The structure of immunoglobulin variable regions in the horned shark,Heterodontus francisci

The heavy and light chains of pooled antibodies of the hybodont shark,Heterodontus francisci (horned shark), were subjected to amino acid sequence analysis. Yield determinations showed that more than 90% of the available polypeptides in the respective pools were sequenced. The heavy chains were homogeneous in the initial framework segment and showed a sequence homology of approximately 70% with the corresponding region of the more recently evolved nurse shark and a 45% homology with a human myeloma heavy chain. The light chains were less homogeneous and not identifiable as either kappa or lambda chains as known in higher species. The first half-cystine characteristics of the variable domain intrachain disulfide bridge of immunoglobulins was present in the same position (22 for heavy chains; 23 for light chains) in the horned shark as in mammalian species. The sequence analysis also suggested the presence of a hypervariable region in the horned shark light chains. The combined data imply that the antigen-binding function of immunoglobulins is mediated in much the same manner in this primitive shark as in more recently evolved species, including mammals.     

Food capture kinematics of the suction feeding horn shark, Heterodontus francisci

The goal of this study was to examine the feeding kinematics of the horn shark, Heterodontus francisci, a member of the most basal clade of galeomorph sharks, the Heterodontiformes. The accessibility of the food was manipulated to determine if the horn shark modulated capture. Three different methods of presenting food were used to mimic the different positions of prey items found in the natural diet of the horn shark. Food was presented unattached to the substrate, securely attached, or fitted snugly in a tube. Using high-speed video kinematic analysis, capture events were examined. Heterodontus francisci uses inertial suction facilitated by rapid mandible depression and labial cartilage protrusion to capture food. The horn shark conforms to a capture kinematic profile characteristic of both basal and derived inertial suction feeding sharks. Unusual post-capture behaviors include body leveraging, use of the mouth to form a seal over food, and chisel-like palatoquadrate protrusion. When presented with food of different accessibility, Heterodontus francisci used one consistent kinematic pattern for capture that was not modulated. Only post-capture behaviors varied according to food accessibility.     

Feeding habits of the horn shark Heterodontus francisci (Girard, 1855) in the northwest of Baja California Sur,Mexico

The feeding habits of the horn shark, Heterodontus francisci (Girard, 1855), were examined in the area of Bahía Tortugas, Baja California Sur, Mexico in the spring, summer and fall of 2014. A total of 78 stomachs were collected, of which 46 (59%) contained food and 32 (41%) were empty. According to the percent Index of Relative Importance (%IRI), the most important prey categories in H. francisci's diet were anomurans (66%), cephalopods (7.2%), lobsters (4.7%), fishes (4.2%) and sea urchins (2.3%). The main prey were the anomuran Blepharipoda occidentalis (65.2%), the octopus Octopus bimaculatus (5.4%), the lobster Panulirus interruptus (4.7%) and the sea urchin Echinometra vanbrunti (2.6%). According to the Levin standardized Index (Bi), the trophic niche breadth in H. francisci is low (B_i = 0.21), making it a specialist predator. The species was classified as a tertiary consumer (trophic position = 4.06).     

Structure and function of the horn shark (Heterodontus francisci) cranium through ontogeny: development of a hard prey specialist

The horn sharks (Heterodontidae: Chondrichthyes) represent one of four independent evolutions of durophagy in the cartilaginous fishes. We used high-resolution computed tomography (CT scanning) to visualize and quantify the mineralized tissue of an ontogenetic series of horn sharks. CT scanning of neonatal through adult California horn sharks (Heterodontus francisci) confirmed that this technique is effective for examining mineralized tissue in even small (<10 mm) specimens. The jaw joint is among the first areas to become mineralized and is the most heavily mineralized area in the cranium of a neonatal horn shark. The hyoid is also well mineralized, although the poorly mineralized molariform teeth indicate that the neonatal animal may be a suction feeder on softer prey. The symphysis of the jaws never mineralizes, in sharp contrast to the condition in the hard prey-crushing stingrays. Digitally reslicing the CT scans along the jaws allowed measurement of the second moment of area (Ina). Assuming that the jaws are made of the same material at all ages, Ina is an indicator of the flexural stiffness of the jaws. In all sizes of shark the lower jaws were stiffer than the upper and the stiffness increased in the area of the molariform teeth. The central region of the jaws, where the rami meet, support cuspidate grasping teeth and has the lowest Ina. The spotted eagle ray (Aetobatus narinari), a hard prey-crushing stingray, shows a different pattern of flexural stiffness, with the peak at the central part of the jaws where the prey is reduced between flattened tooth plates. Although the eagle ray jaws have a higher Ina than the horn shark, they are also far more heavily mineralized. When the relative amounts of mineralization are taken into account, horn sharks do better with what mineral they have than does the eagle ray. With a tight jaw joint and loose mandibular symphysis, as well as nearly opposite patterns of stiffness in the jaws, it is clear that two of the clades of hard prey specialists use very different methods for cracking the hard prey problem.     

Haemoglobins of the shark, Heterodontus portusjacksoni. III. Amino acid sequence of the beta-chain

The amino acid sequence of the beta-chain of the principal haemoglobin from the shark H. portusjacksoni has been determined. The chain has 141 residues, the same as that of mammalian alpha-chains and less than the 146 residues of mammalian beta-chains or the 148 residues of the alpha-chain from the tetrameric shark haemoglobin. The sequence was deduced from the sequences of peptides obtained by digestion of the globin or its cyanogen bromide fragments with trypsin, chymotrypsin, pepsin and papain. The difference in length of the beta-chain is most readily accounted for by the absence of the D helix. This small helical section is normally present in myoglobins and beta-globins but absent in alpha-chains. The deduction that it is absent from shark beta-chain is based on consideration of homology. The beta-chain shows the insertion of histidine beta2 and the deletions corresponding to residues A17 and AB1 relative to alpha-and myoglobin chains. The reactive thiol group in shark haemoglobin was shown by radioactive labelling to be residue 51 in the beta-chain, immediately preceding the E helix. The amino acid sequence of shark beta-chain shows 92 differences from human beta-chain, significantly more differences than shown by chicken or frog beta-chains, in line with its earlier time of divergence. If the tertiary structure of the shark beta-chain is the same as that of the horse then there are two changes in the alpha1beta2 contact site in oxyhaemoglobin and an additional one in deoxyhaemoglobin. When both alpha- and beta-chain contacts are considered there is a total of nine changes in residues involved in the alpha1beta2 contacts. There is no Bohr effect in shark haemoglobin, and of the residues normally involved in this effect the C-terminal histidine residue of the beta-chain is present, but the aspartyl (FG1) residue to which it is salt-linked is not, being replaced by a glutamyl residue.     

Myoglobin of the shark Heterodontus portusjacksoni: isolation and amino acid sequence

Myoglobin isolated from red muscle of the shark H. portusjacksoni was purified by ion-exchange chromatography on sulfopropyl-Sephadex and gel-filtration. Amino acid analysis and sequence determination showed 148 amino acid residues. The amino terminal residue is acetylated as shown by mass spectrographic analysis of N-terminal peptides. There is a deletion of four residues at the amino terminal end as well as one residue in the CD interhelical area relative to other myoglobins. The complete amino acid sequence has been determined following digestion with trypsin, chymotrypsin, pepsin and staphylococcal protease. Sequences of the purified peptides were determined by the dansyl-Edman procedure. The amino acid sequence showed approximately 85 differences from mammalian, monotreme and bird myoglobins. The date of divergence of the shark H. portusjacksoni from these other orders was estimated at 450 +/- 16 million years, based on the number of amino acid differences between species and allowing for multiple mutations during the evolutionary period. This estimate agrees well with similar estimates made using alpha- and beta-globin sequences, in contrast to widely differing estimates of dates of divergence for monotremes using the same three globin chains. Compared with myoglobins from species previously studied, there are many more differences in amino acid sequences, and in many positions residues are found that are more characteristic of alpha- and beta-globins, suggesting a conservation of residues over a long period of evolutionary time. There are fewer stabilizing hydrogen bonds and salt-linkages than in other myoglobins.     

The requirement for macrophages in the in vitro immune response

Previous work from this laboratory indicated that an adherent, light-density, radiation resistant “accessory” cell was required for an in vitro response of mouse spleen lymphocytes to sheep erythrocytes (SRC) but not to another antigen, polymerised bacterial flagellin (POL). This paper confirms this observation, and demonstrates that the “accessory cell” for a SRC response is a macrophage.     

Production of auto-anti-idiotype antibody during the normal immune response. XI. Ficoll-induced variations in auto-anti-idiotype production during the response to 2,4,6-trinitrophenyl-Ficoll

The responses to 2,4,6-trinitrophenyl conjugates of different Ficoll preparations differ with respect to the magnitude of the accompanying auto-anti-idiotype (Id) response in both mice and chickens. Evidence is presented that reduced auto-anti-Id production in the chicken is due to the activation of suppressor activity by some preparations of Ficoll.     

Mutagenic activity of oxathiolane steroids: structural requirement for the genotoxic activity in Salmonella and E. coli.

Oxathiolanes and disulfonyl derivatives of steroids were tested for mutagenic activity in the Ames tester strains. The test compounds exhibited mutagenic activity without metabolic activation although metabolic activation markedly enhanced their activity. A significant decrease in the survival of the radiation-sensitive mutants recA, lexA and rer of E. coli was observed as compared to their wild-type counterpart in the presence of the test steroid. Structural features which appear to be crucial for the mutagenic activity in these steroidal drugs are: (i) an electron-donating group at position 3, and (ii) a bulky group anchored at the 5th and 6th positions. The test steroids appear to damage DNA which in turn initiates the SOS repair with the concomitant induction of mutation.     

Expression, purification, and structural characterization of the bacteriorhodopsin-aspartyl transcarbamylase fusion protein.

We are testing a strategy for creating three-dimensional crystals of integral membrane proteins which involves the addition of a large soluble domain to the membrane protein to provide crystallization contacts. As a test of this strategy we designed a fusion between the membrane protein bacteriorhodopsin (BR) and the catalytic subunit of aspartyl transcarbamylase from Escherichia coli. The fusion protein (designated BRAT) was initially expressed in E. coli at 51 mg/liter of culture, to yield active aspartyl transcarbamylase and an unfolded bacterio-opsin (BO) component. In Halobacterium salinarum, BRAT was expressed at a yield of 7 mg/liter of culture and formed a high-density purple membrane. The visible absorption properties of BRAT were indistinguishable from those of BR, demonstrating that the fusion with aspartyl transcarbamylase had no effect on BR structure. Electron microscopy of BRAT membrane sheets showed that the fusion protein was trimeric and organized in a two-dimensional crystalline lattice similar to that in the BR purple membrane. Following solubilization and size-exclusion purification in sodium dodecyl sulfate, the BO portion of the fusion was quantitatively refolded in tetradecyl maltoside (TDM). Ultracentrifugation demonstrated that BR and BRAT-TDM mixed micelles had molecular masses of 138 and 162 kDa, respectively, with a stoichiometry of one protein per micelle. High TDM concentrations (>20 mM) were required to maintain BRAT solubility, hindering three-dimensional crystallization trials. We have demonstrated that BR can functionally accommodate massive C-terminal fusions and that these fusions may be expressed in quantities required for structural investigation in H. salinarum.     

Structure and functions of the genital ducts of the male Port Jackson shark,Heterodontus portusjacksoni

Synopsis The genital ducts ofHeterodontus portusjacksoni consist of the sperm carrying ducts (the rete testis, ductuli efferentes, and initial and terminal segments of the ductus epididymidis) and the Leydig glands (anterior opisthonephros). The ducts are lined by a ciliated epithelium which maintains a barrier to the transport of solute between blood and the lumen of the duct. Spermatozoa, Sertoli cell bodies, Sertoli cell cytoplasts and cellular debris are released from spermatocysts into the longitudinal canal of the rete testis. However, only the Sertoli cell cytoplasts persist throughout the sperm ducts. The epithelia lining the initial segment of the ductus epididymidis and secretory tubules of the Leydig glands are specialized for protein secretion and (particularly the Leydig glands) must be the main source of luminal protein in the ductus epididymidis. The epithelium lining the terminal segment of the ductus epididymidis also secretes protein, reabsorbs fluid and sodium, and may carry out heterophagic digestion. Spermatozoa develop the capacity for motility in the extratesticular sperm ducts, but do not undergo structural changes. However, they form spherical bundles in the terminal segment of the ductus epididymidis. It is suggested that the reduction in ratio of sodium:potassium from 48:8 in the ductuli efferentes to 3:4 in the distal end of the terminal segment of the ductus epididymidis may favour sperm survival.