A comparison of dogfish and bovine chymotrypsins

Bovine and dogfish chymotrypsins were compared to determine if chymotrypsin from a poikilothermic organism (spiny dogfish (Squalus acanthias] adapted to low temperatures possessed catalytic properties different from those of the same enzyme from a warm-blooded animal. An improved procedure was developed for isolating dogfish pancreatic chymotrypsin. The least hydrophobic and smallest substrate used, p-nitrophenyl acetate, had similar enthalpies of association (delta Ha) with both enzymes, whereas larger, more hydrophobic substrates had delta Ha values that were of opposite sign for the two enzymes. As the temperature increased, the association constants (1/Ks) for p-nitrophenyl valerate and p-nitrophenyltrimethyl acetate increased for dogfish chymotrypsin and decreased for bovine chymotrypsin, while the free energies of association (delta Ga) remained relatively constant. Acylation of chymotrypsin was 1.5-2.5 times slower in the dogfish enzyme than in the bovine enzyme except below 15 degrees C with p-nitrophenyltrimethyl acetate. delta H++ for acylation by p-nitrophenyltrimethyl acetate were 2.0 kcal/mol for the dogfish enzyme and 10.2 kcal/mol for the bovine, whereas delta H++ values were only slightly lower in the dogfish enzyme for the other two substrates. For all substrates, the deacylation rate constant (kcat) was greater with dogfish chymotrypsin than bovine. However, the free energies of activation (delta G++) for deacylation were nearly equal between the two enzymes for each of the substrates.     

Diet of spiny dogfish, Squalus acanthias Linnaeus, on the east coast, South Island, New Zealand

Stomach content data from 7283 spiny dogfish, Squalus ucunthius Linnaeus, from east coast South Island of New Zealand are presented. The major dietary components based on occurrence were 60% crustaceans and 15% fish. The principal food items were the post-larval phase of the squat lobster, Munida greguria Fabricius (30%), and the euphausiid, Nyctiphanes australis Sars (20%). Commercial fish species contributed to less than 5% of the diet. Diet of dogfish varied with dogfish size and sex. Compared to northern hemisphere studies, spiny dogfish in this study area had (1) a high proportion of crustaceans in their diet, (2) a relatively low percentage of empty stomachs, (3) a relatively high incidence of cannibalism.     

Further evidence for ancient role of ACTH peptides at melanocortin (MC) receptors; pharmacology of dogfish and lamprey peptides at dogfish MC receptors

The cloning of melanocortin (MC) receptors in distant species has provided us tools to get insight in how the ligand-receptors interactions in the MC system have evolved. We have however lacked studies on pharmacology of native ancient melanocortin peptides at the ancient MC receptors. In this paper we synthesized melanocortin peptides from both the sea lamprey (Petromyzon marinus) and spiny dogfish (Squalus acanthias) and tested them on the MC3 and MC4 receptors from spiny dogfish. The results show that both the dogfish and lamprey ACTH peptides have similar or higher affinity than the dogfish alpha-, beta- and gamma-MSH peptides to the dogfish MC3 and MC4 receptors. Moreover, both the dogfish and lamprey ACTH peptides have more than 10-fold higher affinity than alpha-MSH to the dogfish MC4 receptor. We also show that dogfish delta-MSH is able to bind to MC receptors and its potency is higher than of dogfish beta-MSH, which is considered to be its precursor. Our results provide the first evidence that native ACTH ligands from dogfish and lamprey have a preference above native MSH peptides to ancient version of the MC3 and MC4 receptors. This further strengthens the hypotheses that the ligand contributing to the first version of the melanocortin ligand-receptor system resembled ACTH.     

Bilirubin metabolism in the spiny dogfish, Squalus acanthias, and the small skate, Raja erinacea.

1. The main bilirubin conjugate in bile of spiny dogfish (Squalus Acanthias) and small skate (Raja Erinacea) is bilirubin monoglucuronide. 2. Microsomal preparations from dogfish and small skate liver have similar bilirubin UDPglucuronyltransferase (UDPGT) activity and catalyze the conjugation of bilirubin with glucose from UDPglucose. 3. The activity of bilirubin glucosidation (UDPGT) was 0.5 times UDPG1T activity in dogfish and 0.15 times in skate liver microsomes. 4. Sodium cholate increased UDPGT and UDPG1T activities in dogfish and skate liver microsomal preparations only minimally, but the detergent markedly increased thermolability of UDPGT in skate liver microsomes.     

Extracellular carbonic anhydrase in the dogfish, Squalus acanthias: a role in CO2 excretion.

In Pacific spiny dogfish (Squalus acanthias), plasma CO(2) reactions have access to plasma carbonic anhydrase (CA) and gill membrane-associated CA. The objectives of this study were to characterise the gill membrane-bound CA and investigate whether extracellular CA contributes significantly to CO(2) excretion in dogfish. A subcellular fraction containing membrane-associated CA activity was isolated from dogfish gills and incubated with phosphatidylinositol-specific phospholipase C. This treatment caused significant release of CA activity from its membrane association, a result consistent with identification of the dogfish gill membrane-bound CA as a type IV isozyme. Inhibition constants (K(i)) against acetazolamide and benzolamide were 4.2 and 3.5 nmol L(-1), respectively. Use of a low dose (1.3 mg kg(-1) or 13 micromol L(-1)) of benzolamide to selectively inhibit extracellular CA in vivo caused a significant 30%-60% reduction in the arterial-venous total CO(2) concentration difference, a significant increase in Pco(2) and an acidosis, without affecting blood flow or ventilation. No effect of benzolamide on any measure of CO(2) excretion was detected in rainbow trout (Oncorhynchus mykiss). These results indicate that extracellular CA contributes substantially to CO(2) excretion in the dogfish, an elasmobranch, and confirm that CA is not available to plasma CO(2) reactions in rainbow trout, a teleost.     

Chemical characterisation of eggs from deep-sea sharks

The chemical characterisation and nutritional value of eggs from the five deep-sea sharks leafscale gulper shark (Centrophorus squamosus), greater lantern shark (Etmopterus princeps), longnose velvet dogfish (Centroscymnus crepidater), Portuguese dogfish (Centroscymnus coelolepis) and black dogfish (Centrocyllium fabricii) captured at Hatton Bank in the North Atlantic were examined. The chemical composition was quite similar for all the eggs studied. The dominant fatty acid in all the eggs was the monounsaturated fatty acid C18:1, which varied from 27-39%. The eggs had a relatively high content of C16:0 (13.0-18.5%) and C22:6n-3 (10.3-15.1%). The two main lipid classes in the eggs were triacylglycerols (36-55%) and phospholipids (34-41%). The eggs had high amounts of vitamin A and E. The shark eggs were particularly rich in the amino acids aspartic acid, glutamic acid, leucine and arginine.     

Class-specific epitopes detected by polyclonal antibodies against the secretory products of the subcommissural organ of the dogfish Scyliorhinus canicula

We have raised antisera against extracts of the subcommissural organ (SCO) of the dogfish, Scyliorhinus canicula L. Brains of 2900 specimens were collected in acetone, and the region containing the SCO and posterior commissure was removed and extracted in three different media. Antisera against these crude extracts were raised in rats and rabbits. Sequential absorptions of the antisera with extracts from different regions of the dogfish brain were performed to eliminate unwanted antibodies. When used to immunostain sections of the whole central nervous system of the dogfish, these purified antisera reacted selectively with the SCO-Reissner's fiber complex. An antiserum against bovine Reissner's fiber was also used. The antisera against the dogfish SCO and bovine Reissner's fiber showed the same staining pattern in the SCO and the Reissner's fiber of the dogfish. For comparative purposes, the brains of 15 vertebrate species from all vertebrate classes were immunostained with both antisera. The anti-dogfish SCO serum reacted with the SCO of the dogfish and that of other phylogenetically related elasmobranch species. Neither the SCO of a primitive elasmobranch species, Heptranchias perlo, nor the SCO of the other classes of vertebrates reacted with the anti-dogfish SCO serum. However, the antiserum against bovine Reissner's fiber reacted with the SCO of all the investigated species. It is concluded that some epitopes (or compounds) in the secretory material of the SCO are class-specific, whereas others are conserved and are synthesized by the SCO in most vertebrate species.     

Isolation and characterization of the erythrocyte surface membrane of the smooth dogfish, Mustelus canis.

1. The plasma membrane of the dogfish erythrocyte is characterized. 2. Surface membranes were isolated from dogfish red cells. 3. Cells suspended in a hypotonic zinc chloride buffer (0.5 mM ZnCl2, 5 mM Tris-HCl, pH 8.0) tended to enucleate spontaneously. 4. By gentle homogenization relatively high yields of red cell "ghosts" were formed; these were purified using discontinuous gradients of sucrose and of glycerol solutions. Analyses of protein, carbohydrates and lipids were carried out. 5. There did not appear to be marked overall differences in the composition of the surface membrane of the nucleated dogfish erythrocyte compared to those of other species.     

Type IV carbonic anhydrase is present in the gills of spiny dogfish (Squalus acanthias)

Physiological and biochemical studies have provided indirect evidence for a membrane-associated carbonic anhydrase (CA) isoform, similar to mammalian type IV CA, in the gills of dogfish (Squalus acanthias). This CA isoform is linked to the plasma membrane of gill epithelial cells by a glycosylphosphatidylinositol anchor and oriented toward the plasma, such that it can catalyze the dehydration of plasma HCO(3)(-) ions. The present study directly tested the hypothesis that CA IV is present in dogfish gills in a location amenable to catalyzing plasma HCO(3)(-) dehydration. Homology cloning techniques were used to assemble a 1,127 base pair cDNA that coded for a deduced protein of 306 amino acids. Phylogenetic analysis suggested that this protein was a type IV CA. For purposes of comparison, a second cDNA (1,107 base pairs) was cloned from dogfish blood; it encoded a deduced protein of 260 amino acids that was identified as a cytosolic CA through phylogenetic analysis. Using real-time PCR and in situ hybridization, mRNA expression for the dogfish type IV CA was detected in gill tissue and specifically localized to pillar cells and branchial epithelial cells that flanked the pillar cells. Immunohistochemistry using a polyclonal antibody raised against rainbow trout type IV CA revealed a similar pattern of CA IV immunoreactivity and demonstrated a limited degree of colocalization with Na(+)-K(+)-ATPase immunoreactivity. The presence and localization of a type IV CA isoform in the gills of dogfish is consistent with the hypothesis that branchial membrane-bound CA with an extracellular orientation contributes to CO(2) excretion in dogfish by catalyzing the dehydration of plasma HCO(3)(-) ions.     

Activation of liver carnitine palmitoyltransferase-1 and mitochondrial acetoacetyl-CoA thiolase is associated with elevated ketone body levels in the elasmobranch Squalus acanthias

Elasmobranch fishes are an ancient group of vertebrates that have unusual lipid metabolism whereby storage lipids are mobilized from the liver for peripheral oxidation largely as ketone bodies rather than as nonesterified fatty acids under normal conditions. This reliance on ketones, even when feeding, implies that elasmobranchs are chronically ketogenic. Compared to specimens sampled within 2 d of capture (recently captured), spiny dogfish Squalus acanthias that were held for 16-33 d without apparent feeding displayed a 4.5-fold increase in plasma concentration of d- beta -hydroxybutyrate (from 0.71 to 3.2 mM) and were considered ketotic. Overt activity of carnitine palmitoyltransferase-1 in liver mitochondria from ketotic dogfish was characterized by an increased apparent maximal activity, a trend of increasing affinity (reduced apparent K(m); P=0.09) for l-carnitine, and desensitization to the inhibitor malonyl-CoA relative to recently captured animals. Acetoacetyl-CoA thiolase (ACoAT) activity in isolated liver mitochondria was also markedly increased in the ketotic dogfish compared to recently captured fish, whereas no difference in 3-hydroxy-3-methylglutaryl-CoA synthase activity was found between these groups, suggesting that ACoAT plays a more important role in the activation of ketogenesis in spiny dogfish than in mammals and birds.     

Spontaneous contractions in elasmobranch vessels in vitro

Isolated vessels from four elasmobranchs, yellow stingray (Urolophus jamaicensis), clearnose skate (Raja eglanteria), ghost shark (Hydrolagus novaezelandiae), and spiny dogfish (Squalus acanthias), were examined for the presence of spontaneous contractions (SC). SC were observed in otherwise unstimulated dorsal aortas (DA) from stingray and ghost shark, but not in skate DA. Unstimulated ventral aortas (VA) did not exhibit SC. After treatment of VA with a contractile agonist, SC appeared in stingray and skate but not ghost shark or dogfish. SC in stingray VA were subsequently inhibited by either epinephrine (10(-5) M) or indomethacin (10(-4) M). Agonist contraction also elicited strong SC in ductus Cuvier from stingray, but not from ghost shark or dogfish. SC in dogfish hepatic portal veins (HPV) produced a rhythmical oscillation in tension. The frequency of HPV SC was highest (approximately 1 min(-1)) in intact veins and lower (approximately 3 min(-1)) in vein segments, indicative of a dominant pacemaker in the intact vessel. SC in HPV were depressed during the first 30 min of hypoxia, but there was substantial recovery over an additional 30 min of hypoxia and complete recovery upon return to normoxia. Addition of 80 mM KCl completely inhibited HPV SC and lowered resting tone. These results show that SC are a common feature of elasmobranch vessels and there appears to be a correlation between swimming behavior and the propensity for SC. KCl inhibition of SC and tonus in HPV is highly unusual for vascular smooth muscle.     

Bombesin-like immunoreactive material in the gut, and the effect of bombesin on the stomach circulatory system of an elasmobranch fish, Squalus acanthias

The distribution, nature and amount of bombesin-like immunoreactivity (IR) in the gastrointestinal canal and its afferent vessels was investigated in the spiny dogfish (Squalus acanthias) together with the in vitro effect of synthetic bombesin on perfusion flow through the vascularly perfused dogfish stomach. Nerve fibres showing bombesin-like IR frequently occurred in the walls of the anterior mesenteric and coeliac arteries and the intrinsic vessels of the gut. Chromatographic studies revealed that multiple peaks of bombesin-like IR material were present in extracts of the spiny dogfish gastrointestinal vessels. Bombesin-like IR was also present in muscle and mucosal layers of the gut with higher levels in muscle compared with mucosa, and higher levels in the stomach than in the intestine and the rectum. Exogenous bombesin increased the flow through the vasculary perfused spiny dogfish stomach in a dose-dependent manner. Studies with tetrodotoxin and atropine showed that bombesin probably exerts its effect directly on the vascular musculature. It is concluded from this study that bombesin-like material is present in nerves innervating the gut circulatory system of the spiny dogfish. Bombesin may affect the blood-flow to the gastrointestinal canal, possibly via a direct effect on vascular smooth muscle.     

Changes in chloride secretion rate and vascular perfusion in the rectal gland of the European lesser-spotted dogfish in response to environmental and hormonal stimuli

Chloride secretion rates of rectal glands taken from the European lesser-spotted dogfish Scyliorhinus canicula adapting to 70% and 120% sea water (SW) were significantly greater and less than, respectively, those in the control 100% SW group. C-type natriuretic peptide (CNP) significantly increased chloride secretion rates above basal values in 100% SW although angiotenisn II (ANG II) had no effect. Perfusion of the secretory epithelia in rectal glands from 70% SW lesser-spotted dogfish was significantly higher than in rectal glands from 100% and 120% SW lesser-spotted dogfish. Perfusion of rectal glands with ANG II had no effect on perfusion of the secretory epithelia, although CNP perfusion induced significantly greater perfusion of the secretory epithelia than all other treatments. It remains to be determined if a reduction in environmental salinity induces an increase in plasma concentration of CNP and hence an increase in rectal gland activity.     

Elasmobranch tissue culture: in vitro growth of brain explants from a shark (Rhizoprionodon) and dogfish (Squalus)

Brain explants from a Caribbean sharpnose shark (Rhizoprionodon porosus) and six spiny dogfish (Squalus acanthias) were cultured at 22 degrees C in Leibovitz L-15 medium, 1000 mOsmol, with 350 mM urea, 20 mM added NaCl, and either 2% fetal bovine serum, 2% bull shark (Carcharhinus leucas) serum, or 2% spiny dogfish serum. Cell outgrowth from explants appeared after 1 month. Cells were subcultured once with 20% survival, and were viable at termination of the trials 6 months later. Cell morphology appeared normal throughout the experiment.     

Glutamine-dependent carbamoyl-phosphate synthetase and other enzyme activities related to the pyrimidine pathway in spleen of Squalus acanthias (spiny dogfish).

The first two steps of urea synthesis in liver of marine elasmobranchs involve formation of glutamine from ammonia and of carbamoyl phosphate from glutamine, catalysed by glutamine synthetase and carbamoyl-phosphate synthetase, respectively [Anderson & Casey (1984) J. Biol. Chem. 259, 456-462]; both of these enzymes are localized exclusively in the mitochondrial matrix. The objective of this study was to establish the enzymology of carbamoyl phosphate formation and utilization for pyrimidine nucleotide biosynthesis in Squalus acanthias (spiny dogfish), a representative elasmobranch. Aspartate carbamoyltransferase could not be detected in liver of dogfish. Spleen extracts, however, had glutamine-dependent carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, dihydro-orotase, and glutamine synthetase activities, all localized in the cytosol; dihydro-orotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine-5'-decarboxylase activities were also present. Except for glutamine synthetase, the levels of all activities were very low. The carbamoyl-phosphate synthetase activity is inhibited by UTP and is activated by 5-phosphoribosyl 1-pyrophosphate. The first three enzyme activities of the pyrimidine pathway were eluted in distinctly different positions during gel filtration chromatography under a number of different conditions; although complete proteolysis of inter-domain regions of a multifunctional complex during extraction cannot be excluded, the evidence suggests that in dogfish, in contrast to mammalian species, these three enzymes of the pyrimidine pathway exist as individual polypeptide chains. These results: (1) establish that dogfish express two different glutamine-dependent carbamoyl-phosphate synthetase activities, (2) confirm the report [Smith, Ritter & Campbell (1987) J. Biol. Chem. 262, 198-202] that dogfish express two different glutamine synthetases, and (3) provide indirect evidence that glutamine may not be available in liver for biosynthetic reactions other than urea formation.     

Global population structure of the spiny dogfish Squalus acanthias,a temperate shark with an antitropical distribution

The spiny dogfish (Squalus acanthias) is a temperate, coastal squaloid shark with an antitropical distribution in the Atlantic and Pacific oceans. The global population structure of this species is poorly understood, although individuals are known to undergo extensive migrations within coastal waters and across ocean basins. In this study, an analysis of the global population structure of the spiny dogfish was conducted using eight polymorphic nuclear microsatellite markers and a 566‐bp fragment of the mitochondrial ND2 gene region. A low level of genetic divergence was found among collections from the Atlantic and South Pacific basins, whereas a high level of genetic divergence was found among Pacific Ocean collections. Two genetically distinct groups were recovered by both marker classes: one exclusive to North Pacific collections, and one including collections from the South Pacific and Atlantic locations. The strong genetic break across the equatorial Pacific coincides with major regional differences in the life‐history characters of spiny dogfish, suggesting that spiny dogfish in areas on either side of the Pacific equator have been evolving independently for a considerable time. Phylogeographic analyses indicate that spiny dogfish populations had a Pacific origin, and that the North Atlantic was colonized as a result of a recent range expansion from the South American coast. Finally, the available data strongly argue for the taxonomic separation of the North Pacific spiny dogfish from S. acanthias and a re‐evaluation of the specific status of S. acanthias is warranted.     

Identification and localization of a dogfish homolog of human cystic fibrosis transmembrane conductance regulator.

Chloride channels in the apical plasma membrane of cells in the dogfish rectal gland have served as a model system for the study of regulation of chloride flux by changes in intracellular cyclic AMP levels. Similar regulation by cyclic AMP has been described for channels in cells of human secretory epithelia where defective regulation by cyclic AMP-dependent protein phosphorylation is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). We have isolated a cDNA clone from the rectal gland encoding a protein that is 72% identical to the human CFTR. One of the major phosphorylation sites in CFTR is absent in the dogfish protein. The dogfish protein has, however, four additional putative substrate sites for the cyclic AMP-dependent protein kinase. A peptide antibody, which was raised against an amino acid sequence common to both the human and dogfish CFTR sequences, recognizes proteins with similar molecular masses (160 kDa) in the dogfish gland and in mammalian lung. Immunolocalization studies with this antibody show that the putative dogfish CFTR is localized to the apical membrane of cells lining the lumen of the rectal gland.     

Molecular cloning and sequencing of the banded dogfish (Triakis scyllia) interleukin-8 cDNA

The dogfish (Triakis scyllia) interleukin-8 (IL-8) cDNA was isolated from mitogen-stimulated peripheral white blood cells (WBCs) utilising the polymerase chain reaction (PCR). The cDNA sequence showed that the dogfish IL-8 clones contained an open reading frame encoding 101 amino acids. A short 5' untranslated region (UTR) of 70 nucleotides and a long 3' UTR of 893 nucleotides were also present in this 1.2-kb cDNA. Furthermore, the 3' UTR of the mRNA contained the AUUUA sequence that has been implicated in shortening of the half-life of several cytokines and growth factors. The predicted IL-8 peptide had one potential N-linked glycosylation site (Asn-72-Thr-74) that is not conserved in other vertebrates. It also contained four cysteine residues (Cys-34, 36, 61 and 77), which are characteristic of CXC subfamily cytokines and found in all vertebrates, to date. The dogfish IL-8 lacked an ELR motif as found in the lamprey and trout. Comparison of the deduced amino acids showed that the dogfish IL-8 sequence shared 50.5, 41.2, 37.1 and 40.4-45.5% identity with the chicken, lamprey, trout and mammalian IL-8 sequences, respectively.     

The effects of dogfish MSH''s and of corticotrophin-like intermediate lobe peptides (CLIP''s) on avoidance behavior in rats

Recently purified melanocyte-stimulating hormones (MSH's) from dogfish pituitary tissue were tested on extinction of a conditioned avoidance response (CAR). Corticotrophin like intermediate lobe peptides (CLIP's) from dogfish and porcine origin were tested for an effect on avoidance extinction as well. All peptides appeared to delay extinction of the CAR.The results suggest that the pituitary contains various peptides which influence adaptive behavior. The observation that MSH is more potent in delaying extinction of the CAR then CLIP leads to the conclusion that the behavioral active sequence of the ACTH molecules is located in the N terminal part rather than in the C terminal part of the polypetide.