Primitive Vertebrates Research Articles

Maturation of hagfish gland thread cells: Composition and characterization of intermediate filament polypeptides

Previous studies with the hagfish, a primitive vertebrate, have shown that the gland thread cells (GTCs) each contain a single thread (approximately 60 cm long in average-sized cells) in the form of a concisely coiled cytoskeletal entity destined for export by holocrine secretion. The thread in relatively immature GTCs consists almost entirely of intermediate filaments (IFs) bundled in parallel alignment with far fewer microtubules (MTs). The three thread polypeptides described earlier (alpha, basic; beta, acidic; gamma, most acidic; each with a Mr of 63-64 kD) are now further evaluated with respect to in vitro assembly, cross-reactivity with IF polypeptides from higher vertebrates, and peptide sequence homology with known IF polypeptides. The overall results mainly suggest that the hagfish polypeptides are keratinlike substances but lamins or a new type of IF is not ruled out. However, cross-reactivity is weak with mammalian keratins; the 8-11-nm filaments formed from mixtures of alpha and gamma in vitro are generally linear rather than the curvilinear structures usually formed by keratin and nonkeratin IFs; and mixtures of alpha and beta tend to yield 9-12-nm granules or granular strings. Polypeptide analyses on GTCs segregated on the basis of maturational stage show a progressive increase in beta/gamma values which correlates with cell maturation, but the alpha/(beta + gamma) ratios remain near 1. Inasmuch as beta and gamma have many similar properties, the documented increase in the amount of the beta component in aging GTCs might in part be the result of a failure in a posttranslational modification system and may contribute to the ultrastructural changes that accompany thread maturation in preparation for holocrine secretion and subsequent modulation of the viscoelastic properties of mucus.

Immunocytochemical studies on the islet and the gut of the arctic lamprey, Lampetra japonica.

The endocrine cells and nerves in the islet and the gut of the arctic lamprey Lampetra japonica were examined immunocytochemically by using antisera against brain-gut peptides and amine. The cellular composition of the islets as reported by previous researchers in European species of the lamprey was confirmed in the present study. The islet consisted exclusively of insulin immunoreactive cells in the larvae (ammocoetes), whereas in the adult somatostatin immunoreactive cells were added to the insulin immunoreactive cells; the gut epithelium in the adult was now devoid of somatostatin cells. In the gut of the lamprey, the endocrine cells--which were flask-shaped with a cytoplasmic process extended to the lumen--were classified into three types in the larvae, but were represented by a single type in the adult. In the larval lamprey, the first type was immunoreactive for somatostatin, the second one for gastrin/cholecystokinin (CCK) and the third cell type was immunoreactive for glucagon, pancreatic polypeptide and FMRFamide, simultaneously. In the gut of the adult lamprey, the single type of endocrine cell reacted simultaneously to C-terminal specific anti-glucagon serum, N-terminal specific anti-glucagon serum, anti-bovine PP serum, anti-neuropeptide Y serum and anti-FMRFamide serum. These cells occurred most frequently in the upper intestine, their distribution decreasing from the middle to the lower intestine. Two types of peptide containing nerves were identified in the islet and the gut of the larval and adult lamprey. The first type of neurons (perikarya and fibers) was immunoreactive for serotonin and calcitonin gene-related peptide (CGRP), and was located in the mucous and muscular layer of the intestine and in the islet. The second type of neurons contained both serotonin- and gastrin releasing peptide (GRP)-like immunoreactivities and was scattered exclusively in the muscular layer of the gut. In larval and adult lampreys, a few serotonin/CGRP immunoreactive nerve cell bodies and beaded fibers were found in the connective tissue around the islet cell cords. These nerve fibers were sometimes closely apposed to the blood capillaries and to the islet cells. These findings indicate that a neuroendocrine correlation comparable with that in mammals may have been established in the islet of this most primitive vertebrate.

Fibril-forming collagens in lamprey.

Five types of collagen with triple-helical regions approximately 300 nm in length were found in lamprey tissues which show characteristic D-periodic collagen fibrils. These collagens are members of the fibril forming family of this primitive vertebrate. Lamprey collagens were characterized with respect to solubility, mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, carboxylmethyl-cellulose chromatography, peptide digestion patterns, composition, susceptibility to vertebrate collagenase, thermal stability, and segment long spacing-banding pattern. Comparison with fibril-forming collagens in higher vertebrates (types I, II, III, V, and XI) identified three lamprey collagens as types II, V, and XI. Both lamprey dermis and major body wall collagens had properties similar to type I but not the typical heterotrimer composition. Dermis molecules had only alpha 1(I)-like chains, while body wall molecules had alpha 2(I)-like chains combined with chains resembling lamprey type II. Neither collagen exhibited the interchain disulfide linkages or solubility properties of type III. The conservation of fibril organization in type II/type XI tissues in contrast to the major developments in type I and type III tissues after the divergence of lamprey and higher vertebrates is consistent with these results. The presence of type II and type I-like molecules as major collagens and types V and XI as minor collagens in the lamprey, and the differential susceptibility of these molecules to vertebrate collagenase is analogous to the findings in higher vertebrates.

The Brain of the Basking Shark <i>(Cetorhinus maximus)</i>

The brain of a basking shark is described for the first time. Allometrical analysis of brain-body weight relationships indicates that Cetorhinus maximus shows by far the lowest degree of cerebralization of any shark investigated to date, possibly reflecting its plankton-feeding mode of life. Furthermore, some external morphological features of the brain appear to be species-specific. The proportions of brain parts indicate a primitive vertebrate brain organization. The size of the telencephalon, 34% of the total brain, equals that in some other sharks, whereas the cerebellum, 30% of the total brain in the basking shark, is significantly larger than in any other shark investigated. Furthermore, C. maximus shows some peculiarities concerning the nuclear expansion in the telencephalon. A rather large caudal interhemispheric region is especially striking. The relation of brain volume to brain cavity volume is approximately 1:16.

Identification of a single sinusoidal bile salt uptake system in skate liver.

To identify the sinusoidal bile acid uptake system(s) of skate liver, photoaffinity labeling and kinetic transport studies were performed in isolated plasma membranes as well as intact hepatocytes. In both preparations photoaffinity labeling with the photolabile bile salt derivative (7,7-azo-3 alpha, 12 alpha-dihydroxy-5 beta-[3 beta-3H]cholan-24-oyl)-2-aminoethanesulfonate revealed the presence of a predominant bile salt binding polypeptide with an apparent molecular weight of 54,000. The labeling of this polypeptide was inhibited by taurocholate and cholate in a concentration-dependent manner and was virtually abolished by 1 mM of the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Kinetic studies of hepatic uptake with taurocholate, cholate, and the photoreactive bile salt derivative indicated the involvement of a single transport system, and all three substrates mutually competed with the uptake of each other. Finally, irreversible inhibition of the bile salt uptake system by photoaffinity labeling of hepatocytes with high concentrations (250 microM) of photolabile derivative reduced the Vmax but not the Km of taurocholate uptake. These findings strongly indicate that a single polypeptide with an apparent molecular weight of 54,000 is involved in sinusoidal bile salt uptake into skate hepatocytes. These findings contrast with similar studies in rat liver that implicate both a 54,000- and 48,000-K polypeptide in bile salt uptake and are consistent with a single Na+-independent transport mechanism for hepatic bile salt uptake in this primitive vertebrate.

Urinary bile alcohols in liver dysfunction

Bile alcohols are polyhydroxy steroids chemically related to bile acids and have long been regarded as major constituents of bile of primitive vertebrates [l]. Interest in bile alcohols increased when these compounds were found in both healthy and diseased humans as intermediates and side products of the normal pathway in bile acid biosynthesis [2,3]. In 1982, Karlaganis et al. [4] found an increased urinary excretion of bile alcohols in children with juvenile cirrhosis and cholestasis. LudwigKijhn et al. [5] also found an elevated amount of bile alcohols in the urine of patients with various liver diseases such as cirrhosis and hepatitis. We have measured urinary bile alcohols in various stages of cirrhosis particularly in cirrhosis with hepatic encephalopathy (HE), and observed their changes in the clinical course of acute hepatitis.

Extensive families of constant region genes in a phylogenetically primitive vertebrate indicate an additional level of immunoglobulin complexity.

A homologous probe for the constant region of the Heterodontus francisci (horned shark) immunoglobulin heavy chain was used to screen a genomic DNA library constructed in bacteriophage lambda, and a large number of independent clones were recovered. Their hybridization patterns with segment-specific probes are consistent with the close linkage of heavy-chain constant (CH), joining (JH), and variable (VH) gene segments. Differences in the nucleotide sequences of the first CH exon of five genes primarily are localized to 5' positions; extended regions of sequence identity are noted at 3' positions. The predicted amino acid sequences of each gene are different and are related distantly to the corresponding regions of higher vertebrate immunoglobulins. Gene-specific oligodeoxynucleotide probes were used to establish that at least three of the five genes are transcriptionally active. Quantitative gene titration data are consistent with the large numbers of genes suggested by the library screening analyses. In this representative early vertebrate, it appears that (VH-diversity-JH) segments are associated with individual constant region genes that can differ at the predicted protein level.

Phylogeny of C4b-C3b cleaving activity: similar fragmentation patterns of human C4b and C3b produced by lower animals.

Functional and structural studies of the activated proteins of the complement system C4b and C3b have led to the identification of cleavage products resulting from the effect of the regulatory proteins, factor I, H, and C4b binding protein (bp). In this paper we report the results of studies that investigated the capacity of plasma or serum from a wide range of phylogenetic species to yield similar cleavage products. Sera and plasma from mammals, reptiles, amphibia, and fishes are capable of cleaving fluid phase human C4b and C3b, generating apparently the same fragments as observed using normal human serum: alpha 2, alpha 3, alpha 4 from the alpha' chain of C4b: and alpha-68, alpha-46, alpha-43, and alpha-30 from the alpha' chain of C3b. When C3b bound to a cell membrane is used C3c and C3dg are generated. The generation of these fragments from C3bi is a dose-dependent reaction. There is no correlation between the evolution of the species and the quantitative capability to degrade the substrates. Birds possess only a limited capability to degrade the alpha' chain of C4b and have no cleaving activity for C3b, whereas sera from more primitive vertebrate species (chondrichthyes and agnatha) fail to participate in the reaction. Contrary to other species, the proteins in fish serum or plasma responsible for the degradation of C4b and C3b show a unique requirement for Ca2+ ions. Magnesium and barium are less effective, and in their presence a 65,000 dalton intermediate product is observed. These results demonstrate that protein(s) displaying proteolytic activity for products of complement activation, probably related to I, H, and C4bp, are present in plasma of species whose evolution have preceded humans by 300 million years. Moreover, the recognition of human substrates and the generation of fragments identical to those produced by human serum suggests that human C4b and C3b share structural characteristics with their evolutionary ancestors in the serum or plasma of the species studied.

Conservation among vertebrate immunoglobulin chains detected by antibodies to a synthetic joining segment peptide

We used affinity purified antibodies produced against a synthetic peptide sequence corresponding to the entire J β of a human T cell receptor gene to screen sera of man, mouse and other vertebrates to determine the presence of cross-reactive molecules. Little evidence for free α β heterodimers was found, but the antibody reacted with light chains of many vertebrate species, including characterized myeloma proteins of man and mouse. Some vertebrate orders, notably Aves, lacked polypeptide chains cross-reactive with J β, but detectable determinants occurred in primitive vertebrates such as the galapagos shark ( Carcharhinus galapagensis ). In addition to the strong cross-reaction with purified light chains, human heavy chains reacted weakly with the antibody. The cross-reaction correlated with the sequence of the denatured immunoglobulins and was inhibitable with free peptide. These results establish the similarity of T cell receptor β chains to immunoglobulin chains and support the conclusion that J region sequences were conserved, not only within mammalian immunoglobulins and T cell receptors, but in vertebrate evolution.

Acetylcholinesterase from the skeletal muscle of the lamprey Petromyzon marinus exists in globular and asymmetric forms.

To obtain information about the evolution of acetylcholinesterase (AChE), we undertook a study of the enzyme from the skeletal muscle of the lamprey Petromyzon marinus, a primitive vertebrate. We found that the cholinesterase activity of lamprey muscle is due to AChE, not pseudocholinesterase; the enzyme was inhibited by 1,5-bis(4-allyldimethylammonium phenyl) pentane-3-one (BW284C51), but not by tetramonoisopropyl pyrophosphortetramide (iso-OMPA) or ethopropazine. Also, the enzyme had a high affinity for acetylthiocholine and was inhibited by high concentrations of substrate. A large fraction of the AChE was found to be glycoprotein, since it was precipitated by concanavalin A-agarose. Optimal extraction of AChE was obtained in a high-salt detergent-containing buffer; fractional amounts of enzyme were extracted in buffers lacking salt and/or detergent. These data suggest that globular and asymmetric forms of AChE are present. On sucrose gradients, enzyme that was extracted in high-salt detergent-containing buffer sedimented as a broad peak of activity corresponding to G4; additionally, there was usually a peak corresponding to A12. Sequential extraction of AChE in conjunction with velocity sedimentation resolved minor forms of AChE and revealed that the G1, G2, G4, A4, A8, and A12 forms of AChE could be obtained from the muscle. The identity of the forms was confirmed through high-salt precipitation and collagenase digestion. The asymmetric forms of AChE were precipitated in low ionic strength buffer, and their sedimentation coefficients were shifted to higher values by collagenase digestion.(ABSTRACT TRUNCATED AT 250 WORDS)

Bioassayable cholecystokinin in the brain of the goldfish, [formula omitted][formula omitted

Cholecystokinin (CCK)-gastrin peptides are shown to be present in the brain of all the species ranging from coelenterates to mammals. Differentiation between CCK and gastrin, presumed to be evolved from a common ancestral cerulein-like peptide, has been suggested to occur at the level of the amphibians. We examined the presence of bioassayable CCK, as determined by its potency to stimulate enzyme secretion from isolated rat pancreatic acini, in the brain of the goldfish Carassius auratus , a more primitive vertebrate than the amphibian. Among the various regions tested, the brain stem, telencephalon and spinal cord possess the highest levels of bioassayable CCK followed in decreasing order by gustatory lobes, optic tectum and duodenum. No detectable levels of CCK were found in the cerebellum. The observed bioactivity was not due to gastrin because: 1) radioimmunnoassay of the brain homogenates for gastrin revealed very low or nondetectable levels of gastrin; 2) amylase release dose-response curves for standard CCK 8 and the brain homogenate were identical; and 3) proglumide, a competitive antagonist of CCK 8, inhibited homogenate CCK-induced enzyme release with a parallel rightward shift in the dose-response curve. These observations provide evidence for the distinct presence of CCK in the brain of the goldfish suggesting that the differentiation of CCK as a distinct neuropeptide, from that of gastrin, occurs at the level of Osteichthyes (bony fish).

Cardiopulmonary Gas Exchange in the Turtle: A Model Analysis

The performance of the cardiorespiratory gas exchange system of the turtle, Pseudemys scripla, at rest and at three body temperatures (15, 25, 35°C), was assessed in reference to theory and experimental data. The primary processes governing gas exchange(ventilation, perfusion, diffusion) were found to be uniformly balanced in regard to CO2 exchange. This was not true for O2 transport where, at higher body temperature, the systemis characterized by underventilation and overperfusion in conjunction with declining O2 saturation levels. Adjustments in convection of air or blood, which favor improvement in arterial O2 saturation levels, are incompatible with stability of the CO2stores and acid-base state. The latter were preserved across the temperature range. Comparisons between bird, mammal, and turtle illustrate close similarity in the relative roles of the primary processes governing CO2 exchange. This persists in the face of wide differences inmetabolic intensity, cardiopulmonary architecture and complexity, and body temperatures characterizing the species which were compared. The close control of carbon dioxide transport may reflect a fundamental adjustment to air breathing by lungs as primitive vertebrates emerged from water to the terrestrial domain. From a regulatory point of view the study emphasizes the importance of both structural and functional control of CO2 homeostasis. It does not rule out regulatory features which may insure more effective O2 transport; however, the latter are not evident within the context of the steady state assumptions underlying this analysis.

Antibody diversity in amphibians. Noninbred axolotls used the same unique heavy chain and a limited number of light chains for their anti-2,4-dinitrophenyl antibody responses.

Noninbred axolotls (Ambystoma mexicanum, amphibia, urodela) were immunized with trinitrophenylated sheep red blood cells (TNP-SRBC) and anti-2,4-dinitrophenyl (DNP)/TNP antibodies were individually purified by affinity chromatography. The isolated IgM-like antibodies were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) under reducing conditions. The SDS-PAGE and IEF-separated heavy (H) and light (L) chains were electroblotted onto nitrocellulose, probed with mouse monoclonal antibodies specific for H or L axolotl Ig chains and stained by a rabbit anti-mouse Ig horseradish peroxidase conjugate. The specific detection of axolotl anti-DNP/TNP H chain spectrotypes shows for each of the 14 individually analyzed samples a very similar pattern of 4-5 ordered spaced bands. This suggests that all animals express the same VH chain segment representing the germinal expression of a unique VH gene. When the same analysis was performed starting from a pool of nonimmunized axolotl sera, a low background of natural anti-DNP antibodies was detected. When analyzed by IEF, the H chains of the pooled anti-DNP natural antibodies display the same pattern of restricted heterogeneity when compared to the H chain spectrotypes of the individual immune anti-DNP/TNP antibodies. The specific detection of the axolotl anti-DNP/TNP L chain spectrotypes indicates at the individual level more heterogeneous and polymorphic patterns compared with H chains, although most animals share the majority of their bands. Our experiments indicate that in axolotl, the production of antibodies to DNP results from the germinal expression of a very limited set of V genes, already expressed as naturally occurring anti-DNP antibodies before immunization. This seriously restricts the possible extension of the antibody repertoire and perhaps even the nature of antibody "specificity" in this primitive vertebrate.

Insulin-like immunoreactive material in the brain of two primitive vertebrates

Insulin-like immunoreactive material in the brain of two primitive vertebrates

The phylogeny of macrophage function: Antigen uptake and degradation by peritoneal exudate cells of two amphibian species and CAF 1 mice

Equal numbers of thioglycollate mobilized peritoneal exudate cells (PEC) of the newt, Notophthalamus viridescens, the South African clawed toad, Xenopus laevis, and CAF 1 mice were compared with respect to their capacity to take up and degrade soluble 14C-ovalbumin (OVA). PEC of the newt failed to take up the labeled antigen, while those of the toad incorporated only one-half as much as those of the mice. Moreover, the toad PEC degraded only 42% of the immunogen which was taken up, while PEC of the mice degraded 78% of the immunogen they had ingested during the 60-min period. Paraformaldehyde treatment of the PEC prevented antigen uptake, while chloroquine treatment prevented degradation with both species, and thus, active processes were involved. While newt PEC were unable to ingest soluble OVA, they were able to ingest and degrade OVA conjugated to sepharose during the same time period. The failure of primitive vertebrates to respond immunologically to soluble proteins appears to be due to their failure to ingest soluble immunogen.

A spinal projection of 5-hydroxytryptamine neurons in the lamprey brainstem; evidence from combined retrograde tracing and immunohistochemistry

To investigate whether there is a descending contribution to the spinal 5-hydroxytryptamine (5-HT) innervation in the lamprey, a primitive vertebrate, a study using retrograde transport of the fluorescent tracer Fast blue combined with 5-HT immunohistochemistry was conducted. Two to 4 weeks after an injection of Fast blue into the rostral spinal cord, retrogradely labelled cells were seen throughout the brainstem. Two groups of these cells, one within the posterior reticular nucleus of the rhombencephalon and another rostral to the trigeminal motor nucleus, were labelled after incubation with 5-HT antiserum. These findings suggest that in addition to the well-described local intraspinal 5-HT system, there is also, as in higher vertebrates, a descending 5-HT projection from the brainstem which extends at least 20 segments into the spinal cord.

Conservation among the immunoglobulins of carcharhine sharks and phylogenetic conservation of variable region determinants

To study primitive vertebrate recognition molecules we have purified the immunoglobulins of three species of carcharhine sharks. The tiger ( Galeocerdo cuvieri), the sandbar ( Carcharhinus plumbeus) and the galapagos sharks ( Carcharhinus galapagensis) possess 18S high molecular weight and 7S low molecular weight immunoglobulin forms. Both within and between species these forms closely resemble each other in polypeptide chain composition, heavy chain mass, carbohydrate content, amino acid composition, antigenic character and amino terminal sequence. These carcharhine species are separated by at least 30 million years of evolutionary time and it is remarkable that so little diversification of immunoglobulin structure has occurred during their evolution. By several criteria carcharhine immunoglobulins most closely resemble mammalian IgM. Studies of serological cross-reactions indicate that some immunoglobulin determinants have been conserved over a broad phylogenetic range of vertebrate classes; most notably J H and V Ha-related markers are shared between forms as diverse as sharks and mammals.

Ultrastructure of spermatozoa of the crested tinamou

We have examined the ultrastructure of spermatozoa of the crested tinamou, which is believed to be one of the most primitive avian species. Tinamou spermatozoa are unusual in that a single long centriole extends the entire length of the midpiece. The motile apparatus does not display dense fibers. However, the flagellum is surrounded by a prominent fibrous sheath. The fibrous sheath is in turn circumscribed by a region of glycogen. The head of tinamou spermatozoa is unusual in possessing a long tube which extends through the center of the nucleus from the acrosome to the base of the nucleus. Such structures have not been described in birds or mammals, although they occur in more primitive vertebrates.

Morphology and function of the feeding apparatus of the lungfish, Lepidosiren paradoxa (Dipnoi).

The feeding mechanism of the South American lungfish, Lepidosiren paradoxa retains many primitive teleostome characteristics. In particular, the process of initial prey capture shares four salient functional features with other primitive vertebrates: 1) prey capture by suction feeding, 2) cranial elevation at the cranio-vertebral joint during the mouth opening phase of the strike, 3) the hyoid apparatus plays a major role in mediating expansion of the oral cavity and is one biomechanical pathway involved in depressing the mandible, and 4) peak hyoid excursion occurs after maximum gape is achieved. Lepidosiren also possesses four key morphological and functional specializations of the feeding mechanism: 1) tooth plates, 2) an enlarged cranial rib serving as a site for the origin of muscles depressing the hyoid apparatus, 3) a depressor mandibulae muscle, apparently not homologous to that of amphibians, and 4) a complex sequence of manipulation and chewing of prey in the oral cavity prior to swallowing. The depressor mandibulae is always active during mouth opening, in contrast to some previous suggestions. Chewing cycles include alternating adduction and transport phases. Between each adduction, food may be transported in or out of the buccal cavity to position it between the tooth plates. The depressor mandibulae muscle is active in a double-burst pattern during chewing, with the larger second burst serving to open the mouth during prey transport. Swallowing is characterized by prolonged activity in the hyoid constrictor musculature and the geniothoracicus. Lepidosiren uses hydraulic transport achieved by movements of the hyoid apparatus to position prey within the oral cavity. This function is analogous to that of the tongue in many tetrapods.

Characterization of the changes in the state of aggregation induced by ligand binding in the hemoglobin system of a primitive vertebrate, the hagfish Eptatretus cirrhatus

1. 1. Hemoglobin (Hb) from the hagfish, Eptatretus cirrhatus, is composed of subunits of approx. 20,000 mol. wt. 2. 2. Aggregation of the deoxy subunits occurred, particularly at low pH and at high protein concentration. 3. 3. Oxygen equilibrium studies indicated slight cooperativity and the presence of a small, phosphateindependent Bohr effect. 4. 4. Equilibrium properties were protein concentration dependent indicating an oxygen-linked dissociation in the millimolar concentration range. 5. 5. Kinetic studies indicated a dimer to monomer transition in the micromolar concentration range. 6. 6. The ligand-binding character of hagfish Hb was similar to that of lampreys, but was governed by different kinetic and equilibrium parameters.