron microscopic studies of liver biopsy specimens revealed steatosis in 3 patients and mitochondrial abnormalities in 2. Skeletal muscle biopsies disclosed nonspecific degenerative changes in 2 patients and were normal in the remaining 2. Ultrastructural abnormalities in mitochondria were found in 3 patients. A review of the literature combined with the data from our series (total 22 patients) disclosed acute clinical onset in 77% of cases and subacute in 23%. In the combined series, the average age at onset was 11 months, family history was positive in 32% of patients and overall mortality was 50%. We describe the clinical spectrum of this disease and emphasize that, among patients with suspected beta-oxidation defects the finding of pigmentary retinopathy should lead to the suspicion of long-chain 3-hydroxyacyl-coenzyme A-dehydrogenase deficiency.     

Chinese hamster ovary cells expressing a novel type of acetylated low density lipoprotein receptor. Isolation and characterization

Macrophage scavenger receptors mediate the recognition of a wide range of negatively charged macromolecules including acetylated low density lipoproteins (AcLDL). Chinese hamster ovary (CHO) cells were cultured in the presence of increasing concentrations of simvastatin, a cholesterol biosynthesis inhibitor, and AcLDL as the sole source of exogenous lipoproteins. The cells surviving under these conditions specifically bound 125I-labeled AcLDL with high affinity and degraded them via an endocytic pathway. Unexpectedly, the association and degradation of 125I-labeled AcLDL by these CHO cells were not inhibited by dextran sulfate, fucoidan, and polyinosinic acid, competitors of macrophage scavenger receptors, but were completely inhibited by maleylated bovine serum albumin. Furthermore, these cells effectively took up negatively charged liposomes containing acidic phospholipids such as phosphatidylserine and phosphatidic acid, whereas CHO cells expressing macrophage scavenger receptors did not. AcLDL and negatively charged liposomes were cross-competed with each other. Northern blot analysis using the cDNA for the macrophage scavenger receptor revealed that these CHO cells did not express this receptor. From these observations, we conclude that the isolated CHO cells express a novel type of AcLDL receptor, which is distinct from macrophage scavenger receptors with respect to ligand specificity and competitor sensitivity.     

Excretion of HCO3- by the urinary bladder of Bufo marinus in metabolic alkalosis

We studied the role of the urinary bladder of Bufo marinus in the excretion of bicarbonate into the urine. The toads were in metabolic alkalosis, produced by administering 120 mM NaHCO3 by stomach tube or by soaking the toads in 120 mM NaHCO3 solution for 48 to 72 hr. In vitro 10 cannulated whole bladders from toads in alkalosis transported bicarbonate from the serosal to mucosal medium. The average gradient created by this transport was 5.7 meq/1. In 15 whole bladders from toads in metabolic acidosis studied under identical conditions, there was no transport of bicarbonate into mucosal medium.     

Archive contributions to a molecular phylogeography of the toad Bufo calamita in Britain

A complete phylogeographic analysis of any species requires sampling throughout its biogeographical range. In the case of the natterjack toad Bufo calamita in Britain, recent local extinctions have left substantial areas of its historical range without extant populations. We therefore obtained tissue samples of archived Bufo calamita from four museums in the United Kingdom. A range of tissues (tongue, liver, skin, lung, and larval tail) was sampled from a total of 33 individual animals. DNA was extracted and eight polymorphic microsatellite loci were scored. One or more loci were amplified successfully from 27 individuals, and sufficient data were obtained from regions with few or no surviving populations to supplement a phylogeographic analysis based on extant populations.     

Isolation, sequence determination, physical and physiological characterization of the neuroparsins and ovary maturing parsins of Schistocerca gregaria

Neurosecretory products immunologically related to either neuroparsin (NP) or ovary maturing parsin (OMP) of Locusta migratoria (Lom) were purified from the nervous corpora cardiaca of Schistocerca gregaria (Scg). The determination of both their molecular masses by mass spectrometry and their sequences by automated Edman degradation established that they are members of the NP and OMP families respectively. NP molecules of Schistocerca (Scg NPs) consisted of two major forms having about the same molecular masses as NPA and NPB of Locusta and 88% primary structure similarity. They had also the same antidiuretic activity. OMP molecules of Schistocerca (Scg OMPs) were composed in young adults of four isoforms: two long isoforms corresponding to Lom OMP, and differing by a tripeptide insertion (Pro-Ala-Ala) at position 21 and two short isoforms deprived of the 13-residue N-terminal peptide of Lom OMP and differing by the same tripeptide insertion. The PAA isoforms were observed in low amounts as compared to the other isoforms. In mature adults, only the two short isoforms were present. The complete sequence of PAA Scg OMP presents a large degree of sequence homology with Lom OMP (83%). The mixed Scg OMPs had the same biological effects as Lom OMPs. They induced precocious occurrence of both ecdysteroids and vitellogenin in the haemolymph and stimulated o?cyte growth.