Primitive Vertebrates Research Articles

Characterization of adrenergic receptors and related transduction pathways in the liver of the rainbow trout

Epinephrine (EPI) is thought to act by stimulating adenylyl cyclase (ACase) and cAMP production through β-adrenoceptors in the liver of more primitive vertebrates. Recent observations, however, point to an involvement of α 1-adrenoceptors in EPI action, at least in some fish species. The role of the α 1- and β-adrenergic transduction pathways has been investigated in rainbow trout (Oncorhynchus mykiss) hepatic tissue. Radioligand-binding assays with the β-adrenergic antagonist 3H-CGP-12177 using hepatic membranes purified on a discontinuous sucrose gradient confirmed the presence of β-adrenoceptors ( K d0.36 nM, B max 8.61 fmol · mg −1 protein). We provide the first demonstration of α 1-adrenoceptors in these same membranes; analysis of binding data with the α 1-adrenergic antagonist 3H-prazosin demonstrated a single class of binding sites with a K dof 15.4 nM and a B max of 75.2 fmol · mg −1 protein. There is a straight correlation between β-adrenoceptor occupancy, ACase activation and cAMP production. On the contrary, the role of inositol 1,4,5-trisphosphate (IP 3) has to be elucidated; in fact, despite the presence of specific microsomal binding sites for IP 3 ( K d 6.03 nM, B max 90.2 fmol · mg −1 protein), its cytosolic concentration was not modulated by EPI. On the other hand, we have previously shown in American eel and bullhead hepatocytes that α 1-adrenergic agonists are able to increase intracellular concentrations of IP 3 and Ca 2+ and to activate glycogenolysis. These data suggest a marked variation in the liver of different fish both in terms of α 1-binding sites affinity and of α 1-adrenoceptor/IP 3/Ca 2+ transduction systems.

The structure and physical properties of invertebrate and primitive vertebrate arteries.

Light and electron microscopy and in vitro inflation experiments were conducted on the aortae of three different invertebrate species: the lobster Homarus americanus, the horseshoe crab Limulus polyphemus and the whelk Busycon contrarium. Inflation experiments were also performed on the aortae of two species of primitive vertebrates, the sea lamprey Petromyzon marinus and the Atlantic hagfish Myxine glutinosa. The inflation experiments demonstrated similar overall biomechanical properties in each case, despite the existence of differences in tissue structure. The vessels were compliant at low strains, but demonstrated nonlinear elasticity, increasing in stiffness as strains increased; this property could act as protection against artery wall rupture. The vessels of the lamprey, hagfish and lobster are capable of acting as fairly efficient elastic reservoirs and of smoothing blood flow during circulation as they had low hysteresis values (13-18%). The aortae of the horseshoe crab and whelk, if performing this function, have much higher energy losses, up to more than 30% per cycle. The microscopy studies of the aortae of the lobster, horseshoe crab and whelk revealed tissue structures which differ widely from each other as well as from the structures of the lamprey and hagfish. None of these arteries contained elastin, but all contained fibrillar material which differed in appearance, size and arrangement between species. These materials were conjectured to contribute to the elastic properties of the tissue.

Macrophage‐lymphocyte cell clusters in the hypothalamic ventricle of some elasmobranch fish: Ultrastructural analysis and possible functional significance

Previous studies have demonstrated the existence of lympho-haemopoietic tissue in the meninges and choroid plexuses of various primitive vertebrates, including the stingray Dasyatis akajei and in early human embryos. In the present study, we extend these results analyzing macrophage-lymphocyte cell clusters found in the floor of the hypothalamic ventricle of several specimens of elasmobranchs. After aseptical isolation of the brain from several specimens of smooth dogfish Triakis scyllia, cloudy dogfish Scyliorhinus torazame, gummy shark Mustelus manazo, and stingray Dasyatis akajei their hypothalamic regions were processed routinely by light, scanning, and transmission electron microscopy. The study of serial histological sections demonstrated that the macrophage-lymphocyte cell clusters proceeded from the meningeal lymphohaemopoietic tissue, reaching the ventricular lumen along large blood vessels. In this tissue, macrophages, different sized lymphocytes, lymphoblasts, granulocytes, monocytes, and developing and mature plasma cells were closely packed among a meshwork of fibroblastic reticular cell processes. It never invaded the brain parenchyma. A cell layer of glial elements and a continuous basement membrane interposed between the lymphoid tissue and the neural elements although some macrophages had migrated across the ependymal cell layer. In the ventricular lumen very irregular macrophages with long cell processes and containing abundant engulfed material of unknown origin formed big cell clusters with neighboring lymphocytes, lymphoblasts, and plasma cells, similar to those described during the immune response. Moreover, electron lucent cells which resembled the antigen-presenting cells of higher vertebrates established intimate surface cell contacts with the surrounding lymphocytes. In the third ventricle of several specimens of gummy shark, Mustelus manazo, morphologically similar cell clusters appeared but these were not connected to the meningeal lympho-haemopoietic tissue. No intraventricular cell aggregates were found in the stingray brain. Although we cannot rule out that these macrophage-lymphocyte cell clusters represent a permanent structure in the elasmobranch brain they rather seem to be only established after specific stimulation for preventing the entrance of noxious, foreign materials into the elasmobranch brain parenchyma.

Low‐frequency auditory behavior in sea lampreys, primitive fish, and marine turtles

Sea lampreys (Petromyzon marinus) are one of only two species of living jawless fish. The brains and ears of lampreys are remarkably similar to that of fossil Ostracoderms, a group of fish that existed over 500 millions years ago. Jawed fish are derived from jawless fish which in turn radiated into primitive bony fish as gars (Lepisosteus) and teleosts and into the line leading to development of tetrapods. Marine turtles (Caretta c. caretta; Lepidochelys kempi) are considered primitive in that they share many characteristics with stem reptilian stock from which birds and mammals also radiated. The three species responded behaviorally and electrophysiologically to very low frequencies in a similar fashion despite their obvious morphological differences. All appear to share an ability to detect substrate vibrations, suggesting that the primitive vertebrate ear was also a low‐frequency vibration sensor. It is hypothesized that the first vertebrates evolved an otic organ for both equilibrium and vibration and each function was not separated until jawed vertebrates. In the primitive forms as in living lampreys, the brain must inhibit the ear function when detection of movement and gravity is needed.

Microwear on conodont elements and macrophagy in the first vertebrates

FEEDING mechanisms may hold the key to understanding the selective pressures that led to the evolution of vertebrates1–3. But in the absence of direct evidence, hypotheses of feeding mechanisms in the most primitive extinct vertebrates are somewhat speculative, and scenarios that seek to explain vertebrate origins remain controversial. The recognition that the affinities of conodonts lie among the most primitive vertebrates4–6 shifts the balance in this debate. I illustrate here microscopic wear patterns on conodont elements that provide the first unequivocal evidence that they functioned as teeth. These microwear patterns were produced as food was crushed and sheared between opposed conodont elements brought into bilateral occlusion. The presence of teeth and evidence of macrophagy in such primitive and early vertebrates support hypotheses that the first vertebrates were predators.

Conservation of IGFBP structure during evolution: Cloning of chicken insulin-like growth factor binding protein-5

The insulin-like growth factor binding proteins (IGFBPs) have conserved characteristics of their genomic organization, including similar locations of exon borders relative to nucleotides encoding conserved cysteine residues. Furthermore, the human IGFBP genes, as well as the human homeobox (HOX) genes, are localized to chromosomes 2, 7, 12, and 17. Although little is known about the evolution of the IGFBP genes, the association of human IGFBP and homeobox (HOX) genes at four chromosomal loci may indicate that their ancestral genes were linked prior to the first duplication of chromosomal DNA containing the ancestral HOX cluster. The hypothesis that IGFBPs are ancient proteins is supported by the reported detection of IGFBP activity in serum from the Agnathan species, Geotria australis, a primitive vertebrate. Further studies of IGFBPs in different species are needed to understand the evolution of this protein/gene family. Chicken provides a good intermediate model, since birds diverged from mammals ∼300 million years ago. A complementary DNA (cDNA) clone encoding chicken insulin-like growth factor binding protein-5 (cIGFBP-5) was isolated. The deduced amino acid sequence is 83% identical to human IGFBP-5 and encodes a mature polypeptide of 251 amino acids. The conservation of IGFBP-5 primary structure across vertebrate species suggests maintenance of important functions during evolution.

The neuronal network for locomotion in the lamprey spinal cord: Evidence for the involvement of commissural interneurons

The spinal cord of the lamprey, a primitive vertebrate, has been used as a model system for investigating the cellular basis of rhythmic locomotor activity. Three classes of interneurons have been characterized that are active during locomotor activity in the isolated spinal cord ( ie fictive swimming). The identified synaptic interactions of these neurons form a network which has been proposed to underlie locomotor rhythmogenesis. Modeling studies confirmed that the network can produce oscillatory activity with phase relations among the neurons similar to those found in the spinal cord. Within the network, inhibitory commissural interneurons form reciprocal inhibitory connections and play a key role in rhythmogenesis. Several experiments have been done to test whether these cells participate in the generation of rhythmic activity in the spinal cord. First, midline lesions that sever the axons of commissural interneurons eliminate rhythmic ventral root bursting. Second, photo-ablation of commissural interneurons on one side of the spinal cord alters the symmetry of ventral root bursts, alters the cycle period, and can eliminate rhythmic bursting. Taken together, these experiments support the model that commissural interneurons are involved in rhythmogenesis in the lamprey spinal cord.

Hagfish biopolymer: a type I/type II homologue of epidermal keratin intermediate filaments

In contrast to most intermediate filaments (IF) which function intracellularly or constitute epidermal appendages, the single massive (≈60 cm length, ≈3 μm width) IF-rich ‘thread’ biopolymer synthesized by the specialized hagfish gland thread cell is released extracellularly via holocrine secretion to interact with mucins and seawater, thereby modifying the viscoelastic properties of the copious mucous exudate. Recently, using the Pacific hagfish ( Eptatretus stouti, class Agnatha), a jawless scaleless marine vertebrate of ancient lineage, we determined that the deduced amino acid sequence of one thread IF chain (α, 66.6 kDa, native pl 7.5) contained an atypical, threonine-rich central rod domain of low identity (< 30%) with other vertebrate IF types, but that the N- and C-terminal domains exhibited several keratin-like features. From these and other unexpected characteristics, it was concluded that hagfish α is best categorized as a type II homologue of an epidermal keratin. We now report the deduced sequence of a second thread IF subunit (γ, 62.7 kDa, native pl 5.3) which is co-expressed and co-assembles in vitro with α in a 1:1 ratio. As was found for α, the N- and C-terminal domains of γ have keratin-like parameters, but the central rod has low identity to IFs of types I-V (< 31%), a cephalochordate I F (< 29%) and invertebrate IFs (<20%) and no particular homology to type I or type II keratins. Central rod identity between γ and α is also low (≈23%), as is typical of comparisons between different rod types but atypical of similar rod types (>50%). The central rods of both γ and α lack the 42-residue insert of helix 1B present in lamins and invertebrate IFs, have unusually high threonine contents (γ, 10%; α, 13%) compared to other IF types (2–5%), contain a number of unexpected residues in consensus conserved sites, and employ a L12 segment of 21 residues rather than the 16 or 17 residues found in keratins. Theoretical analyses indicate that the hagfish molecules exist as coiled coil heterodimers (α/γ) in which the chains are parallel, in axial register, and stabilized by significant numbers of ionic interactions. Fast Fourier-transform analyses revealed that the linear distribution period of ≈9.55 for basic and acidic residues in other IF chains is not completely maintained, partly due to the high threonine content. The threonine residues occupy mainly outer sites b, c, f in the heptad substructure, possibly abetting parallel alignment of thousands of IFs within the thread, interactions with mucins at the thread periphery, and hierarchical IF chain assembly. It is suggested that the γ and α chains from this most primitive extant vertebrate are type I and type II homologues of epidermal keratin chains, possibly related to early specialized keratins.

Alcohol Dehydrogenase Class III Contrasted to Class I

Alcohol dehydrogenases of classes I (the classical liver enzyme) and III (formaldehyde dehydrogenase) constitute a pair of moderately related enzymes (63% residue identity between the human forms) that differ fundamentally in many respects. To elucidate the nature of the differences, we have characterized alcohol dehydrogenase from the most primitive vertebrate line (a cyclostome, Atlantic Hagfish), related that to the multiplicity of the human enzyme, and submitted the enzymes to in vitro hybridization for evaluation of subunit interactions. Three findings illustrate important principles of the enzyme system. First, the alcohol dehydrogenase purified from cyclostomes is a class-III protein, compatible with the facts that cyclostomes constitute the earliest extant vertebrate line and that class III has a distant pre-vertebrate origin. Second, the hagfish enzyme shows multiplicity, with acidic forms in decreasing yield and with amino acid sequences identical between two major isoforms, both aspects constituting properties similar to those of the corresponding human forms. The chemically different subunits are present as homodimers and heterodimers of unmodified and modified subunits, suggesting that the class-III multiplicity derives from modification of a type common to lines as divergent as mammals and cyclostomes. Third, the human enzyme can form cross-species hybrid dimers in vitro with the cod and hagfish or Drosophila class-III enzymes (positional identity with the human form of 82, 76 and 70%, respectively). Hence, the results provide experimental evidence for little class-III divergence in the segments of subunit interactions. The extent of conservation of residues directly involved in the formation of the subunit interface also reveals a clearly different pattern between classes I and III. This highlights separation of divergent forms in an enzyme system, with the constant form (class III) resembling house-keeping enzymes, and exhibiting a correlation between subunit-interacting and substrate-interacting segments.

The Nucleotide and Deduced Amino-Acid Sequences of a cDNA Encoding Lactate Dehydrogenase from Caenorhabditis elegans: The Evolutionary Relationships of Lactate Dehydrogenases from Mammals, Birds, Amphibian, Fish, Nematode, Plants, Bacteria, Mycoplasma, and Plasmodium

The nucleotide and deduced amino-acid sequences of a cDNA encoding L-lactate dehydrogenase (LDH) from nematode, Caenorhabditis elegans, were reported. This first invertebrate LDH sequence of 333 amino acids, including the initiation methionine, exhibits 63% identity with that of the most primitive vertebrate lamprey. The evolutionary relationships among 36 LDH isozymes from mammals, birds, amphibian, fish, nematode, plants, bacteria, mycoplasma and plasmodium were analyzed. The invertebrate nematode LDH is evolutionarily positioned between plant LDH and mammalian testicular LDH-C isozymes. The mammalian LDH-C isozyme appears to have arisen after the invertebrate LDH, but prior to the divergence of vertebrate LDH-A (muscle) and LDH-B (heart) isozymes as described previously.

Hsp 90α and Hsp 90β Genes Are Present in the Zebrafish and Are Differentially Regulated in Developing Embryos

We have employed a polymerase chain reaction-based cloning strategy to demonstrate that both hsp 90α and hsp 90β genes are present in the zebrafish. The fact that zebrafish represents the most primitive vertebrate in which hsp 90 genes have been isolated to date has allowed us to determine that the duplication event which generated the hsp 90α and hsp 90β genes occurred shortly before the emergence of the teleosts from the rest of the vertebrate lineage. In expression studies using Northern blot analysis, hsp 90β mRNA was found to be present at control temperatures throughout normal embryonic development whereas hsp 90α mRNA was barely detectable. Upon heat shock, hsp 90α mRNA levels increased dramatically in all developmental stages examined. The levels of hsp 90β mRNA increased 2-3 fold during heat shock of early stage embryos. Thus, the hsp 90α gene is strongly upregulated during heat shock in zebrafish embryos whereas expression of the hsp 90β gene appears to be weakly induced.

T-cell receptor gene homologs are present in the most primitive jawed vertebrates.

The phylogenetic origins of T-cell immunity and T-cell antigen receptor (TCR) genes have not been established. A PCR approach using short, minimally degenerate oligodeoxynucleotide primers complementing conserved variable region segments amplifies TCR-like products from the genomic DNA of Heterodontus francisci (horned shark), a representative phylogenetically primitive cartilaginous fish. One of these products has been used as a probe to screen a Heterodontus spleen cDNA library and a clone was identified that is most related at the nucleotide sequence and predicted peptide levels to higher vertebrate TCR beta-chain genes. Genomic analyses of the TCR homologs indicate that recombining variable and joining region segments as well as constant region exons are encoded by extensive gene families, organized in the multicluster form, characteristic of both the immunoglobulin heavy- and light-chain gene loci in the cartilaginous fishes. Greater numbers of homologous products were identified when a probe complementing the putative constant region of the TCR homolog was used to screen the same cDNA library. A high degree of intergenic variation is associated with the putative variable region segments of these isolates. Direct evidence is presented for TCR-like genes, which presumably are associated with T-cell function, at the earliest stages in the phylogenetic emergence of jawed vertebrates.

A cell-surface opsonic receptor on leucocytes from the phylogenetically primitive vertebrate, Eptatretus stouti.

A humoral recognition molecule that is homologous to the mammalian complement components C3, C4 and C5 has recently been identified in the Pacific hagfish, Eptatretus stouti. One function of this complement-like protein (CLP) is to opsonize foreign material for phagocytosis by hagfish leucocytes. Here, we demonstrate that CLP's opsonic activity can be abrogated by pre-incubating phagocytes with an anti-hagfish leucocyte mAb (1B1). Moreover, antigen-activated CLP can block the binding of the 1B1 antibody to hagfish leucocytes. Flow cytometry and immunoprecipitation indicate that 1B1 recognizes a 105 kDa cell-surface, monomeric protein that is expressed exclusively on phagocytic hagfish leucocytes. It is concluded that this 105 kDa protein represents the cell surface receptor by which CLP mediates the phagocytosis of opsonized targets.

Structure of reticulospinal axon growth cones and their cellular environment during regeneration in the lamprey spinal cord

The large larval sea lamprey is a primitive vertebrate that recovers coordinated swimming following complete spinal transection. An ultrastructural study was performed in order to determine whether morphologic features of regenerating axons and their cellular environment would provide clues to their successful regeneration compared to their mammalian counterparts. Three larval sea lampreys were studied at 3, 4 and 11 weeks following complete spinal transection and compared with an untransected control. Müller and Mauthner cells or their giant reticulospinal axons (GRAs) were impaled and injected with horseradish peroxidase (HRP). Alternating thick and thin sections were collected for light and electron microscopy. A total of 9 neurites were examined. At all times, growth cones of GRAs differed from those of cultured mammalian neurons in being packed with neurofilaments and in lacking long filopodia, suggesting possible differences in the mechanisms of axon outgrowth. Morphometric analysis suggested that GRA growth cones contact glial fibers disproportionately compared to the representation of glial surface membranes in the immediate environment of these growth cones. No differences were found between glial cells in regenerating spinal cords and those of untransected control animals with regard to the size of the cell body and nucleus and the packing density of their intermediate filaments. Glial fibers in control animals and glial fibers located far from a transection were oriented transversely. Glial cells adjacent to the transection site sent thickened, longitudinally oriented processes into the blood clot at the transection site. These longitudinal glial processes preceded the regenerating axons. Desmosomes were observed on glia adjacent to the lesion but were scarce in the lesion during the first four weeks post-transection. These findings suggest that longitudinally oriented glial fibers may serve as a bridge along which axons can regenerate across the lesion. The presence of desmosomes might prevent migration of astrocytes near the transection, thus stabilizing the glial bridge.

Recombinant protein fragments from haemorrhagic septicaemia rhabdovirus stimulate trout leukocyte anamnestic responses in vitro

This work shows that viral protein fragments are capable of stimulating fish anamnestic immunological responses in leukocytes from the rainbow trout (Oncorhynchus mykiss, W.). Recombinant protein fragments of glycoprotein and nucleoprotein from the rhabdovirus causing viral haemorrhagic septicaemia of trout (VHSV), were cloned and expressed in Escherichia coli, Yersinia ruckeri (a trout pathogen) and Saccharomyces cerevisiae. The recombinant protein fragments stimulated anamnestic responses in leukocyte cultures derived from the anterior kidney of survivors of VHSV infection but not from uninfected trout. Two types of stimulatory anamnestic responses were detected, (i) a stimulation of lymphoproliferation as measured by thymidine incorporation assays and (ii) an increase in number, spreading and size of cells as determined by fibrin-clot and/or flow cytometry techniques. The evidence presented suggests that both adherent and non-adherent trout cell populations are needed for the immunological response to VHSV in this primitive vertebrate. The possible use of in vitro lymphoproliferation assays as a preliminary screening method for candidate fish vaccines prior to their testing in vivo is discussed.

The pathophysiology of venous thrombosis

HE PATHOPHYSIOLOGY of venous thrombosis directly relates to the evolutionary developments of the cardiovascular system. All members of the animal kingdom have some form of blood. In the more primitive species, this is called hemolymph. In crustacea, this material contains proteins that can coagulate with trauma but there is no enzymic clotting cascade. In more evolved species such as horseshoe crabs, a series of serine protease zymogens are present, but there is no recognizable fibrinogen. Coagulation proceeds through sticky white blood cells. In contrast, even the more primitive vertebrates, such as Lampreys, have fibrinogen, prothrombin, tissue factor, and plasminogen and, in overall organization, are analogous to the situation in humans. The presence of a coagulation system is required for the maintenance of fluid-derived bodily nutrition and, hence, is essential for life. Unlike the open low pressure circulation system of the invertebrates, in higher animals and, in particular, in mammals, an enclosed high pressure circulation exists. This situation exposes more extreme demands on the hemostatic and flow requirements of the circulatory system. The net result is the concept of a balance between the requirements to allow blood to remain fluid to provide organ nutrition yet, at the same time, to be able to rapidly form blood clots. The biologic divergence of these two competing requirements probably explains the multiple levels of activity and regulation in the hemostatic system. The simplistic approach to the regulation of thrombosis is to divide the process into discrete compartments. These are the constituents of Virchov's classical triad of causes of thrombosis that involved vessel wall factors, blood clotting, and blood flow characteristicsJ In practice, these components have evolved as a highly integrated process, but, for the purpose of discussing their functions, it is convenient to maintain the traditional format and to crossreference interactions.

Development of an immune system.

Minimally, an immune response is an induced cellular and/or humoral defense mechanism specific for the challenging agent. The system is a cognitive one inasmuch as a second stimulus with the same antigen can specifically induce either an enhanced response (memory) or diminished response (tolerance). The cells responsible for the initial antigen-specific recognition in higher vertebrates are clonally restricted T and B lymphocytes. Accessory cells are necessary for the processing and presentation of antigen, and physiologic mediators (cytokines) are essential for proliferation, interaction, and regulation of the system. Although it now appears that the recombination mechanisms essential for the anticipatory immune response occurred late in the deuterostome stream leading to vertebrates, molecules required for cell adhesion and regulation are widely spread in phylogeny. Their emergence must have preceded the divergence between ancestral protostomes and deuterostomes. Genetic mechanisms underlying the generation of diversity in the light and heavy chains of antibodies of mammals may be quite distinct in primitive vertebrates, particularly elasmobranchs, the ancestors of which diverged from those of mammals more than 400 million years ago. Despite this, clonal selection of antigen receptors of lymphocytes is most probably universal within the vertebrates. There is no need to force induced recognition in protostomes (e.g. insects) or lower deuterostomes (e.g. echinoderms) into mammalian models of immunity.

Cell surface recognition and the immunoglobulin superfamily.

Immunoglobulins serve as humoral recognition and effector molecules and as antigen-specific cell surface receptors on B and T cells. These molecules are constructed according to a characteristic domain pattern. Variable and constant domains diverged from one another early in vertebrate evolution, and they are joined by a "switch peptide" specified by the joining gene segments. Peptides specified by J-gene segments are strongly conserved in evolution in comparison among Ig light chains and T-cell receptors. Molecules less strongly related to Ig domains have been assembled into an Ig "superfamily" where the identities to classical IgC or V domains are < or = 20%. Among these are cell surface adhesion molecules, receptors for cytokines, and Fc receptors. Moreover, MHC antigens have an Ig-like membrane-proximal domain significantly related to IgC regions. We will analyze putative evolutionary relationships among canonical Igs and members of the Ig superfamily using highly conserved sequences from light and heavy chains of primitive vertebrates (e.g., the sandbar shark) as prototypes to ascertain similarities between Ig-related molecules of vertebrates and invertebrates.

Molecular cloning of a lamprey homologue of the mammalian MHC class III gene, complement factor B.

To elucidate the origin and evolution of the complement system and the MHC, we isolated cDNA clones for the MHC class III complement factor B (Bf) gene from lamprey, one of the most primitive extant vertebrates. A part of the serine protease domain of the lamprey Bf was amplified by reverse transcriptase-PCR using the degenerated primers corresponding to the conserved amino acid stretches between the mouse Bf and C2 sequences. A full-length lamprey Bf cDNA clone was isolated from the lamprey liver cDNA library using a PCR-amplified DNA clone as a probe. The deduced amino acid sequence of 763 residues showed essentially the same domain structure as mammalian Bf or C2, consisting of three short consensus repeat domains, a von Willebrand domain, and a serine protease domain. Lamprey Bf showed 33 and 29% overall amino acid similarity to mouse Bf and mouse C2, respectively, whereas amino acid similarity between mouse Bf and mouse C2 was 36%, suggesting that the gene duplication of Bf/C2 occurred in the main line of vertebrate evolution after the divergence of cyclostomes. This is the first report of the molecular cloning from cyclostomes of a component of the mammalian MHC that offers the possibility of genetic analysis of the presumably primitive MHC of cyclostomes.

In vitro motor activity of the lamprey ( Lampetra japonica) intestine

The intestine of the lamprey (primitive vertebrate), was divided into four segments (SI-SIV) of about 4cm from oral to anal. The frequency and amplitude of their spontaneous motor activities (intraluminal pressure) were studied in vitro. Frequency gradient from oral SI (1.44 cycle/min) to anal SIV (0.26 cycle/min) was found. In contrast, the amplitude gradient (0.14–0.36 mmHg) was quite the reverse and SIV was the largest and SI was the smallest. Raising the temperature from 5 to 10–15°C almost linearly increased both frequency and amplitude in Arrhenius's plot. Acetylcholine (1–10 mM) and 5-HT (1–10 μM) directly induced the contractions of the longitudinal and circular smooth muscles in the presence of 1 μg/ml tetrodotoxin (TTX). Atropine (5–20 μM) shifted the dose-response curves of acetylcholine to the right but hardly affected the 5-HT responses. Spiperone and ketanserine (10–40 μM) shifted the dose-response curves of 5-HT to the right but hardly affected the acetylcholine responses. Stimulating the anal part of SIII and SIV by 3–9 g balance weights via hook and pulley induced the contractions (0.2–2 g) of both oral and anal site (26.5% of trial) or one side of oral (50.0%) or anal site (23.5%) about 2–6 mm apart from the stimulated point. Intrinsic reflex arcs seem to operate in the caudal lamprey intestine. The intrinsic reflex responses were suppressed by about 50% in the presence of 14–29 μg/ml curare and suppressed by 80–100% in the presence of 20 μM spiperone or ketanserine. Atropine 20 μM had almost no inhibitory effects on the reflex. It was suggested that lamprey intestinal smooth muscles seem to have separate receptors for acetylcholine and 5-HT. Intrinsic reflex may be mediated both by cholinergic and serotonergic interneurones and more probably by serotonergic motoneurones. These characteristics of the lamprey were discussed by comparison with those of other vertebrate intestines.