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Mitochondrial proton leak rates in the slow, oxidative myotomal muscle and liver of the endothermic shortfin mako shark (Isurus oxyrinchus) and the ectothermic blue shark (Prionace glauca) and leopard shark (Triakis semifasciata).

Mitochondrial proton leak was assessed as a potential heat source in the slow, oxidative (red) locomotor muscle and liver of the shortfin mako shark (Isurus oxyrinchus), a regional endotherm that maintains the temperature of both tissues elevated above ambient seawater temperature. We hypothesized that basal proton leak rates in red muscle and liver mitochondria of the endothermic shortfin mako shark would be greater than those of the ectothermic blue shark (Prionace glauca) and leopard shark (Triakis semifasciata). Respiration rate and membrane potential in isolated mitochondria were measured simultaneously at 20 degrees C using a Clark-type oxygen electrode and a lipophilic probe (triphenylmethylphosphonium, TPMP(+)). Succinate-stimulated respiration was titrated with inhibitors of the electron transport chain, and the non-linear relationship between respiration rate and membrane potential was quantified. Mitochondrial densities of both tissues were measured by applying the point-contact method to electron micrographs so that proton leak activity of the entire tissue could be assessed. In all three shark species, proton leak occurred at a higher rate in red muscle mitochondria than in liver mitochondria. For each tissue, the proton leak curves of the three species overlapped and, at a membrane potential of 160 mV, mitochondrial proton leak rate (nmol H(+) min(-1) mg(-1) protein) did not differ significantly between the endothermic and ectothermic sharks. This finding indicates that red muscle and liver mitochondria of the shortfin mako shark are not specialized for thermogenesis by having a higher proton conductance. However, mako mitochondria did have higher succinate-stimulated respiration rates and membrane potentials than those of the two ectothermic sharks. This means that under in vivo conditions mitochondrial proton leak rates may be higher in the mako than in the ectothermic species, due to greater electron transport activity and a larger proton gradient driving proton leak. We also estimated each tissue's total proton leak by combining mitochondrial proton leak rates at 160 mV and tissue mitochondrial density data with published values of relative liver or red muscle mass for each of the three species. In red muscle, total proton leak was not elevated in the mako shark relative to the two ectothermic species. In the liver, total proton leak would be higher in the mako shark than in both ectothermic species, due to a lower proton conductance in the blue shark and a lower liver mitochondrial content in the leopard shark, and thus may contribute to endothermy.

Animals↗

A comparative infrared spectroscopic study of hydroxide and carbonate absorption bands in spectra of shark enameloid, shark dentin, and a geological apatite.

The purpose of the present work was to investigate the infrared (IR) spectrum of shark enameloid, especially with regard to hydroxide and carbonate bands. With thin sections placed directly in the IR beam it was possible to get high concentrations of ions without interfering effects from a dispersion medium (e.g., alkali halides). For comparison, spectra of shark dentin and a geo-apatite were also recorded. In spectra of shark enameloid and geo-apatite medium strong hydroxide absorption bands were found around 3535 cm(-1), and in shark dentin and geo-apatite spectra weak shoulders were observed at about 3570 cm(-1). Hydroxide libration bands at about 740 cm(-1) were found in shark enameloid and geo-apatite spectra; in the latter, also a band at 680 cm(-1). Carbonate bands were found in shark enameloid spectra at 1480 (weak shoulder), 1453, 1423, and 868 cm(-1). In shark dentin spectra there were carbonate bands at 1452, 1417, and 875 cm(-1), and probably also a carbonate band at about 1530 cm(-1) overlapped by an amide II band. Weak carbonate bands were also found in the spectra of the geo-apatite at 1452 cm(-1), and at about 1425 and 880 cm(-1). The relative intensities of the bands at 1453 cm(-1) (contributed from A and B sites) and around 1420 cm(-1) (B sites) changed from shark enameloid to shark dentin, and also from shark enameloid to the geo-apatite. More A sites seem to be occupied by carbonate in shark dentin than in shark enameloid, supposedly owing to fluoride occupation of A sites in shark enameloid. In geo-apatite and shark enameloid there are hydroxide ions hydrogen bonded to fluoride. Both shark enameloid and the geo-apatite are fluoride rich, and geo-apatite seems to have the highest fluoride concentration. There are, however, indications that the hydroxide concentration is also higher in the geo-apatite than in shark enameloid. This can be explained by the much higher carbonate content, and partly also by the higher water content in shark enameloid. There are A sites in geo-apatite and probably also in shark enameloid which are occupied by carbonate, but the proportion of occupied A sites relative to occupied B sites is greater in geo-apatite than in shark enameloid. This difference can be explained by the preference of A sites when the carbonate concentration is very low. On the other hand, for greater amounts of carbonate such as we have in shark enameloid, B sites are preferred.

Animals↗

Comparative studies of high performance swimming in sharks II. Metabolic biochemistry of locomotor and myocardial muscle in endothermic and ectothermic sharks.

Metabolic enzyme activities in red (RM) and white (WM) myotomal muscle and in the heart ventricle (HV) were compared in two lamnid sharks (shortfin mako and salmon shark), the common thresher shark and several other actively swimming shark species. The metabolic enzymes measured were citrate synthase (CS), an index of aerobic capacity, and lactate dehydrogenase (LDH), an index of anaerobic capacity. WM creatine phosphokinase (CPK) activity, an index of rapid ATP production during burst swimming, was also quantified. Enzyme activities in RM, WM and HV were similar in the two lamnid species. Interspecific comparisons of enzyme activities at a common reference temperature (20 degrees C) show no significant differences in RM CS activity but higher CS activity in the WM and HV of the lamnid sharks compared with the other species. For the other enzymes, activities in lamnids overlapped with those of other shark species. Comparison of the HV spongy and compact myocardial layers in mako, salmon and thresher sharks reveals a significantly greater spongy CS activity in all three species but no differences in LDH activity. Adjustment of enzyme activities to in vivo RM and WM temperatures in the endothermic lamnids elevates CS and LDH in both tissues relative to the ectothermic sharks. Thus, through its enhancement of both RM and WM enzyme activity, endothermy may be an important determinant of energy supply for sustained and burst swimming in the lamnids. Although lamnid WM is differentially warmed as a result of RM endothermy, regional differences in WM CS and LDH activities and thermal sensitivities (Q(10) values) were not found. The general pattern of the endothermic myotomal and ectothermic HV muscle metabolic enzyme activities in the endothermic lamnids relative to other active, ectothermic sharks parallels the general pattern demonstrated for the endothermic tunas relative to their ectothermic sister species. However, the activities of all enzymes measured are lower in lamnids than in tunas. Relative to lamnids, the presence of lower WM enzyme activities in the thresher shark (which is in the same order as the lamnids, has an RM morphology similar to that of the mako and salmon sharks and may be endothermic) suggests that other factors, such as behavior and swimming pattern, also affect shark myotomal organization and metabolic function.

Adaptation, Biological↗

Sequence analysis of homogeneous peptides of shark immunoglobulin light chains by tandem mass spectrometry: correlation with gene sequence and homologies among variable and constant region peptides of sharks and mammals.

Morphologically, sharks are living fossils that are remarkably similar to their Devonian ancestors of ca. 400 million years ago. If a parallel conservation in biochemical properties characterizes shark evolution, knowledge of the properties of shark immunoglobulins should provide information on the structure of primordial immunoglobulins and their genes. The problem of polyclonality of shark immunoglobulins has precluded detailed analysis of shark immunoglobulin light polypeptide chains. Here, we approach the problem of obtaining direct sequence information on polyclonal light chains of shark immunoglobulins by isolating homogeneous peptides from tryptic digests of shark light chains and sequencing these by tandem mass spectrometry. To confirm the location of the peptides, we isolated a complementary DNA (cDNA) clone from a sandbar shark cDNA library in the expression vector lambda gt11, identifying the clone by its ability to produce a peptide serologically detectable using rabbit antibody to purified shark light chain. The correspondence between peptide sequence and that derived from gene sequence provided direct proof that the gene studied was that of a major expressed serum light chain. Using this combined approach, we isolated homogeneous peptides from both constant and variable regions. The variable region peptides showed homology to corresponding sequences of mammalian V lambda and V kappa sequences. The constant region gene sequence we obtained was homologous to mammalian C lambda sequence. The four constant region tryptic peptides we sequenced corresponded exactly to stretches of the C lambda sequence derived from the DNA sequence. The combined approach described here shows that shark light chains exhibit heterogeneity at both the protein and gene level, but that the constant regions of these chains can be identified as homologs of mammalian lambda chains and that evolutionary conservation has occurred in V region sequences ranging from elasmobranchs to man.

Amino Acid Sequence↗

Allometric relationships of the dentition of the great White Shark, Carcharodon carcharias, in forensic investigations of shark attacks.

As a result of a systematic morphometric study of shark dentitions, a system of notation for describing the location of shark teeth has been developed and is proposed as a standard to be adopted for use in similar studies in the future. The macroscopic morphology of White Shark teeth has been characterised in order to gain quantitative data which might assist in identification of these sharks from bite marks on victims or objects or from shark carcasses. Using these data, a nomogram has been developed which can be used to estimate the body length of a White Shark from measurements of tooth or bite mark morphology. An example of the forensic application of such allometric data is provided as it applied to a recent fatal attack on a diver by a White Shark.

Animals↗

Identification of shark species composition and proportion in the Hong Kong shark fin market based on molecular genetics and trade records.

The burgeoning and largely unregulated trade in shark fins represents one of the most serious threats to shark populations worldwide. In Hong Kong, the world's largest shark fin market, fins are classified by traders into Chinese-name categories on the basis of market value, but the relationship between market category and shark species is unclear preventing identification of species that are the most heavily traded. To delineate these relationships, we designed a sampling strategy for collecting statistically sufficient numbers of fins from traders and categories under conditions of limited market access because of heightened trader sensitivities. Based on information from traders and morphological inspection, we hypothesized matches between market names and shark taxa for fins within 11 common trade categories. These hypotheses were tested using DNA-based species identification techniques to determine the concordance between market category and species. Only 14 species made up approximately 40% of the auctioned fin weight. The proportion of samples confirming the hypothesized match, or concordance, varied from 0.64 to 1 across the market categories. We incorporated the concordance information and available market auction records for these categories into stochastic models to estimate the contribution of each taxon by weight to the fin trade. Auctioned fin weight was dominated by the blue shark (Prionace glauca), which was 17% of the overall market. Other taxa, including the shortfin mako (Isurus oxyrinchus), silky (Carcharhinus falciformis), sandbar (C. obscurus), bull (C. leucas), hammerhead (Sphyrna spp.), and thresher (Alopias spp.), were at least 2-6% of the trade. Our approach to marketplace monitoring of wildlife products isparticularly applicable to situations in which quantitative data at the source of resource extraction are sparse and large-scale genetic testing is limited by budgetary or other market access constraints.

Animals↗

Predominance of genetic monogamy by females in a hammerhead shark, Sphyrna tiburo: implications for shark conservation.

There is growing interest in the mating systems of sharks and their relatives (Class Chondrichthyes) because these ancient fishes occupy a key position in vertebrate phylogeny and are increasingly in need of conservation due to widespread overexploitation. Based on precious few genetic and field observational studies, current speculation is that polyandrous mating strategies and multiple paternity may be common in sharks as they are in most other vertebrates. Here, we test this hypothesis by examining the genetic mating system of the bonnethead shark, Sphyrna tiburo, using microsatellite DNA profiling of 22 litters (22 mothers, 188 embryos genotyped at four polymorphic loci) obtained from multiple locations along the west coast of Florida. Contrary to expectations based on the ability of female S. tiburo to store sperm, the social nature of this species and the 100% multiple paternity observed in two other coastal shark species, over 81% of sampled bonnethead females produced litters sired by a single male (i.e. genetic monogamy). When multiple paternity occurred in S. tiburo, there was an indication of increased incidence in larger mothers with bigger litters. Our data suggest that sharks may exhibit complex genetic mating systems with a high degree of interspecific variability, and as a result some species may be more susceptible to loss of genetic variation in the face of escalating fishing pressure. Based on these findings, we suggest that knowledge of elasmobranch mating systems should be an important component of conservation and management programmes for these heavily exploited species.

Animals↗

Mercury and zinc differentially inhibit shark and human CFTR orthologues: involvement of shark cysteine 102.

The apical membrane is an important site of mercury toxicity in shark rectal gland tubular cells. We compared the effects of mercury and other thiol-reacting agents on shark CFTR (sCFTR) and human CFTR (hCFTR) chloride channels using two-electrode voltage clamping of cRNA microinjected Xenopus laevis oocytes. Chloride conductance was stimulated by perfusing with 10 microM forskolin (FOR) and 1 mM IBMX, and then thio-reactive species were added. In oocytes expressing sCFTR, FOR + IBMX mean stimulated Cl(-) conductance was inhibited 69% by 1 microM mercuric chloride and 78% by 5 microM mercuric chloride (IC(50) of 0.8 microM). Despite comparable stimulation of conductance, hCFTR was insensitive to 1 microM HgCl(2) and maximum inhibition was 15% at the highest concentration used (5 microM). Subsequent exposure to glutathione (GSH) did not reverse the inhibition of sCFTR by mercury, but dithiothreitol (DTT) completely reversed this inhibition. Zinc (50-200 microM) also reversibly inhibited sCFTR (40-75%) but did not significantly inhibit hCFTR. Similar inhibition of sCFTR but not hCFTR was observed with an organic mercurial, p-chloromercuriphenylsulfonic acid (pCMBS). The first membrane spanning domain (MSD1) of sCFTR contains two unique cysteines, C102 and C303. A chimeric construct replacing MSD1 of hCFTR with the corresponding sequence of sCFTR was highly sensitive to mercury. Site-specific mutations introducing the first but not the second shark unique cysteine in hCFTR MSD1 resulted in full sensitivity to mercury. These experiments demonstrate a profound difference in the sensitivity of shark vs. human CFTR to inhibition by three thiol-reactive substances, an effect that involves C102 in the shark orthologue.

4-Chloromercuribenzenesulfonate↗

Temperature-dependencies of various catalytic activities of membrane-bound Na+/K+-ATPase from ox brain, ox kidney and shark rectal gland and of C12E8-solubilized shark Na+/K+-ATPase.

The temperature dependence of ouabain-sensitive ATPase and phosphatase activities of membrane fragments containing the Na+/K+-ATPase were investigated in tissue from ox kidney, ox brain and from shark rectal glands. The shark enzyme was also tested in solubilized form. Arrhenius plots of the Na+/K+-ATPase activity seem to be linear up to about 20 degrees C, and non-linear above this temperature. The Arrhenius plots of mammalian enzyme (ox brain and kidney) were steeper, especially at temperatures below 20-30 degrees C, than that of shark enzyme. The Na+-ATPase activity showed a weaker temperature-dependence than the Na+/K+-ATPase activity. The phosphatase reactions measured, K+-stimulated, Na+/K+-stimulated and Na+/K+/ATP-stimulated, also showed a weaker temperature-dependence than the overall Na+/K+-ATPase activity. Among the phosphatase reactions, the largest change in slope of the Arrhenius plot was observed with the Na+/K+/ATP)-stimulated phosphatase reaction. The Arrhenius plots of the partial reactions were all non-linear. Solubilization of shark enzyme in C12E8 did not change the curvature of Arrhenius plots of the Na+/K+-ATPase activity or the K+-phosphatase activity. Since solubilization involves a disruption of the membrane and an 80% delipidation, the observed curvature of the Arrhenius plot can not be attributed to a property of the membrane as such.

Animals↗

Comparison of the gamma-crystallins isolated from eye lenses of shark and carp. Unique secondary and tertiary structure of shark gamma-crystallin.

gamma-Crystallin isolated from the shark of cartilaginous fishes was compared with the cognate gamma-crystallin from the carp of bony fishes. Distinct differences in amino acid compositions, primary, secondary and tertiary structures were found. The most salient features of shark gamma-crystallin lie in the fact that this crystallin possessed a significant alpha-helical structure in the peptide backbone as revealed by circular dichroism study, in contrast to those orthologous gamma-crystallins from other vertebrate species including bony fishes which all show a predominant beta-sheet secondary structure. The tertiary structure as reflected in the intrinsic microenvironments of various aromatic amino acids in the native crystallins also shows unambiguous differences between these two classes of gamma-crystallins. N-Terminal sequence analysis corroborates the structural differences between shark and carp gamma-crystallins. gamma-Crystallin from the more primitive shark seems to be more in line with the main evolutionary phylogeny leading to the modern mammalian gamma-crystallin.

Amino Acid Sequence↗

Shark immunity bites back: affinity maturation and memory response in the nurse shark, Ginglymostoma cirratum.

The cartilaginous fish are the oldest phylogenetic group in which all of the molecular components of the adaptive immune system have been found. Although early studies clearly showed that sharks could produce an IgM-based response following immunization, evidence for memory, affinity maturation and roles for the other isotypes (notably IgNAR) in this group remained inconclusive. The data presented here illustrate that the nurse shark (Ginglymostoma cirratum) is able to produce not only an IgM response, but we also show for the first time a highly antigen-specific IgNAR response. Additionally, under appropriate conditions, a memory response for both isotypes can be elicited. Analysis of the response shows differential expression of pentameric and monomeric IgM. Pentameric IgM provides the 'first line of defense' through high-avidity, low-affinity interaction with antigen. In contrast, monomeric IgM and IgNAR seem responsible for the specific, antigen-driven response. We propose the presence of distinct lineages of B cells in sharks. As there is no conventional isotype switching, each lineage seems pre-determined to express a single isotype (IgM versus IgNAR). However, our data suggest that there may also be specific lineages for the different forms (pentameric versus monomeric) of the IgM isotype.

Animals↗

Identification of a second immunoglobulin in the most primitive shark, the frill shark, Chlamydoselachus anguineus.

Three molecular forms of immunoglobulins: pentamer, dimer and monomer, were isolated from serum of the frill shark, Chlamydoselachus anguineus, the most primitive extant shark. A pentamer having an apparent mass of 900 kDa, consisting of 68 kDa heavy (H) chains and 22-24 kDa light (L) chains, was considered to be IgM because of its similarity to mammalian IgM in both molecular form and H chain molecular weight. The dimer and monomer with apparent masses of 300 kDa and 150 kDa, respectively, were composed of the same H chains of 45-50 kDa, a value smaller than that of the H chains from the pentamer, and light chains identical to those of the pentamer. The H chains of 68 kDa and 45-50 kDa represented individual epitopes and were synthesized by different plasma cells. We thus concluded that the frill shark has two distinct classes of immunoglobulins: one a pentameric IgM and the other a second class of immunoglobulin with dimeric and monomeric structure. The presence of the second Ig is of considerable interest with respect to antigen recognition and exclusion.

Animals↗

Characterization of liver flavin-containing monooxygenase of the dogfish shark (Squalus acanthias) and partial purification of liver flavin-containing monooxygenase of the silky shark (Carcharhinus falciformis).

Flavin-containing monooxygenase (FMO) activity as N,N-dimethylaniline (DMA) N-oxygenation was characterized in microsomes from the smooth dogfish shark (Squalus acathias). DMA N-oxygenase activity from the liver of the dogfish shark was linear with increasing protein content and over 60 min. The optimal temperature for catalysis was 25 degrees C with a 76 percent reduction in activity when incubated at 15 degrees C and 99 percent loss of activity at 45 degrees C. Optimal pH was approximately 9.6. The maximum velocity for DMA N-oxygenase activity was calculated to be 1.3 nmol min-1 mg-1 with an apparent Michaelis constant of 44 microM. Methimazole oxidase activity was also observed in dogfish liver microsomes which was inhibited by trimethylamine (TMA). Inhibition of DMA N-oxygenase activity by TMA and thiobenzamide was competitive, while inhibition by methimazole was not competitive. Western blot analysis indicated a single liver protein from both Squalus and Carcharhinus of approximately 50 kDa that bound to antibodies raised against FMO 2. An attempt was made to purify FMO as methimazole oxidase from the liver of the silky shark. A single peak of about 10-fold purity was observed following passage through two chromatographic media (CM-Sepharose and HA-Agarose). However, no activity was recoverable after the FMO-containing fractions were applied to a 2'5' ADP-Sepharose column.

Animals↗

Modulation of CD(4)(+) and CD(8)(+) tumor infiltrating lymphocytes by a fraction isolated from shark cartilage: shark cartilage modulates anti-tumor immunity.

Shark cartilage has proven to have some inhibitory effects on angiogenesis, metastasis, cell adhesion and proteolysis. In this study, we wanted to study some of the effects of shark cartilage on tumor immune response. Firstly, by means of chromatographic methods and delayed type hypersensitivity (DTH) test, we optimized a procedure for isolation and purification of a shark cartilage protein fraction with most immunostimulatory effects. Then, we examined its effect on the infiltration of CD(4)(+) and CD(8)(+) lymphocytes into a murine tumor model. Our fraction was composed of two major proteins with molecular weights (MWs) of about 14 and 15 kDa. This fraction highly increases DTH response against sRBC in mice. Furthermore, intraperitoneal injection of this fraction to tumor-bearing mice could increase T-cell infiltration into the tumor. Also, there was a significant increase in the CD(4)/CD(8) ratio in tumor infiltrating lymphocytes, but no such changes were found in the peripheral blood lymphocytes. According to these results, we suppose that this fraction is a good candidate for further studies in cancer therapy. Also, we concluded that this fraction, with previously proven anti-angiogenic effects, can augment cellular immune response and T-cell infiltration into the tumor and thus, there may be a direct relationship between angiogenesis inhibition and T-cell infiltration.

Adenocarcinoma↗

Bacteriology of the teeth from a great white shark: potential medical implications for shark bite victims.

Bacteria were cultured for the first time from the teeth of a great white shark (Carcharodon carcharias). Isolates included Vibrio alginolyticus, Vibrio fluvialis, Vibrio parahaemolyticus, and other genera. All are common in the marine environment and some may be associated with wound infections in humans. Shark bite lacerations may serve as a source of these potentially infectious bacteria, particularly Vibrio spp., and should be treated immediately. Antibiotic susceptibility patterns are shown for representatives of Vibrio isolates and indicate that a variety of new agents may be appropriate chemotherapy for shark bite victims.

Animals↗

Shark tooth morphogenesis. An SEM and EDX analysis of enameloid and dentin development in various shark species.

The study provides a survey of shark tooth morphogenesis based on SEM and EDX analyses of whole tooth families in six shark species. The teeth, demonstrating different stages of development, were acid-etched and coated with palladium. Calcium content was determined semi-quantitatively by using the palladium coating as an internal standard. Due to the rapid development of the enameloid, all major events took place in the two or three youngest teeth of a tooth family. Enameloid appeared to develop as a transformation of the peripheral part of the dental papilla. Mineralization started immediately. Based on morphological criteria the middle zone of the enameloid was established at an early stage, excluding the possibility of an unambiguous centrifugal or centripetal direction of growth. Substantial mineral increase first occurred in the middle zone, spreading from the tooth tip toward the base. Dentin formed after the enameloid was completely established. Dentin formation started basally as a direct prolongation of the enameloid cap, then spreading toward the tooth tip, first along the edges. It is concluded that shark enameloid has a mesenchymal background, but a role played by the inner dental epithelium can not be excluded.

Amelogenesis↗

Maneuvering in juvenile carcharhinid and sphyrnid sharks: the role of the hammerhead shark cephalofoil.

The peculiar head morphology of hammerhead sharks has spawned a variety of untested functional hypotheses. One of the most intuitively appealing ideas is that the anterior foil acts, as in canard-winged aircraft, to increase maneuverability. We tested this hypothesis by determining whether juveniles of two hammerhead species (Sphyrna tiburo and S. lewini) turn more sharply, more often, and with greater velocity than a juvenile carcharhinid shark (Carcharhinus plumbeus). Although the hammerheads were more maneuverable, further investigation revealed that they do not roll their body during turns, suggesting that the cephalofoil does not act as a steering wing. We also show that hammerhead sharks demonstrate greater lateral flexure in a turn than carcharhinids, and that this flexibility may be due to cross sectional shape rather than number of vertebrae.

Journal Article↗