Dogfish Brain Research Articles

Binding sites for tachykinin peptides in the brain and stomach of the dogfish, Scyliorhinus canicula

In membranes of dogfish brain and stomach, two binding sites for tachykinins were identified. One site specifically bound [ 125I]-Bolton-Hunter substance P (BH-SP) and the rank potency of tachykinins to compete for BH-SP binding revealed similarities with the rank potency of an NK 1 receptor. The pharmacology of the other site, which specifically bound [ 125I]-Bolton-Hunter scyliorhinin II (BH-Scy II), did not resemble any of the mammalian tachykinin receptors. The rank potency to inhibit BH-Scy II binding to this second site was: scyliorhinin II ≈ scyliorhinin I > eledoisin ≈ substance P ≈ neurokinin A > phyllomedusin ≈ physalaemin > [Sar 9Met(O 2) 11]substance P. Neurokinin B and senktide did not displace BH-Scy II binding. In addition, nucleotide analogues inhibited BH-SP binding but not BH-Scy II binding. Our binding data suggest the existence of a mammalian-like NK 1 receptor and of a nonmammalian tachykinin receptor in the dogfish.

Immunocytochemical distribution of FMRFamide-like substance in the brain of the cloudy dogfish, Scyliorhinus torazame

The distribution of the molluscan cardioexcitatory tetrapeptide FMRFamide (Phe-Met-Arg-Phe-NH2) in the brain of the cloudy dogfish, Scyliorhinus torazame, was examined by immunocytochemistry. FMRFamide-like immunoreactivity was demonstrated to occur extensively in various regions of the dogfish brain, except for the corpus cerebelli. Immunoreactive neuronal perikarya were located in the ganglion of the nervus terminalis, the preoptic area, and the hypothalamic periventricular gray matter consisting of the nucleus medius hypothalamicus, the nucleus lateralis tuberis, and the nucleus lobi lateralis. Some of the immunoreactive cells in the hypothalamus were identified as cerebrospinal fluid-contacting neurons. The bulk of the immunostained fibers in the nervus terminalis penetrated into the midventral portion of the telencephalon and ran dorsocaudally toward the basal telencephalon and hypothalamus, showing radial projections or ramifications. The labeled fibers were abundant in the midbasal part of the telencephalon and in the hypothalamus, where some fibers were found in loose networks around the cell bodies of the nucleus septi and hypothalamic periventricular nuclei. The fibers demonstrated in the hypothalamus terminated around the vascular wall of the primary capillary plexus of the median eminence or penetrated deeply into the pars intermedia of the hypophysis. These results suggest that, in the dogfish, an FMRFamide-like substance participates in the regulation of adenohypophysial function. This molecule may have a role as a neurotransmitter and/or neuromodulator in the central nervous system.

Immunohistochemical Localization of Calbindin-D<sub>28</sub><sub>K</sub> in the Brain of a Cartilaginous Fish, the Dogfish (<i>Scyliorhinus canicula</i> L.)

The occurrence and distribution of the vitamin-D-induced calcium-binding protein, calbindin-D28K, has been studied in the brain of a cartilaginous fish using immunohistochemical techniques. A strong immunoreactivity was found in the perikarya, dendrites and axons of neurons located in the nucleus interstitialis commissurae anterioris, the nucleus medialis of the left habenula, the thalamus dorsalis, the thalamus ventralis, the nucleus lobi lateralis, the nucleus interpeduncularis, the lobus vagi and the medial reticular zone. Fibre tracts associated with some of these neuronal groups, such as the fasciculus retroflexus, the stria medullaris and the commissura habenulae, also contained immunopositive fibres. Only a minor immunoreactivity could be detected in other brain areas such as the tectum mesencephali and some telencephalic zones. Interestingly, the cerebellum did not show any immunoreactivity in Purkinje cells nor in other neurons. The distribution of calbindin-D28K in the dogfish brain appears to be mainly related to the viscerosensory centres.

A novel ganglioside in dogfish brain. Occurrence of a trisialoganglioside with a sialic acid linked to N-acetylgalactosamine.

A novel trisialoganglioside has been isolated from dogfish (Squalus acanthias) brain. From the results of gas chromatography-mass spectrometric analysis of methylated sugars, enzymatic hydrolysis, fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance analysis, its structure was concluded to be (formula; see text) This is the first report of the occurrence of a ganglioside with 3 sialic acid residues separately linked to its gangliotetraosyl backbone.

Isolation of heparin-binding growth factors from dogfish ( Mustela canis) brain and retina

We have purified two growth factors from dogfish brain and retina by using their binding affinity for heparin-Sepharose. These growth factors were eluted at 1 and 2 M NaCl similarly to those purified from bovine brain or retina. Their mitogenic activity was assayed in vitro on the same mammalian cells: bovine lens epithelial cells or human fibroblasts. All these data seem to indicate that these growth factors belong to the families of other well defined mammalian growth factors: EDGF I, brain basic FGF, AGF II, on the one hand and EDGF II, brain acidic FGF, AGF I, ECGF, on the other. Thus, these growth factors have been widely distributed during evolution and retain at least a conservative sequence to stimulate cell proliferation, in mammalian cells.

Diverse molecular forms of gonadotropin-releasing hormone in an elasmobranch and a teleost fish

Immunoreactive and biologically active gonadotropin-releasing hormones (GnRHs) in dogfish ( Poroderma africanum) and teleost ( Coris julis) brain extracts were studied by high-performance liquid chromatography (HPLC), radioimmunoassay with region-specific antisera, and assessment of luteinizing hormone (LH)-releasing activity in a chicken dispersed pituitary cell bioassay. In dogfish brain extract, seven GnRH molecular forms with LH-releasing activity were demonstrated. Three of these forms coeluted with synthetic mammalian GnRH; His 5,Trp 7,Tyr 8-GnRH; and Trp 7,Leu 8-GnRH on HPLC. The peaks coincident with His 5,Trp 7,Tyr 8-GnRH and Trp 7,Leu 8-GnRH had immunological and biological properties identical to those of the synthetic peptides. However, the molecular form coeluting with mammalian GnRH had immunological and biological properties different from those of mammalian GnRH and is thus a novel molecular variant of GnRH. The four remaining forms are also novel GnRHs or structurally unrelated peptides with LH-releasing activity. Dogfish systemic blood contained immunoreactive GnRH. In teleost brain extract, three biologically active GnRH forms with LH-releasing activity were present. The major peak of GnRH immunoreactivity coeluted with Trp 7,Leu 8-GnRH, and a second immunoreactive form coeluted with His 5,Trp 7,Tyr 8-GnRH. The third biologically active peak is a novel, early-eluting molecular variant of GnRH or a structurally unrelated peptide with LH-releasing activity.

Microtubule-associated proteins (MAPs) of dogfish brain and squid optic ganglia.

Annals of the New York Academy of SciencesVolume 466, Issue 1 p. 440-443 Microtubule-Associated Proteins (MAPs) of Dogfish Brain and Squid Optic Gangliaa GEORGE M. LANGFORD, GEORGE M. LANGFORD Department of Physiology School of Medicine University of North Carolina Chapel Hill, North Carolina 27514 Marine Biological Laboratory Woods Hole, Massachusetts 02543Search for more papers by this authorERROL WILLIAMS, ERROL WILLIAMS Department of Physiology School of Medicine University of North Carolina Chapel Hill, North Carolina 27514 Marine Biological Laboratory Woods Hole, Massachusetts 02543Search for more papers by this authorDARRYL PETERKIN, DARRYL PETERKIN Department of Physiology School of Medicine University of North Carolina Chapel Hill, North Carolina 27514 Marine Biological Laboratory Woods Hole, Massachusetts 02543Search for more papers by this author GEORGE M. LANGFORD, GEORGE M. LANGFORD Department of Physiology School of Medicine University of North Carolina Chapel Hill, North Carolina 27514 Marine Biological Laboratory Woods Hole, Massachusetts 02543Search for more papers by this authorERROL WILLIAMS, ERROL WILLIAMS Department of Physiology School of Medicine University of North Carolina Chapel Hill, North Carolina 27514 Marine Biological Laboratory Woods Hole, Massachusetts 02543Search for more papers by this authorDARRYL PETERKIN, DARRYL PETERKIN Department of Physiology School of Medicine University of North Carolina Chapel Hill, North Carolina 27514 Marine Biological Laboratory Woods Hole, Massachusetts 02543Search for more papers by this author First published: June 1986 https://doi.org/10.1111/j.1749-6632.1986.tb38418.xCitations: 8 a This work was supported by NIH Grant GM-28107. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume466, Issue1Dynamic Aspects of Microtubule BiologyJune 1986Pages 440-443 RelatedInformation

Characterization of microtubules isolated from dogfish ( Squalus acanthias and Scyliorhinus canicula) brain in the absence of glycerol

1. 1. Microtubules were isolated by two cycles of assembly-disassembly from dogfish brain in the absence of glycerol. 2. 2. The microtubule protein preparation consist mainly of tubulin as characterized by SDS-polyacrylamide gel electrophoresis, but tau and high molecular weight microtubule associated proteins (MAPs) were also identified, in addition to several other MAPs. 3. 3. The microtubules were cold-sensitive and had an assembly temperature optimum at 21–25°C. 4. 4. Ca 2+, at a concentration of 1 mM or higher, induced spirals of several protofilaments, sheets and “macrotubules”. 5. 5. In the presence of colchicine (0.1–1.0 mM) spirals as well as microtubules were seen. These structures were often found to be clustered.

Concerning the efficiency of the treadmilling phenomenon with microtubules.

Radioactive tubulin subunit incorporation into porcine and dogfish shark brain microtubules which are at steady state has been found to result primarily from a diffusional reaction, in which subunits are incorporated although there are an equal number of tubulin subunit additions to and losses from each of the two microtubule ends in a unit of time. Treadmilling is very inefficient, and the Wegner s-values are equal to 0.0005-0.001. At steady state there are approximately 5000 (pig brain) or 2500 (dogfish brain) tubulin subunits lost from the two microtubule ends/s; an equivalent number of subunit addition reactions maintains a constant microtubule mass. The rate constants for subunit loss and addition with porcine brain microtubules, determined from analysis of the steady state rate for radioactive subunit incorporation, are much larger than those measured previously, when the rate constants were determined from the disassembly rate following perturbation of the steady state by dilution (Zeeberg, B., Reid, R., and Caplow, M. (1980) J. Biol. Chem. 255, 9891-9899). To account for this discrepancy it is suggested that at steady state the microtubule is capped by a short finite length of tubulin-GTP subunits, which undergo extremely facile association and dissociation (2500-5000 subunits/microtubule/s). This cap would be rapidly lost following dilution so that the observed rate only measures the relatively slow loss of tubulin-GDP subunits (120 subunits/microtubule/s) which had been in the interior of the microtubule; this is not equal to the rate constant for subunit loss (and addition) from the ends at steady state. Because of this, previous estimates of the Wegner s-value for treadmilling which utilized dilution for determining the steady state molecular rate constants for subunit loss are believed to be too high.

Concentration, composition and structure of gangliosides from the central nervous systems of dogfish, squalus acanthia, and cod, gadus callarias

1. 1. Gangliosides were isolated from the cerebral grey and white matter and spinal cord, and from the brain and spinal cord myelin of dogfish and cod. In the cod the highest ganglioside concentration, expressed as lipid- N-acetylneuraminic acid, was found in cerebral (forebrain) grey matter, 1.67 μmol/g. It was lower in cerebral white matter, 1.13 μmol/g, and spinal cord, 0.70 μmol/g. In dogfish the relations between the concentrations were unique, with the lowest concentration in cerebral grey matter, 0.93 μmol/g, higher in cerebral white matter, 1.13 μmol/g, and the highest in spinal cord, 2.36 μmol/g. 2. 2. Cod brain and spinal cord and dogfish brain had a high proportion of tetra- and pentasialogangliosides, 50–60% of total sialic acid. Of these, the tetrasialogangliosides were the largest fraction in the cod nervous tissues while the pentasialogangliosides were the largest fraction in the dogfish brain. In spinal cord of dogfish GM2 was the predominant ganglioside and accounted for 60% of the total sialic acid. In addition to GM2, spinal cord also contained 12% of the total sialic acid in GD2 and approximately 2% in GT2. 3. 3. The major portions of ganglioside GM2 and GD2 of dogfish were isolated in the myelin fraction which contained a uniquely high ganglioside proportion. The molar ratio of the lipids in spinal cord myelin was: phospholipid: cholesterol:cerebroside:ganglioside-NeuAc (1.00:1.02:0.22:0.021). In the cod myelin the corresponding values were: 1.00:0.76:0.033:0.0033. In the cod myelin the gangliosides with three or more sialic acids accounted for the main part of the total sialic acids. 4. 4. In the nervous system of the dogfish, all the higher gangliosides of the G1 series, GD1, GT1, GQ1 and GP1, had a fatty acid composition similar to gangliosides in mammalian brain, with stearic acid as the dominating fatty acid. In ganglioside GM2 and GD2 the fatty acid 22:1 was more than 60% of the total acids. In cod, stearic acid was the major fatty acid in all the gangliosides.

Chapter 5 Purification and Reassembly of Tubulin from Outer Doublet Microtubules

The chapter discusses the purification and reassembly of tubulin from outer doublet microtubules. The most commonly used microtubule system is that of vertebrate brain. This system has the advantage of yielding large quantities of native tubulin; however, the tubulin derives from the total brain microtubule population, which shows considerable heterogeneity. The optimal in vitro assembly requirements of outer doublet and vertebrate brain tubulins are very similar, as are the properties of the assembled microtubules. This appears to be generally true for all tubulins so far examined, irrespective of the properties, functions, cellular location, or phylogeny of the microtubules from which the tubulin derives. The reconstituted microtubules are “labile” singlet microtubules, archetypical of which are reconstituted vertebrate brain microtubules. Structurally bonafide singlet microtubules can be formed in vitro from outer doublet and dogfish brain tubulins in the absence of associated proteins. Direct evidence that tubulin conformational changes alter the microtubule lattice is lacking. Studies on the mechanism of colchicine poisoning of microtubule assembly are consistent with this hypothesis. Colchicine poisons microtubule assembly by first complexing with tubulin dimers, which then copolymerize with uncomplexed tubulin into the microtubules and decrease the apparent rate of subsequent drug-free dimer addition.

Laser-light scattering diffusion measurements on brain microtubule protein from dogfish (Squalus acanthus) and beef.

Laser photon correlation spectroscopy and analytical ultracentrifugation were used to compare the equilibrium properties of disassembled, assembly-competent, brain microtubule protein from dogfish (Squalus acanthus) and beef. Analytical ultracentrifugation confirmed that assembly-competent bovine material at 4 degrees C, purified in the presence of glycerol through 2 cycles of assembly and disassembly, consisted of 6 S (dimer) and 35 S (ring) components, whereas assembly-competent dogfish material prepared in the same way was composed primarily of 6 S protein. By means of photon correlation spectroscopy, z-average diffusion coefficients (D'20, w) of 0.55 (+/- 0.04) x 10(-11) m2 s-1 (beef) and 1.27 (+/- 0.11) x 10(-11) m2 s-1 (dogfish) were measured at 4 degrees C for the twice-cycled assembly competent microtubule protein. Although D'20, w values for dogfish material are significantly higher than those for beef, they depart from the calculated value of 5.0 x 10(-11) m2 s-1 for a pure 6 S (dimer) solution. Microtubule accessory proteins were detectable on sodium dodecyl sulphate/polyacrylamide gels of dogfish microtubule protein. By fitting photocount autocorrelation functions to a 2-particle (dimer-ring) model it was estimated that the presence in a solution of assembly-competent dogfish microtubule protein of less than 1 ring per 250 dimers could account for the measured z-average diffusion coefficient. The temperature sensitivity of D'20, w showed that dogfish brain microtubule protein polymerized at lower temperatures than samples prepared by the same methods from beef brain.

Comparison of proteolytic cleavage patterns of α-tubulins and β-tubulins from taxonomically distant species

The α and β-chains of tubulins from several taxonomically distant species were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and isolated by a procedure that avoided stains and acidic conditions. All the β-chains had the same electrophoretic mobility, but the α-chains of some of the lower eukaryotes migrated faster than the rest. Limited proteolytic digestion of the subunits with Staphylococcus aureus protease, followed by electrophoresis on 15% acrylamide gels, resulted in very similar peptide patterns for all the β-chains, but a variety of patterns for the α-chains. The peptide patterns of α-tubulins from sea urchin egg and sperm flagellar outer doublets were markedly different, whereas those of bovine brain and kidney were identical. Bovine and dogfish brain α-tubulin peptide patterns were also identical, in contrast to the very different one of squid brain. Strong similarities were found between the α-chain peptide patterns of sperm flagellar tubulin from the echiurid worm Urechis caupo and the sea urchins Lytechinus pictus and Strongylocentrotus purpuratus, indicating that functionally similar tubulins from very different species can be more closely related than functionally different tubulins from the same organism. Evidence for the evolution of plant sperm flagella from protistan flagella was provided by the distinctive and very similar α-chain peptide patterns of tubulin from sperm flagella of the bracken fern Pteridium aquilinum and the flagella of the unicellular biflagellate alga Chlamydomonas reinhardtii.

In vitro assembly of dogfish brain tubulin and the induction of coiled ribbon polymers by calcium

Dogfish brain tubulin exhibits properties which differ from mammalian and chick brain tubulin. Polymerization of dogfish brain tubulin occurs optimally between 18 and 21 °C. The high molecular weight proteins which typically co-purify with tubulin are nearly absent as shown by SDS-polyacrylamide gel electrophoresis. A “dynein-like” protein occasionally co-purifies with dogfish brain tubulin, but its absence does not affect polymerization. At 0 °C, tubulin exists almost exclusively in the form of 6S dimers as revealed by analytical ultracentrifugation. Samples negatively stained and observed in the electron microscope have insignificant numbers of ring and spiral structures present. In some preparations, short thin filaments, approx. 200 nm in length, are seen. During polymerization, large numbers of coiled sheet polymers appear. These coiled ribbons are present throughout the early stages of polymerization, but not after polymerization reaches a plateau. The helical ribbons have an average width of 35 nm and contain approx. 8 protofilaments. The pitch angle of the helix is 33 ° and two or three turns are usually seen. The addition of 10 mM CaCl 2 to a polymerized or polymerizing solution of tubulin induces the formation of rings which transform into stable coiled sheet polymers. Some of the coiled sheets form tubules when adjacent gyres of the spirals join together. The diameter of these “macrotubules” range between 50 and 100 nm. The data suggest that ring structures are not obligate intermediates of polymerization and bring into question the role of the microtubule-associated proteins.

EFFECTS OF HYPOTHALAMIC EXTRACTS, NEUROTRANSMITTERS AND SYNTHETIC HYPOTHALAMIC RELEASING HORMONES ON ADENYLYL CYCLASE ACTIVITY IN THE LOBES OF THE PITUITARY OF THE DOGFISH (SCYLIORHINUS CANICULA L.)

Neutralized acid extracts of the median eminence of the dogfish hypothalamus were found to cause a dose-related activation of adenylyl cyclase in all lobes of the dogfish pituitary. Equal concentrations of extracts of extrahypothalamic areas of the dogfish brain did not activate the enzyme. The putative neurotransmitters melatonin, serotonin, adrenaline, noradrenaline, dopamine and acetylcholine were without effect, as were the prostaglandins E1 and E2. The effects of synthetic mammalian hypothalamic hormones were also studied. Both thyrotrophin releasing hormone and gonadotrophin releasing hormone activated the ventral lobe enzyme, but had no effect on the adenylyl cyclase of the other three lobes. The tripeptide, Pro-Leu-Gly-NH2, a possible melanocyte-stimulating hormone release-inhibiting factor had no effect on the enzyme of the neurointermediate lobe. It is suggested that all four lobes of the dogfish pituitary may be under hypothalamic control and that this control is likely to be mediated by peptide hormones, as in mammals.

Ventricular fluid production rates and turnover in elasmobranchii

1. 1. Ventricular fluid (VF) production rates were measured by frontocerebellar perfusion with buffered inulin solution in three species of sharks; the spiny dogfish ( Squalus acanthia). the nurse shark ( Ginglymostoma cirratum) and the lemon shark ( Negaprion brevirostris). 2. 2. Mean VF production rates in dogfish (3–5 kg) were 0.004 ml/min, in nurse sharks (5–18 kg) 0.005 ml/min and in lemon sharks (5 kg) -.004 ml/min. 3. 3. Acetazolamide (30–300 mg/kg) decreased VF production rate 29 per cent in dogfish. 4. 4. In dogfish, bulk removal of VF, as measured by the disappearance of inulin and radioiodinated albumin, occurred at a rate consistent with VF production rates. 5. 5. Anatomical structures that might be responsible for bulk removal of VF were not found in the brain of dogfish. 6. 6. It is concluded that VF in three species of sharks is produced and turns over at a relatively brisk rate, and that a bulk removal system functionally similar to that found in mammals is present in the dogfish.