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PubMed1984

Distribution of different molecular species of collagen in the vertebral cartilage of shark (Carcharius acutus).

It is known that cartilage collagen in higher vertebrates conforms to Type II collagen but very little is known of the nature of shark cartilage. This study was undertaken to determine the differences, if any, between shark cartilage collagen and that of higher vertebrates. Collagen was obtained from shark cartilage by pepsin solubilization and characterized by amino acid analysis and determination of chain composition by SDS-polyacrylamide gel electrophoresis and CM-cellulose chromatography. Results indicated the presence not only of Type II collagen but also of Type I collagen. Type I collagen accounted for about one third of the total collagen content of shark cartilage.

Amino Acids↗
PubMed2002

Proteasome, transporter associated with antigen processing, and class I genes in the nurse shark Ginglymostoma cirratum: evidence for a stable class I region and MHC haplotype lineages.

Cartilaginous fish (e.g., sharks) are derived from the oldest vertebrate ancestor having an adaptive immune system, and thus are key models for examining MHC evolution. Previously, family studies in two shark species showed that classical class I (UAA) and class II genes are genetically linked. In this study, we show that proteasome genes LMP2 and LMP7, shark-specific LMP7-like, and the TAP1/2 genes are linked to class I/II. Functional LMP7 and LMP7-like genes, as well as multiple LMP2 genes or gene fragments, are found only in some sharks, suggesting that different sets of peptides might be generated depending upon inherited MHC haplotypes. Cosmid clones bearing the MHC-linked classical class I genes were isolated and shown to contain proteasome gene fragments. A non-MHC-linked LMP7 gene also was identified on another cosmid, but only two exons of this gene were detected, closely linked to a class I pseudogene (UAA-NC2); this region probably resulted from a recent duplication and translocation from the functional MHC. Tight linkage of proteasome and class I genes, in comparison with gene organizations of other vertebrates, suggests a primordial MHC organization. Another nonclassical class I gene (UAA-NC1) was detected that is linked neither to MHC nor to UAA-NC2; its high level of sequence similarity to UAA suggests that UAA-NC1 also was recently derived from UAA and translocated from MHC. These data further support the principle of a primordial class I region with few class I genes. Finally, multiple paternities in one family were demonstrated, with potential segregation distortions.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

J chain in the nurse shark: implications for function in a lower vertebrate.

J chain is a small polypeptide covalently attached to polymeric IgA and IgM. In humans and mice, it plays a role in binding Ig to the polymeric Ig receptor for transport into secretions. The putative orthologue of mammalian J chain has been identified in the nurse shark by sequence analysis of cDNA and the polypeptide isolated from IgM. Conservation with J chains from other species is relatively poor, especially in the carboxyl-terminal portion, and, unlike other J chains, the shark protein is not acidic. The only highly conserved segment in all known J chains is a block of residues surrounding an N-linked glycosylation site. Of the eight half-cystine residues that are conserved in mammalian J chains, three are lacking in the nurse shark, including two in the carboxyl-terminal segment that have been reported to be required for binding of human J chain-containing IgA to secretory component. Taken together with these data, the relative abundance of J chain transcripts in the spleen and their absence in the spiral valve (intestine) suggest that J chain in nurse sharks may not have a role in Ig secretion. Analysis of J chain sequences in diverse species is in agreement with accepted phylogenetic relationships, with the exception of the earthworm, suggesting that the reported presence of J chain in invertebrates should be reassessed.

Amino Acid Sequence↗
PubMed2001

Understanding the mechanism of action of cytochrome c oxidase in normal and disease states: shark heart enzyme, a potential model.

Cytochrome c oxidase, the final member of the electron transport chain, is crucial to respiration and also contributes to the synthesis of cellular ATP. The total absence of this enzyme is incompatible with life and its deficiency or malfunction leads to a number of serious disease states. Understanding the mechanism of action of this enzyme, which is an important prerequisite to unravelling its role in the pathogenesis of disease states, is hampered by the lack of suitable enzyme models. The bovine enzyme, which is commonly used, is enormously complex and the bacterial enzymes, which are structurally simple, appear to follow a different mechanism of action. The hammer head shark is a seasonal resident of the warm waters of the Caribbean Sea. The work presented here indicates that, like the bovine enzyme, the enzyme of the heart of this shark (i) possesses thirteen subunits and two substrate binding sites and (ii) exhibits biphasic kinetics. The work also confirms that, unlike the bovine enzyme which is dimeric, the shark enzyme functions as a monomer. Given this latter simplifying feature, in conjunction with its kinetic and structural similarities to the more complex mammalian varieties, we propose that shark heart cytochrome c oxidase replace the bovine and bacterial forms as the enzyme of choice for model studies.

Animals↗
PubMed1990

Shark repellent: not yet, maybe never.

An effective life jacket shark repellent packet remains an officially stated, unsatisfied requirement of the sea and air military services. Shark Chaser of World War II, a useful psychological crutch for the times, has been deemed inefficacious, and its procurement has been cancelled. Systems analyses clearly indicate inherent, irreconcilable incompatability between stringent military packet specifications and limited capabilities of known candidate chemicals to evoke rapid diminution of aggressive shark behavior. Guidelines for realistic estimates of minimum acceptable potencies are prescribed for future research on shark repellents. Prospective use of semiochemicals is considered, and corresponding changes in current research directions are suggested.

Animals↗
PubMed1989

Gastric evacuation of the lemon shark Negaprion brevirostris (Poey) under controlled conditions.

Gastric evacuation of the juvenile lemon shark, Negaprion brevirostris, a tropical inshore apex predator, was studied in the laboratory under conditions of 25 degrees C and 32% salinity. Sharks were starved for 48-96 h after which they were fed a 2.7% body weight meal of fish fillet. Gastric evacuation was assessed by stomach lavage every 3 h for 24 h thereafter. Results showed that the gut is emptied in about 24 h with fillets being removed from the stomach following the equation Y = Yoe-bt, where Y is percent of meal recovered at time t, Yo is the initial meal size, and b is the instantaneous rate of evacuation. The exponential function of gastric evacuation in the lemon shark is similar to that in teleosts, but the kinetics are slower. The caloric density of the meal remaining in the stomach increased, approaching that of protein in the first 12 h after feeding, as carbohydrates (glycogen) were digested. After 12 h the caloric value dropped as protein was digested, suggesting an orderly and efficient intake of food energy by the lemon shark.

Animals↗
PubMed1993

Fine structure of the term umbilical cord in the Atlantic sharpnose shark, Rhizoprionodon terraenovae.

The fine structure of the umbilical cord and appendiculae in the Atlantic sharpnose shark, Rhizoprionodon terraenovae, is examined by light, scanning and transmission electron microscopy. During ontogeny of placental sharks, the yolk sac and stalk become progressively modified as a functional hematrophic placenta and umbilical cord respectively. In most placental sharks the umbilical cord is smooth. In the Atlantic sharpnose shark, the epithelial ectoderm of the somatopleure forms richly vascularized extensions termed appendiculae. Scanning electron microscopy reveals that the base and shaft of appendiculae are flattened while the distal portion may be expanded to form one to three lobes. The surface of appendiculae is composed of two distinct cell types, the most plentiful are microvillar cells. The second cell type contains prominent granules. These cells are much larger than the former and are partially submerged below the surface, except for the cell apex. These cells undergo secretory cycles ending in expulsion of their contents. The possible function of the granulated cells is discussed. The umbilical cord contains an umbilical vein, umbilical artery, ductus vitellointestinalis and extraembryonic coelom. The endodermal ductus initially conveys yolk from the yolk sac to the fetal gut by activity of ciliated cells lining it. The ductus persists in the adult. Microvillar cells, also present in the ductus, may play a role in the absorption of yolk metabolites early in development, prior to yolk depletion. Enteroendocrine cells are wedged between the ciliated and microvillar cells. These cells may exert paracrine regulation of local areas of the ductus.

Animals↗
PubMed1996

Shark-cartilage containing preparation protects cells against hydrogen peroxide induced damage and mutagenesis.

Natural products from flora and fauna are frequently used as nutritional supplements and medicaments. Two short-term assays were carried out and negative results were obtained for shark-cartilage containing preparation. The tests employed were the Salmonella/mammalian microsome assay using tester strains TA97, TA98, TA100, TA102 and TA1535 with or without S9 mix and the SOS-Chromotest with Escherichia coli strain PQ37. Evidence for shark-cartilage containing preparation functioning as an antimutagen was detected. Using bacterial survival assays with Escherichia coli fpg (BH20) and xthA (BW9091), we investigated the putative role of shark-cartilage containing preparation in protecting cells against lesions induced by hydrogen peroxide in normal and low iron level conditions. Our data suggest that shark-cartilage containing preparation can play a scavenger role for reactive oxygen species and protect against DNA lesions in both conditions.

2,2'-Dipyridyl↗
PubMed2004

Ionic strength and the polyvalent cation receptor of shark rectal gland and artery.

The dogfish shark Squalus acanthias regulates plasma osmolality and extracellular volume by secreting a fluid from its rectal gland which has a higher NaCl and lower urea concentration than plasma. We have previously identified the presence of a calcium-sensing receptor or polyvalent cation sensing receptor (CaSR) on vascular smooth muscle of the rectal gland artery (RGA) and rectal gland tubules (RGT). Activity of the CaSR is influenced by changes in ionic strength. This led us to speculate that the ingestion of invertebrate sea animals increased plasma ionic strength, resulting in inhibition of the receptor, relaxation of RGA, and reversal of inhibition of chloride secretion by the RGT. In contrast, ingestion of fish could diminish ionic strength and have the opposite effect. To study the effect of changes in extracellular ionic strength, shark Ringers solutions were adjusted to three different ionic strengths with NaCl, but the osmolarities were kept constant by varying the concentration of urea. High ionic strength inhibited and low ionic strength enhanced the response to increasing external Ca2+ from 2.5 to 4.7 mM in RGT. The increase in cytosolic Ca2+ ([Ca2+]i) of cells in low, normal, and high ionic strength Ringers solution was 344 +/- 60, 201 +/- 26, and 114 +/- 15 nmol/L, respectively. The [Ca2+]i responses of RGA to external Ca2+ in Ringers of three different ionic strengths were 323 +/- 43, 231 +/- 14, and 56 +/- 11 nmol/L, respectively. Activation of the CaSR by spermine was reduced by more than 50% by high ionic strength in both RGT and RGA. Whether the small changes in shark plasma ionic strength that occur after a shark ingests marine animals with lower and higher ionic strength modulates salt secretion by the rectal gland is not yet known.

Animals↗
PubMed2006

Mercury toxicity in the shark (Squalus acanthias) rectal gland: apical CFTR chloride channels are inhibited by mercuric chloride.

In the shark rectal gland, basolateral membrane proteins have been suggested as targets for mercury. To examine the membrane polarity of mercury toxicity, we performed experiments in three preparations: isolated perfused rectal glands, primary monolayer cultures of rectal gland epithelial cells, and Xenopus oocytes expressing the shark cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. In perfused rectal glands we observed: (1) a dose-dependent inhibition by mercury of forskolin/3-isobutyl-1-methylxanthine (IBMX)-stimulated chloride secretion; (2) inhibition was maximal when mercury was added before stimulation with forskolin/IBMX; (3) dithiothrietol (DTT) and glutathione (GSH) completely prevented inhibition of chloride secretion. Short-circuit current (Isc) measurements in monolayers of rectal gland epithelial cells were performed to examine the membrane polarity of this effect. Mercuric chloride inhibited Isc more potently when applied to the solution bathing the apical vs. the basolateral membrane (23 +/- 5% and 68 +/- 5% inhibition at 1 and 10 microM HgCl2 in the apical solution vs. 2 +/- 0.9% and 14 +/- 5% in the basolateral solution). This inhibition was prevented by pre-treatment with apical DTT or GSH; however, only the permeant reducing agent DTT reversed mercury inhibition when added after exposure. When the shark rectal gland CFTR channel was expressed in Xenopus oocytes and chloride conductance was measured by two-electrode voltage clamping, we found that 1 microM HgCl2 inhibited forskolin/IBMX conductance by 69.2 +/- 2.0%. We conclude that in the shark rectal gland, mercury inhibits chloride secretion by interacting with the apical membrane and that CFTR is the likely site of this action.

1-Methyl-3-isobutylxanthine↗
PubMed1996

Functional expression of recombinant spiny dogfish shark (Squalus acanthias) cytochrome P450c17 (17 alpha-hydroxylase/C17,20-lyase) in yeast (Pichia pastoris).

The cDNA encoding the spiny dogfish shark (Squalus acanthias) testicular form of cytochrome P450c17 (CYP17) was used to direct the heterologous expression of a functional enzyme in yeast (Pichia pastoris). This protein possesses two enzymatic activities: 17 alpha-hydroxylase and C17,20-lyase reactions. Cytochrome P450c17 is a key steroidogenic enzyme for the production of sex steroids in gonadal tissue and for cortisol production in adrenal tissue. This study describes the culture conditions and the enzymatic activity of recombinant shark cytochrome P450c17. The shark enzyme was compatible with the endogenous yeast NADPH-cytochrome P450 reductase and was bioactive within the living yeast cell. Progesterone (at 15 microM) was metabolized (51 pmol/min/10(9) cells) faster than pregnenolone (36 pmol/min/10(9) cells). Both progesterone and pregnenolone were completely metabolized to their respective androgens (androstenedione and dehydroepiandrosterone). Although 11 beta-hydroxy-progesterone was readily 17 alpha-hydroxylated by the shark P450, the lyase reaction was not evident. Alterations to the 2-carbon sidechain of progesterone (21-hydroxylation or 20 beta-reduction) prevented metabolism. High-density cultures (> 1.5 x 10(9) cells/ml) yielded the greatest quantity of recombinant protein but cultures of lower density produced more recombinant protein per cell. This is the first report of heterologous expression in yeast of a steroidogenic cytochrome P450 from a lower vertebrate.

Aldehyde-Lyases↗
PubMed1996

Heterologous expression of the spiny dogfish shark (Squalus acanthias) cytochrome P450c17 (17 alpha-hydroxylase) in Escherichia coli.

Cytochrome P450c17 is a key steroidogenic enzyme for the production of sex steroids in gonadal tissue and for cortisol production in adrenal tissue. This protein possesses two enzymatic activities. The 17-alpha-hydroxylase activity introduces a hydroxyl group into the steroid nucleus. The resultant 17-alpha-hydroxylated, C(21) pregnenes can be converted to a C(19) androgen by the C(17,20)-lyase activity. The cDNA encoding the spiny dogfish shark (Squalus acanthias) testicular form of cytochrome P450c17 was used to direct heterologous expression in Escherichia coli. The wild-type P450c17 protein was not conducive to expression in E. coli using either a pET21 or a pCwori+ vector. However, modification of the amino terminus permitted overexpression of inactive shark protein with the pFT21 vector. This protein was fused with a hexahistidinyl peptide to facilitate purification by column chromatography using a Ni(2+)-chelated resin. Transformed bacteria yielded an average of 1.8 mg of purified, concentrated P450c17 protein per 100-ml culture. This modified protein was used to raise antisera in rabbits and the resultant antisera was used at a working titer of 1:10,000 for Western blot analysis. Culture conditions that result in the accumulation of bioactive recombinant bovine P450c17 failed to demonstrate expression of either the native or the modified form of shark P450c17 using the pCWori+ and pET21 vectors. These results suggest that the amino terminus of the native shark P450c17 was not conducive to synthesis in E. coli. However, modifications of the amino terminus permitted synthesis of an inactive protein that was protected from degradation within inclusion bodies.

Amino Acid Sequence↗
PubMed1991

The origin of high cadmium loads in some bivalve molluscs from Shark Bay, Western Australia: a new mechanism for cadmium uptake by filter feeding organisms.

Although Shark Bay is remote from all known industrial and geological sources of heavy metals, the cadmium content of several species of Shark Bay molluscs may exceed 10 mg/kg. The cadmium load in these molluscs varies geographically within the bay, but possible explanations for cadmium distribution involving variation in salinity, saline groundwater influx, the dissolved cadmium concentration, the cadmium concentration in substrate sediments, species, or an anthropogenic source are not supported by analytical data. The cadmium concentration is normal in Shark Bay seawater (0.04 microgram/L to about 0.35 microgram/L), rarely exceeds 0.25 microgram/L in ground waters, bore waters, and salt lake brines, and very seldom exceeds 1 mg/kg in sediments. No direct link between the cadmium loads in molluscs and its concentration in the water or substrate sediment is evident, but the cadmium load in molluscs is usually highest where turbulence is high and the substrate sediment contains fine hematite. Over about 2,000 km2, the water depth in Shark Bay is less than 1 m and fine sediment is readily suspended by strong winds. The iron-oxides (superfine hematite) are eroded from the Peron Sandstone exposed in some coastal cliffs and constitute up to 2% of substrate sediments near these cliffs. This study reveals that cadmium in the water adsorbs extremely efficiently onto the surface of the hematite, which is negatively charged at the prevailing seawater pH of 8.15, and that suspended hematite particles are ingested by the molluscs. Inside the molluscs, lower pH conditions cause reversal of the hematite charge and the cadmium is released and accumulated by the organism.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2006

Cell and molecular biology of SAE, a cell line from the spiny dogfish shark, Squalus acanthias.

Cartilaginous fish, primarily sharks, rays and skates (elasmobranchs), appeared 450 million years ago. They are the most primitive vertebrates, exhibiting jaws and teeth, adaptive immunity, a pressurized circulatory system, thymus, spleen, and a liver comparable to that of humans. The most used elasmobranch in biomedical research is the spiny dogfish shark, Squalus acanthias. Comparative genomic analysis of the dogfish shark, the little skate (Leucoraja erincea), and other elasmobranchs have yielded insights into conserved functional domains of genes associated with human liver function, multidrug resistance, cystic fibrosis, and other biomedically relevant processes. While genomic information from these animals is informative in an evolutionary framework, experimental verification of functions of genomic sequences depends heavily on cell culture approaches. We have derived the first multipassage, continuously proliferating cell line of a cartilaginous fish. The line was initiated from embryos of the spiny dogfish shark. The cells were maintained in a medium modified for fish species and supplemented with cell type-specific hormones, other proteins and sera, and plated on a collagen substrate. SAE cells have been cultured continuously for three years. These cells can be transfected by plasmids and have been cryopreserved. Expressed Sequence Tags generated from a normalized SAE cDNA library included a number of markers for cartilage and muscle, as well as proteins influencing tissue differentiation and development, suggesting that SAE cells may be of mesenchymal stem cell origin. Examination of SAE EST sequences also revealed a cartilaginous fish-specific repetitive sequence that may be evidence of an ancient mobile genetic element that most likely was introduced into the cartilaginous fish lineage after divergence from the lineage leading to teleosts.

Animals↗
PubMed2002

Use of the 'shark-fin' osteotome in separation of the pterygomaxillary junction in Le Fort I osteotomy: a clinical and computerized tomography study.

Le Fort I osteotomy fails in many cases to completely separate the pterygomaxillary junction and often results in fractures of the pterygoid bone and the tuberosity, which subsequently can cause complications. The objectives of this study were to describe the specifically developed Laster 'shark-fin' osteotome and to compare its use to other methods of pterygomaxillary dysjunction. Pterygomaxillary dysjunction was performed in 10 adult patients requiring Le Fort I osteotomy. In one randomly chosen side of the maxilla, the Obwegeser osteotome was used, while the Laster 'shark-fin' osteotome was used on the opposite side. A postoperative computerized tomography of the separation at the pterygomaxillary junctions revealed that in all sites treated with the Laster 'shark-fin' osteotome, a complete or almost complete separation was obtained, whereas the use of the Obwegeser osteotome resulted in five sites with fractures of the maxillary tuberosity and three with high-level fractures of the pterygoid plates (P<0.001). Comparing these findings with the literature, we concluded that the Laster 'shark-fin' osteotome is preferable for separating the pterygomaxillary junction in Le Fort I osteotomy.

Adolescent↗
PubMed1999

Toxic effects of cadmium and copper on the isolated heart of dogfish shark, Squalus acanthias.

The adverse effects of heavy metal ions on the heart functions of lower vertebrates are largely unknown. In the present study, the effects of Cd2+, Cu2+, and Cu+ on the cardiac functions of the heart isolated from dogfish shark, Squalus acanthias, including the epicardial electrocardiogram, ventricular developed pressure (VDP), and heart beating rate, were studied. Cadmium (10 to 100 microM) significantly decreased VDP of the isolated shark hearts in a reversible manner. However, heart beating rate was not affected by cadmium. Cadmium also induced a transient modification of the amplitude and the form of the QRS complex. Cupric ion transiently increased VDP in a concentration-dependent manner, whereas cuprous ion (1 to 100 microM) did not markedly alter the cardiac functions of shark. Cupric or cuprous ions did not change heart beating rate and electrocardiogram at concentrations of 10 to 100 microM. Our results, for the first time, demonstrated the effects of cadmium on shark heart and indicated that the cardiac effects of copper are valence dependent. An elucidation of heavy metal effects on fish cardiac functions will help to understand the complex toxicological properties of heavy metals in different species and tissues, and will provide information for management of pollution control and marine resource protection.

Animals↗
PubMed1991

High expression of the HNK-1/L2 carbohydrate epitope in the major glycoproteins of shark myelin.

The major 24- and 28-kDa glycoproteins in shark PNS and CNS myelin express high levels of the adhesion-associated HNK-1/L2 carbohydrate epitope. The 28-kDa protein, but not the 24-kDa protein, cross-reacts strongly with one of two anti-bovine P0 antisera not previously tested against fish myelin proteins. Shark PNS and CNS myelin also contains smaller amounts of high-molecular-weight HNK-1-positive proteins, including a prominent broad band in the 65-85-kDa range. Although myelin-associated glycoprotein (MAG) is well known to react with HNK-1 in some mammals, monoclonal and polyclonal anti-MAG antibodies did not react with the high-molecular-weight HNK-1-positive material in shark myelin, a result suggesting that it is not a MAG-like protein. The high expression of the HNK-1/L2 epitope in glycoproteins of shark myelin, including the major P0-related ones, suggests that this adhesion-related carbohydrate structure may have had an important role in the molecular evolution of the myelinating process.

Animals↗
PubMed2004

Endothelin B receptor Ca2+ signaling in shark vascular smooth muscle: participation of inositol trisphosphate and ryanodine receptors.

In mammals, endothelin receptors are sub-classified into ET(A) receptors (ET(A)R), which are purely constrictive in vascular smooth muscle (VSM), and ET(B)R, which may produce constriction in VSM or dilatation by stimulating the production of nitric oxide (NO) from endothelial cells. In contrast, previous studies suggested that shark VSM is stimulated exclusively by ET(B)R. The Ca(2+) signaling pathways utilized by shark VSM in response to stimulation by endothelin-1 (ET-1) have not previously been investigated. We measured cytosolic Ca(2+) concentration ([Ca(2+)](i)) in fura-2-loaded VSM of anterior mesenteric artery of Squalus acanthias and show that the ET(B)R agonists IRL 1620 and sarafotoxin S6c (SRX) increase [Ca(2+)](i) in VSM to the same extent as ET-1 and ET(B)R appears to be the only ETR subtype in sharks. To investigate the participation of the inositol trisphosphate (IP(3)) receptors (IP(3)R), we utilized two inhibitors of the mammalian IP(3)R, TMB-8 and 2-APB. In Ca(2+)-free Ringer, these agents inhibit the response to ET(B)R agonist stimulation by 71%. The ryanodine-sensitive receptor (RyR) may be activated by low concentrations of ryanodine, by abrupt local increases of [Ca(2+)](i), (calcium-induced calcium release) or by cyclic adeninediphosphate ribose (cADPR). We employed three inhibitors of activation of the RyR, Ruthenium Red, 8-Br cADPR and high concentrations of ryanodine; these agents blocked the [Ca(2+)](i) response to ET(B)R agonist stimulation by a mean of 39%. These data show for the first time that in VSM of the shark, ET(B)R activation stimulates both IP(3)R and RyR, and that cADPR is involved in RyR activation.

Analysis of Variance↗