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.     

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.     

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.     

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.     

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.     

Tachykinin NK2 receptors: characterization in the rat small intestine by the use of a peptide agonist and a nonpeptide antagonist as radioligands

OBJECTIVE: A rare family pedigree is described with a multigenerational history of an early onset, non-Alzheimer's dementia consistent with autosomal dominant inheritance. Information on five generations, with 26 suspected or proven cases of dementia, are presented. METHOD: Previous work on the family was collated and verified. The pedigree was updated. Thirty-three family members agreed to be interviewed for the present study. Standardised clinical information was obtained using the Cambridge Mental Disorders of the Elderly Examination (CAMDEX) National Adult Reading Test (NART), vocabulary and digit substitution subscales of the Wechster adult intelligence scale-revised edition (WARS-R). Abbot samples were taken for biochemical and genetic analysis. RESULTS: Fifteen males and 11 females have been affected. The age of onset of dementia in those for whom data were available (n = 12) ranged from 39 to 64 years with a mean of 53 years. The duration of illness ranged from 4 to 14 years and the age at death ranged from 49 to 69 years (mean 62 years). Autopsy data exist for nine cases. In one case the neuropathology was consistent with Alzheimer's disease. In two cases the diagnosis of Pick's disease was made on the basis of frontal or frontal/temporal lobe atrophy without Pick bodies or cells. One case diagnosed as Pick's disease had frontal/temporal lobe atrophy with cells resembling Pick's bodies. In the five remaining cases there were no distinctive neuropathological features to differentiate the type of dementia. CONCLUSIONS: The importance of recognising familial dementia, collating information on multiple generations and prospectively collecting standardised data 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.     

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.     

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

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.     

Vasotocin and hydrin 2 (vasotocinyl-Gly) in the African toad Bufo regularis: study under various environmental conditions

1. The neurohypophysial osmoregulatory hormones of the African toad Bufo regularis, a species adapted to estivate under dry and hot conditions, have been investigated. Vasotocin and hydrin 2 (vasotocinyl-Gly) have been identified by their retention times in high-pressure reverse-phase liquid chromatography and coelution with synthetic peptides, their pharmacological properties (vasotocin) and microsequencing. 2. Vasotocin-associated neurophysin (MSEL-neurophysin type) has been characterized by its N-terminal amino acid sequence. 3. In toads subjected to dehydration by evaporation (20% weight loss) or to osmotic stress by immersion in 2% NaCl for 3 hr (6% weight loss), the molar ratio hydrin 2/vasotocin (about 2:1) remained similar to the one observed in control animals. 4. In toads exposed to saline solution, there was a large decrease (roughly 30%) in the amounts of both hormones in the neuro-hypophysis. Environmental conditions for distinct secretions of vasotocin and hydrin 2 remain to be found.     

Effects of lesions in the medial pallium on instrumental learning in the toad (Bufo arenarum)

Lesions of the medial pallium in toads significantly retarded extinction of a water-reinforced instrumental response in a runway training situation, relative to sham-operated and intact controls. The lesion had no effect on acquisition or in the amount of water uptake during acquisition. The results suggest that the medial pallium of toads plays a role in response inhibition. This possibility is discussed in relation to the results of analogous experiments with hippocampectomized rats.     

Induction of hepatocellular carcinomas in the Egyptian toad Bufo regularis by an antifungal drug (griseofulvin)

Neoplastic lesions (hepatocellular carcinomas) were induced in the liver in 18 of 100 experimental toads (Bufo regularis) force fed with the antifungal drug griseofulvin, at a dose level of 0.4 mg/50 g every day. Maximal time of exposure and observation was 12 weeks. The first tumors appeared in 2 male toads 4 weeks after the initiation of feeding.     

Promotion of muscle regeneration in the toad (Bufo viridis) gastrocnemius muscle by low-energy laser irradiation

The effect of low-energy laser (He-Ne) irradiation on the process of skeletal muscle regeneration after cold injury to the gastrocnemius muscle of the toad (Bufo viridis) was studied using quantitative histological and morphometric methods. The injured zones in the experimental toads were subjected to five direct He-Ne laser (632.8 nm wavelength) irradiations (6.0 mW for 2.3 min) every alternate day starting on the fourth day postinjury. Muscles that were injured as above, and subjected to red-light irradiation, served as a control group. Morphometric analysis was performed on histological sections of injured areas at 9, 14, and 30 days postinjury. At 9 days postinjury, mononucleated cells populated 69.3% +/- 16.8% of the total area of injury. Thereafter, their volume fraction (percent of total injured zone) decreased gradually but more rapidly in the laser-irradiated muscle than in the control. The volume fraction of the myotubes in the laser-irradiated muscles at 9 days of muscle regeneration was significantly higher (7.0% +/- 2.2%) than in the control muscle (1.2% +/- 0.4%). Young myofibers in the laser-irradiated muscles populated 15.5% +/- 7.9% and 65.0% +/- 9.5% of the injured area at 9 and 14 days of muscle regeneration, respectively, while in control muscles these structures were not evident at 9 days and made up only 5.3% +/- 2.9% of the traumatized area at 14 days postinjury. The volume fraction of the young myofibers further increased by 30 days of muscle regeneration making up 75.7% +/- 13.2% of the traumatized area, while in the laser-irradiated muscles most of the injured zone was filled with mature muscle fibers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Agonist action at D2(long) dopamine receptors: ligand binding and functional assays

Clinical pathways are being introduced by hospitals to reduce costs and control unnecessary variation in care. We studied 766 inpatients to measure the impact of a perioperative clinical pathway for patients undergoing knee replacement surgery on hospital costs. One hundred twenty patients underwent knee replacement surgery before the development of a perioperative clinical pathway, and 63 patients underwent knee replacement surgery after pathway implementation. As control groups, we contemporaneously studied 332 patients undergoing radical prostatectomy (no clinical pathway in place for these patients) and 251 patients undergoing hip replacement surgery without a clinical pathway (no clinical pathway and same surgeons as patients having knee replacement surgery). Total hospitalization costs (not charges), excluding professional fees, were computed for all patients. Mean (+/-SD) hospital costs for knee replacement surgery decreased from $21,709 +/- $5985 to $17,618 +/- $3152 after implementation of the clinical pathway. The percent decrease in hospitalization costs was 1.56-fold greater (95% confidence interval 1.02-2.28) in the knee replacement patients than in the radical prostatectomy patients and 2.02-fold greater (95% confidence interval 1.13-5.22) than in the hip replacement patients. If patient outcomes (e.g., patient satisfaction) remain constant with clinical pathways, clinical pathways may be a useful tool for incremental improvements in the cost of perioperative care. Implications: Doctors and nurses can proactively organize and record the elements of hospital care results in a clinical pathway, also known as "care pathways" or "critical pathways." We found that implementing a clinical pathway for patients undergoing knee replacement surgery reduced the hospitalization costs of this surgery.     

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.     

Eosinophilia and sarcoma of the small intestine (author's transl)

Eosinophilia occurs as a concomitant sign in multiple diseases of different etiology. Especially its occurrence in malignant diseases is still unknown. In three cases of primary sarcoma of the small intestine a constant eosinophilia is described. In one case this pronounced eosinophilia was present long before the final diagnose was made. The eosinophilic count was highest in the presence of clear symptoms of obstruction and normalized after surgical removal of the tumor. In one case a relapse was indicated by an increasing eosinophilic count. It is difficult to diagnose the sarcoma of the small intestine especially in its early stage because of the lack of typical symptoms. Concerning the described cases it is therefore pointed out, that in connection with undifferentiated abdominal complaints an obscure eosinophilia should be considered to be due to sarcomatous small bowel disease.