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PubMed1981

Lower vertebrate collagen. Evidence for type I-like collagen in the skin of lamprey and shark.

The soluble skin collagens of the lamprey, Entosphenus japonicus, and the great blue shark, Prionace glauca, have been isolated and characterized with respect to their chain composition. Chromatography on CM-cellulose of the denatured skin collagens and agarose gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and chemical analysis of the chromatographic fractions revealed that the two distinct subunits, alpha 1 and alpha 2, were present in a molar ratio of about 2:1. Thus, the chain composition of both lower vertebrate collagens is designated by the formula (alpha 1)2 alpha 2, similar to that of Type I collagen in higher vertebrate tissues. However, electrophoresis of the collagens in sodium dodecyl sulfate showed mostly a single type of alpha component. This seems to be due to the preferential crosslinking of alpha 1 into beta 11 dimers for the lamprey collagen and of alpha 2 into beta 12 dimers for the shark protein. These composite findings indicate that Type I-like collagen is widely distributed in the skin of vertebrates ranging from cyclostomes to mammalians.

Amino Acids↗
PubMed1988

The shark GABA-benzodiazepine receptor: further evidence for a not so late phylogenetic appearance of the benzodiazepine receptor.

Whilst the brain-specific benzodiazepine receptor has been assumed to show a late evolutionary appearance, we present evidence for the presence of a central benzodiazepine binding site in sharks, which shows a high affinity for [3H]Ro 15-1788. However, the receptor density and the affinities of several benzodiazepine receptor ligands are lower than in mammals, thus presumably explaining why the benzodiazepine binding sites had previously escaped detection in elasmobranchs. Additionally, radio- and immunohistochemistry were performed to localize the radioligand binding sites and the antigenic sites of the shark gamma-aminobutyric acid (GABA)-benzodiazepine receptor. In cerebellum, the granular layer reveals a high density of [3H]muscimol binding sites. The immunoreaction obtained with the beta-subunit-specific monoclonal antibody bd-17 seemingly parallels the distribution of high-affinity GABA binding sites. In contrast, [3H]Ro 15-1788 binding sites are evenly distributed in the molecular and granular layers, thus the results are similar to those previously described for rat cerebellum. Apparently, the respective distributions in this brain region are well conserved throughout vertebrate evolution.

Animals↗
PubMed1989

Characterization of gamma-crystallins from eye lenses of shark: closer structural similarity to mammalian than other piscine gamma-crystallins?

Lens crystallins were isolated and characterized from sharks of the cartilaginous fishes. Four crystallin fractions corresponding to alpha-, beta H-, beta L- and gamma-crystallins, similar to those of mammalian crystallins, were obtained. The native molecular masses and subunit structures of these purified fractions were analyzed by gel permeation chromatography, SDS gel electrophoresis and isoelectric focusing, revealing the typical subunit compositions with various extents of heterogeneity in each orthologous crystallin class. Amino acid and N-terminal sequence analyses corroborate the identification and classification of crystallin classes based on electrophoresis. Unexpectedly, it was found that the amino acid composition and N-terminal sequence of shark gamma-crystallin are more closely related to those of bovine than carp gamma-crystallin. This finding may have some bearing on the divergence and specification of gamma-crystallins between the phylogenetic lines of mammals and fishes.

Amino Acid Sequence↗
PubMed1988

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.

Animals↗
PubMed1986

Macrophage-like effector of spontaneous cytotoxicity from the shark.

The effector of spontaneous cytotoxicity from shark peripheral blood has been shown to be a macrophage-like cell. Effector cells are isolated by centrifugation over Lymphocyte Separation Medium, adherence to glass, Percoll density gradient centrifugation and adherence to fibronectin sequentially. Effector cells are adherent to glass, sediment to densities of 1.048-1.052 g/ml and are adherent to fibronectin. The isolated effectors represent less than 1% of the peripheral blood leukocytes, and exhibit potent cytotoxic capability, both spontaneous and in the presence of phytohemagglutinin. In addition, the activity of these cells is resistant to 3000 rads gamma irradiation. Although nurse sharks have natural antibody to trinitrophenol, spontaneously cytotoxic cells are incapable of killing trinitrophenol modified targets indicating that natural antibody is not required for reactivity, and that natural antibody and spontaneous effectors do not have the same repertoire. However, cold target inhibition studies showed that these effector cells can recognize four of five human lymphomyeloid targets. It is concluded that the spontaneous, extracellular killing by the macrophage-like effector most closely resembles that of activated mammalian tumoricidal macrophages with the exception that they do not appear to require in vitro activation.

Animals↗
PubMed1991

Shark spontaneous cytotoxicity: characterization of the regulatory cell.

Down regulation of shark macrophage-mediated spontaneous cytotoxicity is evident in vitro when animals are maintained at environmental temperatures greater than 26 degrees C. Previous work has shown that inhibition of spontaneous killing is mediated by viable, glass nonadherent, nonphagocytic cells which are sensitive to alterations in environmental temperature. The current report further characterizes the regulatory cell population. Glass nonadherent leukocytes were enriched for regulatory activity by density gradient centrifugation, and the majority of activity sedimented to 34% iso-osmotic Percoll. Two morphologically distinct cells are found in this fraction, lymphocyte-like cells and granulocytes. The 34% fraction was further separated by adherence to SIg-coated dishes, and the ability to inhibit spontaneous killing partitioned with the SIg- subset. In addition, fractionation of cells bearing Fc receptors (FcR) for shark Ig showed the regulator to be devoid of FcR. Sequential depletion of cells expressing SIg and FcR confirmed these data. Inhibition by allogeneic cells indicated that histocompatibility between the cytotoxic effector and the regulator is not a requirement for expression of activity. Thus, down regulation of spontaneous cytotoxicity is mediated by nonadherent, nonphagocytic, SIg-, FcR-cells which are not MHC restricted. The lymphoid or granulocytic lineage of the regulatory cell is discussed.

Animals↗
PubMed1996

NMR study of the active site of shark met-cyano myoglobins.

The myoglobins from the sharks Galeorhinus japonicus and Musterus japonicus possess a distal Gln-E7 instead of the usually found His-E7. The met-cyano form of these shark myoglobins has been studied by 1H- and 15N-NMR in order to gain insight into the functional properties of the Gln-E7. The analysis of paramagnetic relaxation has provided the assignment of the resonance arising from one of the Gln-E7 N epsilon H labile protons, whilst the rate of its chemical exchange has been analyzed in detail by using a saturation transfer method. The hydrogen-bonding interaction between this proton and Fe-bound-CN(-) has been clearly manifested in the hyperfine shift of the Gln-E7 N epsilon H proton resonance as well as its chemical exchange behavior. The resonances of the Gln-E7 side-chain non-labile protons have been partly assigned on a basis of both scalar and dipolar connectivities. The analysis of the dipolar connectivities among the side-chain protons and the iron-proton distances determined from their paramagnetic relaxation rate has revealed that the side chain adopts a conformation with its carbonyl oxygen oriented away from the heme. Although (1)H-NMR spectra of these two myoglobins are essentially similar, a relatively large difference in the shift of Gln-E7 N(epsilon)H proton and Fe-bound C15 N- resonances between the two has been observed which is attributed to a differential hydrogen-bonding interaction between these proteins. The present study demonstrates the sensitivity of NMR parameters to the hydrogen-bonding interaction between coordinated ligand and a distal amino-acid side chain in paramagnetic hemoproteins.

Animals↗
PubMed1986

Shark (Prionace glauca) elastoidin: characterization of its collagen as [alpha 1(E)]3 homotrimers.

A new type of collagen was isolated from elastoidin of great blue shark by limited pepsin digestion. The collagen alpha chain of elastoidin, designated alpha 1(E), was very similar in electrophoretic and chromatographic behavior and amino acid composition to shark skin alpha 1(I) chain, but they were genetically-distinct on the basis of CNBr-peptide maps. The collagen molecule of elastoidin was shown to be an [alpha 1(E)]3 homotrimer.

Amino Acids↗
PubMed2004

Reproductive and feeding ecology of parasitic gnathiid isopods of epaulette sharks (Hemiscyllium ocellatum) with consideration of their role in the transmission of a haemogregarine.

Epaulette sharks Hemiscyllium ocellatum were surveyed on Heron Island, Great Barrier Reef, Australia for gnathiid isopods and protozoan (haemogregarine) parasites to determine the prevalence and intensity of infection and to investigate the potential role of gnathiids as vectors of these haemogregarines, the first such study carried out on elasmobranchs. Juvenile gnathiids were collected and quantified using a novel non-invasive and chemical-free technique and gnathiid squashes were examined for haemogregarine developmental stages. The feeding and reproductive ecology of the Gnathia spp. was investigated to better understand the relationship between gnathiids and haemogregarines. Gnathiids were found on all sharks and intensities ranged between two and 66. Only third-stage gnathiid juveniles were found, which fell into two size groups (A and B). These juveniles remained attached to H. ocellatum for up to 17 days, the longest period of attachment yet recorded for gnathiids. Group A female gnathiids produced broods of 45-187 (median =120) first stage juveniles from between 54 and 82 days (median=63 days) after detachment. First stage juveniles survived for an average of 15.8+/-0.1 (SEM) days without feeding. The prevalence (6.7%) and parasitaemia (usually <0.1% infected erythrocytes) of infections of the haemogregarine Haemogregarina hemiscyllii were relatively low and most stages were immature gamonts. Two undescribed Gnathia spp. were identified by examining adult male gnathiids that metamorphosed from juveniles from each of the two size groups. Our hypothesis that Gnathia spp. transmit H. hemiscyllii is neither supported or refuted, as although intact H. hemiscyllii gamonts were detected in squashes of gnathiids that had engorged on haemogregarine-positive H. ocellatum 24-57 days previously, no further developmental stages were detected.

Animals↗
PubMed2006

In vitro improvement of a shark IgNAR antibody by Qbeta replicase mutation and ribosome display mimics in vivo affinity maturation.

We have employed a novel mutagenesis system, which utilizes an error-prone RNA dependent RNA polymerase from Qbeta bacteriophage, to create a diverse library of single domain antibody fragments based on the shark IgNAR antibody isotype. Coupling of these randomly mutated mRNA templates directly to the translating ribosome allowed in vitro selection of affinity matured variants showing enhanced binding to target, the apical membrane antigen 1 (AMA1) from Plasmodium falciparum. One mutation mapping to the IgNAR CDR1 loop was not readily additive to other changes, a result explained by structural analysis of aromatic interactions linking the CDR1, CDR3, and Ig framework regions. This combination appeared also to be counter-selected in experiments, suggesting that in vitro affinity maturation is additionally capable of discriminating against incorrectly produced protein variants. Interestingly, a further mutation was directed to a position in the IgNAR heavy loop 4 which is also specifically targeted during the in vivo shark response to antigen, providing a correlation between natural processes and laboratory-based affinity maturation systems.

Animals↗
PubMed2006

Dimerisation strategies for shark IgNAR single domain antibody fragments.

Immunoglobulin new antigen receptors (IgNARs) are unique single domain antibodies found in the serum of sharks. The individual variable (VNAR) domains bind antigen independently and are candidates for the smallest antibody-based immune recognition units (approximately 13 kDa). Here, we first isolated and sequenced the cDNA of a mature IgNAR antibody from the spotted wobbegong shark (Orectolobus maculatus) and confirmed the independent nature of the VNAR domains by dynamic light scattering. Second, we asked which of the reported antibody fragment dimerisation strategies could be applied to VNAR domains to produce small bivalent proteins with high functional affinity (avidity). In contrast to single chain Fv (scFv) fragments, separate IgNARs could not be linked into a tandem single chain format, with the resulting proteins exhibited only monovalent binding due solely to interaction of the N-terminal domain with antigen. Similarly, incorporation of C-terminal helix-turn-helix (dhlx) motifs, while resulting in efficiently dimerised protein, resulted in only a modest enhancement of affinity, probably due to an insufficiently long hinge region linking the antibody to the dhlx motif. Finally, generation of mutants containing half-cystine residues at the VNAR C-terminus produced dimeric recombinant proteins exhibiting high functional affinity for the target antigens, but at the cost of 50-fold decreased protein expression levels. This study demonstrates the potential for construction of bivalent or bispecific IgNAR-based binding reagents of relatively small size (approximately 26 kDa), equivalent to a monovalent antibody Fv fragment, for formulation into future diagnostic and therapeutic formats.

Amino Acid Sequence↗
PubMed2006

Rapid isolation of IgNAR variable single-domain antibody fragments from a shark synthetic library.

The immunoglobulin isotype IgNAR (Novel Antigen Receptor) was discovered in the serum of the nurse shark (Ginglymostoma cirratum) and wobbegong shark (Orectolobus maculates) as a homodimer of two protein chains, each composed of a single variable domain (V) domain and five constant domains. The IgNAR variable domain contains an intact antigen-binding site and functions as an independent domain able to react to antigen with both high specificity and affinity. Here we describe the successful construction of a synthetic phage-displayed library based upon a single anti-lysozyme clone HEL-5A7 scaffold, which was previously selected from an immune IgNAR variable domain library. The complementarity-determining region 3 (CDR3) loop of this clone was varied in both length and composition and the derived library was used to pan against two model proteins, lysozyme and leptin. A single anti-lysozyme clone (Ly-X20) and anti-leptin clone (Lep-12E1) were selected for further study. Both clones were shown to be functionally expressed in Escherichia coli, extremely thermostable and bind to corresponding antigens specifically. The results here demonstrate that a synthetic IgNAR variable domain library based on a single framework scaffold can be used as a route to generate antigen binders quickly, easily and without the need of immunization.

Amino Acid Sequence↗
PubMed2004

Maternal serum and yolk hormone concentrations in the placental viviparous bonnethead shark, Sphyrna tiburo.

Among vertebrates, maternal transfer of hormones to offspring has been studied extensively in mammals (placental transfer) and more recently in oviparous birds and reptiles (yolk transfer). The placental viviparous bonnethead shark, Sphyrna tiburo, allows the investigation of both yolk and placental hormone transfers in a single organism. In this species, yolk provides nutrition for the first half of embryonic development and placental transfer provides the second half. As sex determination is complete prior to development of placental connections, it was postulated that yolk hormones would have a prominent role in embryonic regulation. The goal of the current study was to determine serum and yolk hormone concentrations during five reproductive stages, from pre-ovulatory through pre-implantation (pre-placental) stages. Radioimmunoassay was used to determine 17beta-estradiol, progesterone, and testosterone concentrations in both serum and yolk. When yolk and serum concentrations were compared, the yolk had significantly higher concentrations of both estradiol and progesterone during post-ovulation and early pregnancy. Yolk concentrations of testosterone were significantly less than serum at pre-ovulation, but there were no differences after that stage. When yolk concentrations were compared between stages, significantly higher concentrations of estradiol were present in ovulatory, post-ovulatory, and pre-implantation stages, while progesterone was significantly higher in post-ovulatory, early pregnancy, and pre-implantation stages and testosterone was higher in pre-ovulation. Most of these results are consistent with the published findings in birds and reptiles. Further, in the bonnethead shark, they suggest that yolk transfer of hormones is adequate for sexual differentiation in embryonic development and that estradiol probably has a significant developmental role.

Animals↗
PubMed2006

Phylogeny of sharks of the family Triakidae (Carcharhiniformes) and its implications for the evolution of carcharhiniform placental viviparity.

We present a study of inter- and intra-familial relationships of the carcharhiniform shark family Triakidae aimed at testing existing hypotheses of relationships for this group and at improving understanding of the evolution of reproductive traits in elasmobranchs. Our analyses and conclusions are based on evidence from DNA sequences of four protein-coding genes (three from the mitochondrial genome and a single copy nuclear gene) from eight of the nine genera and 20 of the 39 species currently assigned to the Triakidae. The sequence data offer strong support for the following previously proposed triakid clades: Galeorhinini (Hypogaleus+Galeorhinus); a subset of the Iagini (Furgaleus+Hemitriakis but not Iago); and part of the Triakinae (Mustelus, Scylliogaleus and part of Triakis). Interestingly, the molecular data provide considerable evidence of paraphyly of the genera Triakis and Mustelus. Our results suggest that the subgenera Triakis and Cazon of Triakis represent two distinct lineages that are only distantly related and that the genus Mustelus as currently defined does not constitute a monophyletic assemblage unless S. quecketti and some species of Triakis (subgenus Cazon) are included in Mustelus. Within our sample of species of Mustelus (including Cazon and Scylliogaleus), the sequence data support two well-defined clades that can be diagnosed by mode of reproduction (placental vs. aplacental species). The phylogenetic framework presented here is used to infer key events in the evolution and loss of placental viviparity among carcharhiniform sharks.

Animals↗
PubMed2003

Serum relaxin concentrations and reproduction in male bonnethead sharks, Sphyrna tiburo.

Relaxin is a 6-kd polypeptide hormone that is responsible for regulating several reproductive processes in female vertebrates, but its role in male reproduction remains unclear. To aid in clarifying this role, the objective of the present study was to investigate changes in endogenous relaxin levels associated with reproductive events in male elasmobranchs, which represent one of only three vertebrate groups known to possess this hormone. Serum relaxin concentrations were measured in 27 immature and 66 mature male bonnethead sharks (Sphyrna tiburo), a species with a well-characterized, seasonal reproductive cycle. Temporal changes in serum relaxin concentrations of immature male S. tiburo were not observed. In contrast, a temporal cycle in serum relaxin concentrations of mature male S. tiburo was observed in individuals from two sampling locations. Significant increases (P<0.05) in serum relaxin concentrations of mature male S. tiburo from both collection sites occurred during late spermatogenesis and the mating period, two critical stages of the reproductive cycle. The results from this study suggest that relaxin may play an important role in regulating semen quality, or other aspects of reproduction in male sharks. This is the first study to demonstrate a temporal pattern in endogenous serum Rlx concentrations associated with reproductive events in feral vertebrates. As such, it strengthens earlier hypotheses that suggested a role for this hormone in regulating male vertebrate fertility and copulatory success.

Animals↗
PubMed1997

Sheina orri (Myodocopa: Cypridinidae), an ostracod parasitic on the gills of the epaulette shark, Hemiscyllium ocellatum (Elasmobranchii: Hemiscyllidae).

The cypridinid ostracod, Sheina orri, was found on the gills of healthy epaulette sharks, Hemiscyllium ocellatum, collected from the Great Barrier Reef, Australia. Seventeen of the 28 fish examined had ostracods attached to their gills. Detailed investigation of the gills and ostracods using light microscopy and scanning electron microscopy revealed that ostracods anchor themselves to the gill tissues using their mandibular and maxillular claws. These claws appear to be adapted for this purpose and the process of attachment causes some damage to the host tissues. The observation that ostracods were often located in distinct pockets, formed by local distortion of shark respiratory lamellae, strongly suggests that they had been attached to the gills for considerable time.

Animals↗
PubMed1977

Phylogeny of immunoglobulin structure and function; characterization of the cysteine-containing peptide involved in the pentamerization of shark IgM.

Nurse shark (Ginglymostoma cirratum) immunoglobulins were studied in an attempt to further define the relationship between the naturally occurring monomeric 7S and the pentameric 19S forms of extracellular IgM. A peptide containing the cysteine involved in the formation of intersubunit disulfide bonds linking 7S monomers into pentamers was isolated from the H chain of the 19S molecule and characterized. A similar peptide was also isolated from the H chain of the naturally occurring 7S molecule. These observations serve to substantiate previous claims that the two shark proteins belong to the same immunoglobulin class.

Amino Acid Sequence↗