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.     

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.     

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.     

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.     

Conservation, innovation, and the evolution of horned beetle diversity

Beetle horns represent an evolutionary novelty exhibiting remarkable diversity above and below the species level. Here, we show that four typical appendage patterning genes, extradenticle (exd), homothorax (hth), dachshund (dac), and Distal-less (Dll) are expressed in the context of the development of sexually dimorphic thoracic horns in three Onthophagus species. At least two of these genes, Dll and hth, exhibited expression patterns consistent with a conservation of patterning function during horn development relative to their known roles in the development of insect legs. exd, hth, and dac expression patterns during horn development were largely invariable across species or sexes within species. In contrast, Dll expression was far more discrete and exhibited consistent differences between sexes and species. Most importantly, differences in location and domain size of Dll expression tightly correlated with the degree to which prepupal horn primordia were retained or resorbed before the final adult molt. Our results lend further support to the hypothesis that the origin of beetle horns relied, at least in part, on the redeployment of already existing developmental mechanisms, such as appendage patterning processes and that changes in the exact location and domain size of Dll expression may represent important modifier mechanisms that modulate horn expression in different species or sexes. If correct, this would imply that certain components of genetic basis of horn development may be able to diversify rapidly within lineages and largely independent of phylogenetic distance. We present a first model that integrates presently available data on the genetic regulation of horn development and diversity.     

The Na-K-Cl cotransport protein of shark rectal gland. I. Development of monoclonal antibodies, immunoaffinity purification, and partial biochemical characterization.

The Na-K-Cl cotransporter mediates the coupled transport of Na, K, and Cl across the plasma membrane of many animal cell membranes. It is inhibited by loop diuretics such as furosemide, bumetanide, and benzmetanide. We have developed a panel of monoclonal antibodies directed against the 195-kDa shark rectal gland Na-K-Cl cotransport protein. Four representative antibodies (J3, J4, J7, and J25), each of which recognizes a discrete structural domain, were selected for detailed characterization. When a radiolabeled loop diuretic is bound to the cotransporter prior to solubilization, each antibody immunoprecipitates the same diuretic-protein complex. Of the four antibodies, J4 favors the native protein over the denatured one and does not bind well to proteolytic fragments; in contrast, J7 recognizes the cotransporter only after it has been solubilized. J3, J7, and J25 each recognize a unique ensemble of proteolytic fragments of the 195-kDa protein; analysis of the patterns of recognition has yielded a tentative assignment of the approximate location of the epitopes within the peptide. When the cotransport protein is treated with N-glycanase to remove N-linked oligosaccharides, its apparent mass decreases to approximately 135 kDa. The deglycosylated form is recognized by each of the antibodies except J25; this suggests that the J25 epitope is within the oligosaccharide component or in a peptide domain whose folding is disturbed by carbohydrate removal. An immunoaffinity matrix constructed with the J4 antibody permits single-step purification of the 195-kDa protein; other proteins copurify with the large glycoprotein, but none of these appear to be subunits of a stoichiometric complex. The amino acid sequence of four fragments of the 195-kDa cotransport protein is reported. Immunofluorescence and immunoelectron microscopy demonstrates, in agreement with physiological evidence, that the 195-kDa protein is distributed along the basolateral membrane and excluded from the apical membrane of the rectal gland secretory cell.