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Quantitative anatomical and behavioral analyses of regeneration and collateral sprouting following spinal cord transection in the nurse shark (ginglymostoma cirratum).

The spinal cord was transected at the mid-thoracic level in 32 nurse sharks. Four animals per group were sacrificed at intervals of 10, 20, 30, 40, 60 and 90 days postoperative. Two groups of fish underwent a subsequent spinla1 cord retransection at the same site at 90 days and were sacrificed 10 and 20 days later. Three sections of spinal cord were removed from each shark for histological analysis. Behaviorally, timed trials for swimming speed and a strength test for axial musculature contraction caudal to the lesion site were performed at 5 day postoperative intervals. Histological analysis showed little regeneration (9-13 percent) of two descending tracts 90 days following the lesion and no return of rostrally controlled movements caudal to the lesion. However, synaptic readjustment did occur caudal to the lesion. This phenomenon was attributed to local segmental sprouting of adjacent, intact nerve fibers. A close correlation was shown between this synaptic readjustment and the strength of uncontrollable undulatory movements seen caudal to the lesion site following spinal cord transection. The relationship of regeneration and collateral sprouting to quantitative behavioral changes is discussed.

Animals↗

[A macroscopical study of the nervus glossopharyngeus of shark (Mustelus manazo) (author's transl)].

Branches of the Nervus glossopharyngeus of shark Mustelus manazo, one of the most typically retained branchial nerves, is studied macroscopically with a hope of comprehension of its original morphological meanings. There were found some anastomosis among the terminal branches of the Ramus pharyngeus dorsalis of this nerve and those of the Nervus facialis, which, though in the fine caliber of branches, probably could be considered as prototype of the JACOBSON's anastomosis encountered in other vertebrates. In addition to the hitherto reported different content of their nerve components Ramus pretrematicus and posttrematicus of the N. glossopharyngeus of the shark take essentially different courses from each other, especially in relation to the Arteria efferentis branchialis:the former courses always medial to this artery, while the latter is situated laterally to this artery in the branchial arch. The Ramus pretrematicus, in this regard, seemed rather to be equivalent to the Ramus comitans internus a. branchialis of the intermediate branches of the N. glossopharyngeus. Filamentous branches were observed to be emitted from the Ramus externus comitans a. branchialis and distributed to the gill lamellae.

Animals↗

Methods for transport and long-term maintenance of spiny dogfish sharks.

Spiny dogfish shark (Squalus acanthias) embryos were transported from Maine to the Maryland laboratory where they were maintained for 20 months. During this maintenance period, these sharks progressed from a yolk-sac stage to feeding stage. Animals were maintained at 10 degrees C in artificial seawater containing penicillin and streptomycin and were fed frozen brine shrimp and krill. The ease with which these animals could be maintained in the laboratory for an extended period of time greatly enhanced their usefulness as an experimental animal in a variety of studies.

Animal Husbandry↗

A biophysical, morphological and morphometrical survey of the eye of the small shark (Hemiscyllium plagiosum).

The biophysical and morphological characteristics of the small shark's ( Hemiscyllium plagiosum ) eye were studied. The focal lengths of the lens were found to be very short. A small proportion of cones were present in the retina and this proportion varied with the weights of the sharks. The topographic distribution of cones in different segments of the retina was also presented.

Animals↗

[A comparative characteristic of DNA pyrimidine sequences of shark, protopterus and perch].

Pyrimidine sequences of DNA from three fishes: shark, protopterus and perch have been studied. These data together with the evidence from the literature were used to support earlier conclusions that dipnoi and cartilagenous fishes should be distinguished as independent classes. The clustering index, beta, and the total molar percentage of long pyrimidine oligonucleotides (Z) containing greater than or equal to 8 nucleotides--a new parameter offered by us--have been used in comparative investigation of DNAs. The new parameter has permitted to obtain a higher resolution in the analysis of our own and literature data on DNA pyrimidine clusters in fishes. Investigation of pyrimidine clusters and of the base composition of individual isoplits of these clusters using statistical analysis showed that DNA from shark, protopterus, sturgeon and perch significantly differ by many features. Significant differences between these DNAs were found also in the base composition. Thus, new evidence for distinguishing cartilagenous fishes and dipnoi as independent classes have been received.

Animals↗

Production and characterization of monoclonal antibodies to shark cartilage proteoglycan.

1. Two proteoglycans, PG1 and PG2, have been isolated from shark cartilage. Both are highly polydisperse and large (molecular mass: 1-10 x 10(6) Daltons) and contain chondroitin sulfate and keratan sulfate side chains, but PG2 is somewhat smaller than PG1 and contains less keratan sulfate. 2. Monoclonal antibodies were raised against PG1. Many antibodies were obtained and one of them, MST1, was subcloned and further characterized. This monoclonal antibody reacts with PG1 and PG2 from shark cartilage and also with aggrecan from bovine trachea cartilage. Chondroitinase AC-treated proteoglycans react with MST1, indicating that the antibody does not recognize chondroitin sulfate. MST1 also recognizes aggrecan from human cartilage and a proteoglycan from bovine brain (neurocan) but it does not recognize proteoglycans from rat Walker tumor, fetal calf muscle and decorin from human myoma. 3. Using MST1 we were able to demonstrate that both PG1 and PG2 aggregate with hyaluronic acid.

Animals↗

Diversity of Ig light chain clusters in the sandbar shark (Carcharhinus plumbeus).

The genes encoding Ig chains in elasmobranchs are arranged in clusters or cassettes, an organizational pattern dramatically different from the mammalian translocon gene arrangement. Cluster gene arrangements, which have now been found in non-elasmobranchs as well, pose interesting dilemmas for understanding the mechanisms of Ig gene expression and regulation in terms of allelic exclusion and clonal selection. We have sequenced five lambda genomic clones encoding complete sandbar shark (Carcharhinus plumbeus) lambda L chain gene loci. While the coding regions among all five clones are highly homologous, the noncoding regions have significant differences that allowed us to identify two types of lambda L chain clusters. The noncoding regions are < 60% identical between groups, while the three clones belonging to the first group share > 95% identity in their noncoding regions. The second group is more diverse and may be comprised of several related subgroups. The two clones in this group share approximately 85% identity in the noncoding regions. Variations in the promoter region, including octamer and TATA box orientation and position, are identified between the two groups and may have implications for the molecular regulation of Ab production. Our results show the sandbar shark lambda L chain family to be a complex and diverse system.

Amino Acid Sequence↗

A comparison of mode of attachment and histopathogenicity of four tapeworm species representing two orders infecting the spiral intestine of the nurse shark, Ginglymostoma cirratum.

This study was undertaken to compare 2 species of Tetraphyllidea and 2 species of Trypanorhyncha with regard to the relationship between attachment structure morphology, mode of attachment, and tapeworm size, to damage at the sites of attachment in the Atlantic nurse shark, Ginglymostoma cirratum. Regions of the spiral intestine with worms attached were removed from 8 nurse sharks and sectioned according to conventional techniques. Sections of 5-50 specimens of each tapeworm species were examined. Regions of the spiral intestine devoid of worms were processed for characterization of the normal mucosa. The normal mucosa was found to consist of a folded surface covered with round-to-oval primary mucosal crypts. In the first 7 or 8 chambers of the spiral intestine the mucosal surface was thrown into secondary folds, forming ridges and secondary crypts. The primary mucosal crypts were lined with a single layer of columnar epithelium resting on a basement membrane. A highly cellular lamina propria and submucosa were found between the crypts and the muscularis mucosa. The small tetraphyllidean Pedibothrium brevispine was found with its scolex lying within the primary mucosal crypts with its hooks embedded in the basement membrane. Epithelial denudation was evident. The large tetraphyllidean Pedibothrium globicephalum was found with its bothridia engulfing large portions of the mucosa and its hooks embedded into the lamina propria. It was associated with moderate to severe mucosal necrosis. The small trypanorhynch Prochristianella tenuispine was found lying between the mucosal ridges in the secondary crypts with its tentacles either penetrating the epithelium, or occasionally, the lamina propria.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Anti-inflammatory and analgesic activity of a water-soluble fraction from shark cartilage.

The anti-inflammatory and analgesic activities of a water-soluble fraction (WSF), extracted with 0.1 M ammonium bicarbonate, pH 8.0, from shark cartilage were studied in several experimental models. Orally administered WSF (10 mg/kg) caused 25.7 and 23.6% inhibition of the paw edema produced in female Wistar rats (200-250 g) by carrageenan and dextran, respectively, after 3 h, as compared to controls. WSF administered orally had no effect on acetic acid-induced writhings in male Swiss mice (25-30 g) at the dose of 0.01 mg/kg but caused 52.8 and 61.4% inhibition at the doses of 0.1 and 0.5 mg/kg, respectively, compared to controls (No. of writhings/20 min, means +/- SEM: treated groups = 18.6 +/- 2.5, N = 12 and 15.2 +/- 1.4, N = 12, respectively; controls = 39.3 +/- 1.3, N = 77). In the formalin test (male Swiss mice, 25-30 g), orally administered WSF (0.5 and 1 mg/kg) caused 12.0 and 46.6% inhibition of licking time, respectively, only in the 2nd phase (inflammatory) of the test (licking time, means +/- SEM: treated group = 18.3 +/- 4.4 sec, N = 7 and 11.1 +/- 3.4 sec, N = 13; controls = 20.8 +/- 2.4 sec, N = 44). The results suggest that a molecule of a protein nature in shark cartilage is probably responsible for the effects observed.

Administration, Oral↗

Occurrence and morphological comparisons of Campula oblonga (Digenea: Campulidae), including a report from an atypical host, the thresher shark, Alopias vulpinus.

New host records for Campula oblonga Cobbold, 1858 from the common dolphin, Delphinus delphis L., and from the thresher shark, Alopias vulpinus (Bonnaterre), are reported herein. Campulids have not been reported previously from a host that was not a marine mammal. The excellent condition and small size of the gravid specimen and the diet and natural history of the thresher shark lead us to conclude that the digene was acquired from the consumption of infected fish. Specimens of C. oblonga from these 2 hosts and from harbor porpoises, Phocoena phocoena (L.), and from Dall's porpoises, Phocoenoides dalli (True), are compared, and a wide variation in ranges and values for characters is noted. The synonymy of Campula folium Ozaki, 1935 with C. oblonga is further supported by the apparent effect of the host on the size of the trematodes. Due to the degree of variation in measurements and the numerous factors that may impact these values, we recommend the use of qualitative morphologic characters for the identification of C. oblonga.

Animals↗

Chloride secretagogues stimulate inositol phosphate formation in shark rectal gland tubules cultured in suspension.

Neuroendocrine activation of transepithelial chloride secretion by shark rectal gland cells is associated with increases in cellular cAMP, cGMP, and free calcium concentrations. We report here on the effects of several chloride secretagogues on inositol phosphate formation in cultured rectal gland tubules. Vasoactive intestinal peptide (VIP), atriopeptin (AP), and ionomycin increase the total inositol phosphate levels of cultured tubules, as measured by ion exchange chromatography. Forskolin, a potent chloride secretagogue, has no effect on inositol phosphate formation. The uptake of 3H-myo-inositol into phospholipids is very slow, preventing the detection of increased levels of inositol trisphosphate. However, significant increases in inositol monophosphate (IP1) and inositol biphosphate (IP2) were measured. The time course of VIP- and AP-stimulated IP1 and IP2 formation is similar to the effects of these agents on the short-circuit current responses of rectal gland monolayer cultures. In addition, aluminum fluoride, an artificial activator of guanine nucleotide-binding proteins, stimulates IP1 and IP2 formation. We conclude that rectal gland cells contain VIP and AP receptors coupled to the activation of phospholipase C. Coupling may be mediated by G-proteins. Receptor-stimulated increases in inositol phospholipid metabolism is one mechanism leading to increased intracellular free calcium concentrations, an important regulatory event in the activation of transepithelial chloride secretion by shark rectal gland epithelial cells.

Aluminum↗

C-type natriuretic peptides are potent dilators of shark vascular smooth muscle.

Previous studies of the effects of C-type natriuretic peptides (CNP) in intact mammals have demonstrated limited hypotensive responses, in contrast to other natriuretic peptides. Our previous studies, on isolated vascular smooth muscle (VSM) from various fish species, utilizing either mammalian or non-homologous fish atrial natriuretic peptides (ANP), have demonstrated vasodilation with a relatively high sensitivity (EC50 approximately 5 nM). The recent sequencing of a C-type natriuretic peptide from the heart of the dogfish shark, Squalus acanthias, has enabled us to compare the efficacy of this peptide on aortic VSM from that species with two other CNPs (from killifish and pig), as well as rat ANP. The EC50 of dilation for sCNP, as well as kCNP and pCNP, was 0.5 nM, over 15 times lower than the EC50 of the response to rANP. These data suggest that CNP is released from the dogfish shark heart and is a circulating hormone with potent vasodilatory effects, in sharp contrast to the apparent role of CNP predominantly as a brain neuropeptide in mammals.

Animals↗

Purification, characterization, and biological activity of insulins from the spotted dogfish, Scyliorhinus canicula, and the hammerhead shark, Sphyrna lewini.

Insulin was purified from pancreatic extracts of two elasmobranch species belonging to different families in the order Carcharhiniformes, the European spotted dogfish, Scyliorhinus canicula (Scyliorhinidae), and the hammerhead shark, Sphyrna lewini (Carcharhinidae). The amino acid sequence of dogfish insulin was established as A-chain GIVDHCCRNT(10)CSLYDLEGYC(20)NQ and B-chain LPSQHLCGSH(10)LVETLYFVCG(20)QKGFYYVPKV(30). The primary structure of hammerhead shark insulin was similar to that of dogfish insulin with only 2 amino acid substitutions at A8 (R --> H) and B30 (V --> I). The elasmobranch insulins were markedly different from human insulin (17 amino acid substitutions) but all the residues in human insulin that are believed to be important in determining the receptor binding conformation (B6, B8, B11, B13, B23, B24, B25, A2, A3, and A19) have been conserved in the elasmobranch insulins with the exception of the conservative substitution Phe --> Tyr at B25. Consistent with this, dogfish and human insulin showed almost identical binding affinity to the recombinant solubilized human insulin receptor (K(D) values of 14.0 and 18.6 pM, respectively; relative potency 133%). Previous studies have shown that bovine insulin produces severe and sustained hypoglycemia in elasmobranchs but the effect is of slow onset. Bolus arterial injections of dogfish insulin (10 nmol x kg(-1)) into unanesthetized, fasting dogfish (n = 9) produced no changes in blood glucose, 3-hydroxybutyrate, and acetoacetate concentrations over a 4-h period. In a second series of experiments (n = 7), dogfish insulin (10 nmol x kg(-1)) produced a significant (P < 0.05) fall in blood glucose after 12 h that persisted for at least 48 h, but no change in ketone body concentrations. The data indicate that the metabolic actions of an endogenous elasmobranch insulin in an elasmobranch are similar to those previously described for mammalian insulin.

3-Hydroxybutyric Acid↗

ETB-type receptors mediate endothelin-stimulated contraction in the aortic vascular smooth muscle of the spiny dogfish shark, Squalus acanthias.

Using various agonists, and the specific antagonist BQ-123, we have examined the sensitivity to endothelin of the vascular smooth muscle of the ventral aorta of the spiny dogfish shark, Squalus acanthias. Human endothelin-1 produced significant contraction of isolated vascular smooth muscle rings, with an EC50 of 10 nmol.l-1. The presence of an intact endothelium did not alter this response but the magnitude of the contraction was greater in rings with an intact endothelium. The response to 0.2 mumol.l-1 endothelin-1 was equivalent to that of 0.1 mmol.l-1 acetylcholine, and significantly greater than that to 80 mmol.l-1 KCl, suggesting high sensitivity even to the heterologous, mammalian peptide. The Hill plot of the contractile response was a straight line with a slope of 1.12, indicating that a single receptor was mediating the response. Endothelin-1, endothelin-3, and sarafotoxin S6c produced similar concentration-response curves, and the response to endothelin-1 was insensitive to the ETA-specific inhibitor BQ-123. Our data are consistent with the hypothesis that the receptor involved in the contractile response to endothelin of shark aortic vascular smooth muscle is of the ETB-rather than the ETA-type.

Animals↗

Complementary DNA Encoding nm23/NDP Kinase Gene from the Korean Tiger Shark Scyliorhinus torazame.

: A new tumor suppressor gene, snm23, homologous to the gene for human nucleoside diphosphate kinase nm23/NDP was first cloned from Korean tiger shark (Scyliorhinus torazame) skin lambda ZAP-II complementary DNA library. The gene (named snm23) containing the tumor metastasis suppressor protein was sequenced. The nucleotide and deduced amino acid sequences of snm23 revealed an open reading frame of 450 bp that corresponded to a protein of 150 amino acid residues, with a calculated molecular mass of 16.8 kDa. Sequence comparison of snm23 with nm23/NDP kinases was performed. In order to determine tissue specificity, reverse transcription-polymerase chain reaction was used. The expression of snm23/NDP kinase was detected in tissues from skin, cartilage, and liver of Korean tiger shark.

Journal Article↗

Cloning of sgk serine-threonine protein kinase from shark rectal gland – a gene induced by hypertonicity and secretagogues.

Recently, the cell-volume-regulated serine-threonine protein kinase h-sgk was cloned from a human hepatoma cell line. The sgk gene was shown to be induced by cell shrinkage in many different mammalian cell lines. In this study, two highly conserved serine-threonine protein kinases, sgk-1 and sgk-2, were cloned from rectal gland tissue of the spiny dogfish (Squalus acanthias). Both kinases showed a distinct pattern of tissue specificity, with high expression levels in kidney, intestine, liver and heart. In rectal gland slices sgk-1 transcription was induced by exposure to hypertonic solution, reduction of the extracellular urea concentration, and addition of the secretagogues vasoactive intestinal polypeptide (VIP) and carbachol. The shark sgk-1 serine-threonine protein kinase may therefore provide a link between cell volume, Cl–secretion and protein phosphorylation state in shark rectal gland cells.

Amino Acid Sequence↗

Occlusion of Rb+ by detergent-solubilized (Na+ + K+)-ATPase from shark salt glands.

Occlusion of Rb+ by C12E8-solubilized (Na+ + K+)-ATPase from shark salt glands has been measured. The rate of de-occlusion at room temperature is about 1 s-1, which is the same as for the membrane-bound enzyme. The amount of Rb+ occluded is 3 moles Rb+ per mole membrane-bound shark enzyme, whereas only about 2 moles Rb+ are occluded by the C12E8-solubilized enzyme.

Animals↗

Properties of oligomycin-induced occlusion of Na+ by detergent-solubilized Na,K-ATPase from pig kidney or shark rectal gland.

Oligomycin induces occlusion of Na+ in membrane-bound Na,K-ATPase. Here it is shown that Na,K-ATPase from pig kidney or shark rectal gland solubilized in the nonionic detergent C12E8 is capable of occluding Na+ in the presence of oligomycin. The apparent affinity for Na+ is reduced for both enzymes upon solubilization, and there is an increase in the sigmoidicity of binding curves, which indicates a change in the cooperativity between the occluded ions. A high detergent/protein ratio leads to a decreased occlusion capacity. De-occlusion of Na+ by addition of K+ is slow for solubilized Na,K-ATPase, with a rate constant of about 0.1 s-1 at 6 degrees C. Stopped-flow fluorescence experiments with 6-carboxyeosin, which can be used to monitor the E1Na-form in detergent solution, show that the K(+)-induced de-occlusion of Na+ correlates well with the fluorescence decrease which follows the transition from the E1Na-form to the E2-form. There is a marked increase in the rate of fluorescence change at high detergent/protein ratios, indicating that the properties of solubilized enzyme are subject to modification by detergent in other respects than mere solubilization of the membrane-bound enzyme. The temperature dependence of the rate of de-occlusion in the range 2 degrees C to 12 degrees C is changed slightly upon solubilization, with activation energies in the range 20-23 kcal/mol for membrane-bound enzyme, increasing to 26-30 kcal/mol for solubilized enzyme. Titrations of the rate of transition from E1Na to E2K with oligomycin can be interpreted in a model with oligomycin having an apparent dissociation constant of about 2.5 microM for C12E8-solubilized shark Na,K-ATPase and 0.2 microM for solubilized pig kidney Na,K-ATPase.

Animals↗