Calcitonin-like substance in the plasma of Cyclostomata and its putative role

The present study indicated that in the lamprey, Lampetra japonica, which undergoes a spawning migration from seawater to freshwater in its life cycle, a calcitonin (CT)-like substance was not detected in the plasma of seawater-adapted specimens (both sexes), but it was detected in the plasma of freshwater-adapted specimens (both sexes) by enzyme-linked immunosorbent assay using anti-salmon CT serum. Furthermore, this substance increased with the gonad somatic index (GSI) in both sexes. In lamprey, therefore, this CT-like substance may have some relationship with gonadal maturation. In the plasma of the hagfish, Eptatretus burgeri, on the other hand, the CT-like substance was always extremely high (47.2+/-4.2 ng/ml) independently of GSI in both sexes. However, a correlation was found between this substance and plasma calcium. In hagfish, the CT-like substance may participate in calcium homeostasis, as does calcium excretion via bile, because the hagfish has the highest bile calcium concentration determined to date among vertebrates, with a level corresponding to 12-fold that in plasma. This speculation is supported by reports that, in rat and cartilaginous fish, CT acts on calcium excretion via bile. In both fishes, moreover, the molecular weight of these substances was equal to that of salmon CT, which strongly suggests that these substances detected here are their CTs.     

Genome biology of the cyclostomes and insights into the evolutionary biology of vertebrate genomes

The jawless vertebrates (lamprey and hagfish) are the closest extant outgroups to all jawed vertebrates (gnathostomes) and can therefore provide critical insight into the evolution and basic biology of vertebrate genomes. As such, it is notable that the genomes of lamprey and hagfish possess a capacity for rearrangement that is beyond anything known from the gnathostomes. Like the jawed vertebrates, lamprey and hagfish undergo rearrangement of adaptive immune receptors. However, the receptors and the mechanisms for rearrangement that are utilized by jawless vertebrates clearly evolved independently of the gnathostome system. Unlike the jawed vertebrates, lamprey and hagfish also undergo extensive programmed rearrangements of the genome during embryonic development. By considering these fascinating genome biologies in the context of proposed (albeit contentious) phylogenetic relationships among lamprey, hagfish, and gnathostomes, we can begin to understand the evolutionary history of the vertebrate genome. Specifically, the deep shared ancestry and rapid divergence of lampreys, hagfish and gnathostomes is considered evidence that the two versions of programmed rearrangement present in lamprey and hagfish (embryonic and immune receptor) were present in an ancestral lineage that existed more than 400 million years ago and perhaps included the ancestor of the jawed vertebrates. Validating this premise will require better characterization of the genome sequence and mechanisms of rearrangement in lamprey and hagfish.     

Crystal structures of deoxy- and carbonmonoxyhemoglobin F1 from the hagfish Eptatretus burgeri

Hagfish are extremely primitive jawless fish of disputed ancestry. Although generally classed with lampreys as cyclostomes ("round mouths"), it is clear that they diverged from them several hundred million years ago. The crystal structures of the deoxy and CO forms of hemoglobin from a hagfish (Eptatretus burgeri) have been solved at 1.6 and 2.1 A, respectively. The deoxy crystal contains one dimer and two monomers in a unit cell, with the dimer being similar to that found in lamprey deoxy-Hb, but with a larger interface and different relative orientation of the partner chains. Ile(E11) and Gln(E7) obstruct ligand binding in the deoxy form and make room for ligands in the CO form, but no interaction path between the two hemes could be identified. The BGH core structure, which forms the alpha1beta1 interface of all vertebrate alpha2beta2 tetrameric Hbs, is conserved in hagfish and lamprey Hbs. It was shown previously that human and cartilaginous fish Hbs have independently evolved stereochemical mechanisms other than the movement of the proximal histidine to regulate ligand binding at the hemes. Our results therefore suggest that the formation of the alpha2beta2 tetramer using the BGH core and the mechanism of quaternary structure change evolved between the branching points of hagfish and lampreys from other vertebrates.     

Anatomical and Physiological Peculiarities of the Heart in Jawless and Jawed Fish

Journal of Evolutionary Biochemistry and Physiology - The heart of jawless fish (Cyclostomata; lamprey, hagfish) and jawed fish (Teleostei) is homologous to the heart of higher vertebrates. A study...     

Repression of rat kidney L-arginine:glycine amidinotransferase synthesis by creatine at a pretranslational level

The first committed reaction in the biosynthesis of creatine is catalyzed by the enzyme L-arginine:glycine amidinotransferase, commonly called transamidinase. Creatine, the end product of the biosynthetic pathway, is known to alter the levels of kidney transamidinase activity. Rats fed a diet containing 0.3% creatine had 26% of the kidney transamidinase activity of the rats fed a creatine-free diet. This reduction in transamidinase activity was correlated with a decrease in transamidinase protein in the creatine-fed rats. The relative synthetic rates and mRNA functional activities of transmidinase were measured in control and creatine-fed rats. The relative synthetic rate of transamidinase in creatine-fed rats was 21% of that found in the control animals. The functional transamidinase mRNA in creatine-fed rats was correspondingly reduced to 37% of the amount in the control animals. Thus, creatine affects transamidinase activity by altering its rate of synthesis at a pretranslational step and represents an example of end-product repression in a higher eukaryote.     

Immunochemical evidence that the single lactate dehydrogenase of lampreys is more similar to LDHB4 than to LDHA4 of hagfish

The tetrameric lactate dehydrogenases (LDH) of vertebrates contain several different subunits that arose by gene duplication. While the A and B subunits occur in all classes of gnathostomes, the enzymes of agnathans appear to represent two stages in the evolution of vertebrate LDH. Lampreys of the family Petromyzontidae have a single enzyme classified as LDHA4, while hagfish possess both A and B subunits which form only the two homopolymers LDHA4 and LDHB4. It is generally assumed that the original vertebrate LDH was an A4 type, that duplication to give the B subunit occurred prior to the divergence of lampreys and hagfish, and that modern lampreys subsequently lost expression of the B gene. Lactate dehydrogenases were purified from representatives of all three lamprey families, and it was confirmed that members of the Mordaciidae and Geotriidae also possess single tetrameric LDH enzymes containing one subunit type. The kinetic properties of the lamprey LDH enzymes were compared with the LDH homopolymers of hagfish, skate, and sardine. These properties did not allow the lamprey enzymes to be unequivocally identified as either LDHA4 or LDHB4. Immunochemical titration using antisera against lamprey and hagfish LDH homopolymers demonstrated that the lamprey LDH enzymes showed greater immunochemical similarity to LDHB4 than to LDHA4 of hagfish. It is concluded that there is little evidence for the claim that the original vertebrate LDH was an A4 rather than B4 type.     

Vasoactivity of the ventral aorta of the American eel (Anguilla rostrata), Atlantic hagfish ( Myxine glutinosa), and sea lamprey (Petromyzon marinus)

To determine if vascular smooth muscle from teleost and agnathan fishes expresses receptors for signaling agents that are important in vascular tension in other vertebrates, we exposed rings of aortic vascular smooth muscle from the eel (Anguilla rostrata), the hagfish (Myxine glutinosa), and the lamprey (Petromyzon marinus) to a suite of putative agonists, including: acetylcholine, endothelin, nitric oxide, natriuretic peptides, and prostanoids. Acetylcholine constricted aortic rings from the eel, but had no effect on the rings from lamprey. On the other hand, endothelin constricted rings from all three species. Use of receptor-specific ET agonists demonstrated that only ET(A) receptors are expressed in the eel and lamprey aorta. The nitric oxide donor sodium nitroprusside or nitric oxide itself dilated rings from the eel, but both agonists constricted rings from the hagfish and NO produced a biphasic response (constriction followed by dilation) in the lamprey. Two natriuretic peptides, eel ANP and porcine CNP, produced marginally significant dilation in the eel aorta, human ANP dilated the hagfish rings, and pCNP and eANP dilated the lamprey rings. The prostanoids PGE(1) and PGE(2) both dilated the eel aortic rings, and PGE(1) and carbaprostacyclin (stable PGI(2) agonist) dilated the hagfish and lamprey rings. Our results suggest that receptors for a variety of vasoactive signaling agents are expressed in the aortic smooth muscle of the earliest vertebrates (lamprey and hagfish), as well as the more advanced teleosts (eel).     

Early Evolution of Conserved Regulatory Sequences Associated with Development in Vertebrates

Comparisons between diverse vertebrate genomes have uncovered thousands of highly conserved non-coding sequences, an increasing number of which have been shown to function as enhancers during early development. Despite their extreme conservation over 500 million years from humans to cartilaginous fish, these elements appear to be largely absent in invertebrates, and, to date, there has been little understanding of their mode of action or the evolutionary processes that have modelled them. We have now exploited emerging genomic sequence data for the sea lamprey, Petromyzon marinus, to explore the depth of conservation of this type of element in the earliest diverging extant vertebrate lineage, the jawless fish (agnathans). We searched for conserved non-coding elements (CNEs) at 13 human gene loci and identified lamprey elements associated with all but two of these gene regions. Although markedly shorter and less well conserved than within jawed vertebrates, identified lamprey CNEs are able to drive specific patterns of expression in zebrafish embryos, which are almost identical to those driven by the equivalent human elements. These CNEs are therefore a unique and defining characteristic of all vertebrates. Furthermore, alignment of lamprey and other vertebrate CNEs should permit the identification of persistent sequence signatures that are responsible for common patterns of expression and contribute to the elucidation of the regulatory language in CNEs. Identifying the core regulatory code for development, common to all vertebrates, provides a foundation upon which regulatory networks can be constructed and might also illuminate how large conserved regulatory sequence blocks evolve and become fixed in genomic DNA.     

Cyclostome embryology and early evolutionary history of vertebrates

Modern agnathans include only two groups, the lampreys and thehagfish, that collectively comprise the group Cyclostomata.Although accumulating molecular data support the cyclostomesas a monophyletic group, there remain some unsettled questionsregarding the evolutionary relationships of these animals inthat they differ greatly in anatomical and developmental patternsand in their life histories. In this review, we summarize recentdevelopmental data on the lamprey and discuss some questionsrelated to vertebrate evolutionary development raised by thelimited information available on hagfish embryos. Comparisonof the lamprey and gnathostome developmental patterns suggestssome plesiomorphic traits of vertebrates that would have alreadybeen established in the most recent common ancestor of the vertebrates.Understanding hagfish development will further clarify the,as yet, unrecognized ancestral characters that either the lampreysor hagfishes may have lost. We stress the immediate importanceof hagfish embryology in the determination of the most plausiblescenario for the early history of vertebrate evolution, by addressingquestions about the origins of the neural crest, thyroid, andadenohypophysis as examples.     

Plasma guanidino compounds are altered by oral creatine supplementation in healthy humans.

Although creatine is one of the most widely used nutritional supplements for athletes as well as for patients with neuromuscular disorders, the effects of oral creatine supplementation on endogenous creatine synthesis in humans remains largely unexplored. The aim of the present study was to investigate the metabolic consequences of a frequently used, long-term creatine ingestion protocol on the circulating creatine synthesis precursor molecules, guanidinoacetate and arginine, and their related guanidino compounds. For this purpose, 16 healthy young volunteers were randomly divided to ingest in a double-blind fashion either creatine monohydrate or placebo (maltodextrine) at a dosage of 20 g/day for the first week (loading phase) and 5 g/day for 19 subsequent wk (maintenance phase). Fasting plasma samples were taken at baseline and at 1, 10, and 20 wk of supplementation, and guanidino compounds were determined. Plasma guanidinoacetate levels were reduced by 50% after creatine loading and remained approximately 30% reduced throughout the maintenance phase. Several circulating guanidino compound levels were significantly altered after creatine loading but not during the maintenance phase: homoarginine (+35%), alpha-keto-delta-guanidinovaleric acid (+45%), and argininic acid (+75%) were increased, whereas guanidinosuccinate was reduced (-25%). The decrease in circulating guanidinoacetate levels suggests that exogenous supply of creatine chronically inhibits endogenous synthesis at the transamidinase step in humans, supporting earlier animal studies showing a powerful repressive effect of creatine on l-arginine:glycine amidinotransferase. Furthermore, these data suggest that this leads to enhanced utilization of arginine as a substrate for secondary pathways.     

Actin-related intercellular adhesion junctions in the germinal compartment of the testis in the hagfish (Eptatretus stouti) and lamprey (Lampetra tridentatus)

The germinal epithelium of male vertebrates consists of Sertoli cells and spermatogenic cells. Intercellular junctions formed by Sertoli cells assume critical roles in the normal functions of this epithelium. While Sertoli cell junctions have been well characterized in mammals, similar junctions in nonmammalian vertebrates have received little attention. We examined the intercellular junctions found within the germinal epithelium of the hagfish (Eptatretus stouti) and lamprey (Lampetra tridentatus). Ultrastructurally, Sertoli cells were seen to form filament-associated junctions in both species. Adjacent Sertoli cells formed microfilament-related junctions near their apices. Filaments of these junctions were arranged in loose networks and were not associated with cisterns of endoplasmic reticulum. In fixed, frozen sections of hagfish testis, similar areas labeled with rhodamine phalloidin, indicating the filament type is actin. In the lamprey, desmosomes were observed immediately below the microfilament-related junctions. In appearance and location, the Sertoli cell junctions observed in these species resembled those of the typical junctional complex of other epithelial cell types. No junctions were observed between Sertoli cells and elongating spermatids. In the hagfish, but not the lamprey, an additional zone of microfilaments occurred near the base of Sertoli cells in areas of association with the basal lamina. Our observations are consistent with the proposal that the unique forms of intercellular attachment found in the testes of higher vertebrates evolved from a typical epithelial form of intercellular junction.     

Genomic Evidence for a Simpler Clotting Scheme in Jawless Vertebrates

Mammalian blood clotting involves numerous components, most of which are the result of gene duplications that occurred early in vertebrate evolution and after the divergence of protochordates. As such, the genomes of the jawless fish (hagfish and lamprey) offer the best possibility for finding systems that might have a reduced set of the many clotting factors observed in higher vertebrates. The most straightforward way of inventorying these factors may be through whole genome sequencing. In this regard, the NCBI Trace database ( http://www.ncbi.nlm.nih.gov/Traces/trace.cgi ) for the lamprey (Petromyzon marinus) contains more than 18 million raw DNA sequences determined by whole-genome shotgun methodology. The data are estimated to be about sixfold redundant, indicating that coverage is sufficiently complete to permit judgments about the presence or absence of particular genes. A search for 20 proteins whose sequences were determined prior to the trace database study found all 20. A subsequent search for specified coagulation factors revealed a lamprey system with a smaller number of components than is found in other vertebrates in that factors V and VIII seem to be represented by a single gene, and factor IX, which is ordinarily a cofactor of factor VIII, is not present. Fortuitously, after the completion of the survey of the Trace database, a draft assembly based on the same database was posted. The draft assembly allowed many of the identified Trace fragments to be linked into longer sequences that fully support the conclusion that lampreys have a simpler clotting scheme compared with other vertebrates. The data are also consistent with the hypothesis that a whole-genome duplication or other large scale block duplication occurred after the divergence of jawless fish from other vertebrates and allowed the simultaneous appearance of a second set of two functionally paired proteins in the vertebrate clotting scheme.     

The unusual cartilaginous tissues of jawless craniates, cephalochordates and invertebrates

A collagenous extracellular matrix was previously considered to be a requirement for classification of true cartilage. Data from the lamprey and hagfish now clearly indicate that both of these jawless craniates have extensive non-collagenous, yet cartilaginous endoskeletons. Non-collagenous cartilages are present in the cephalochordates (amphioxus) and in the invertebrates, although collagen-containing cartilages also are found in the invertebrates. This review summarizes current knowledge of the morphological, biochemical and molecular characteristics of the unusual non-collagenous cartilages in jawless craniates and the cartilaginous tissues in amphioxus and invertebrates. A least two types of non-collagenous cartilage matrix proteins are found in both the hagfishes and the lampreys, all of which are resistant to digestion by cyanogen bromide (CNBr). Although all four of these matrices show some similarities with each other, suggesting a family of non-collagenous, elastin-like proteins, it is clear that the major matrix proteins of each are different. New morphological and biochemical information on the cartilaginous tissues in squid, horseshoe crab and amphioxus reveals the presence of CNBr-insoluble, non-collagenous matrix proteins, potentially extending the jawless craniate family of cartilaginous proteins into the invertebrates. Details of the evolutionary relationships between these non-collagenous matrix proteins and the significance of the occurrence of these proteins as the major components of the cartilaginous tissues of jawless craniates, amphioxus, horseshoe crab and squid, all of which are capable of producing a variety of collagens in other tissues, remain to be investigated.     

Elements of the major myofibrillar binding peptide motif are present in the earliest of true muscle type creatine kinases

Most vertebrates possess two genes for cytoplasmic creatine kinase (CK) coding for muscle (M-CK) and brain (B-CK) isoforms which assemble into homo-dimeric (MM, BB) and hetero-dimeric (MB) active enzymes. In mammals and birds, a significant fraction of MM-CK is bound to the myofibrillar M-line where it is thought to facilitate energy buffering and transport. Myofibrillar binding is mediated by major and minor lysine charge clamp motifs (K104/K115 [major] and K8/K24 [minor] in chicken M-CK) located in the N-terminal region [J. Cell Biol. 149 (2000) 1225]. We have obtained the cDNA and deduced amino acid sequences for cytoplasmic CKs from two hagfish, Myxine glutinosa and Eptatretus stoutii, non-vertebrate craniates, and the sequences for two cytoplasmic CKs from the lamprey Lampetra japonica, a jawless true vertebrate. All four cDNAs code for CKs consisting of approximately 380 residues. Phylogenetic analyses showed that the hagfish and lamprey CKs are coded for by genes which are clearly muscle type (M) creatine kinases. Two of these four M-CKs have the K104/K115-equivalent residues of the major myofibrillar binding region while the other two have the K115 equivalent but lack the corresponding K104 residue. All four M-CKs lack the K8/K24 equivalent elements of the minor myofibrillar binding region. Comparison of these sequences to corresponding sequences of cytoplasmic CKs from two protochordates (tunicate, amphioxus) and M- and B-CKs from true fish and above reveal a pattern of acquisition (and loss) of key lysine residues consistent with the physiological context in which these enzymes operate.     

Changes in Creatine and Urea Formation in Rats Fasted and Fed Low Protein Diets

Changes in the time course of the urinary excretion of creatinine, creatine and urea, and the activities of kidney transamidinase and liver urea-cycle enzymes were investigated in rats fasted and fed on a 10% casein diet and 10% casein diets supplemented with 10% glycine and/or 1.4% arginine.

The urinary total-creatinine of the fasted rats increased extremely during fasting for 7 days, while that of the animals given the 10% casein diet supplemented with glycine and arginine rose exceedingly on the 3rd day and thereafter no significant change was observed. Most of the increase of total-creatinine could be accounted for by the increase of creatine. The activity of kidney transamidinase in the fasted rats decreased in the 3rd day and thereafter kept nearly constant. The transamidinase activity of rats fed on the 10% casein diet after giving a protein-free diet for 5 days increased in the 3rd day. An inverse relation was observed between the urinary creatine and the transamidinase activity. The urinary urea increased in the rats fasted or fed on the 10% casein diets with the supplement of glycine and/or arginine. In fasting, the activities of liver urea-cycle enzymes, except arginase, had a tendency of increasing with the lapse of time. The arginase activity remained more or less constant. The reason of the extreme increase of urinary creatine during starvation was discussed.     

The Vertebrate 7S K RNA Separates Hagfish (Myxine glutinosa) and Lamprey (Lampetra fluviatilis)

7S RNA sequences from the hagfish (Myxiniformes) and lamprey (Petromyzontiformes) were cloned and analyzed. In both species, 7S L RNA (also designated SRP RNA, since it represents the RNA constituent of the signal recognition particle) was clearly detectable. The sequence similarity between the two species was 86%, compared with about 75% similarity between either of these species and mammals. 7S K RNA was also cloned from the lamprey. The similarity between the 7S K RNA of the lamprey and that of mammals was 68%. Interestingly, several interspersed elements were found with nearly 100% similarity compared with mammals. In contrast to the lamprey, no 7S K RNA-related sequences were detectable among hagfish RNA, neither in northern blots nor with the PCR assay. In view of the significant conservation between the 7S K RNA of lamprey and that of mammals (human), this unexpected result clearly separates lamprey and hagfish. In addition, the lack of detectable 7S K RNA sequences in an outgroup, such as amphioxus, indicates that these results do not reflect an autapomorphy of hagfish. Therefore, our data provide additional support to the notion of a sister group relationship between Petromyzontiformes and gnathostomous vertebrates to the exclusion of Myxiniformes. Received: 24 September 1999 / Accepted: 9 February 2000     

The origin of developmental mechanisms underlying vertebral elements: implications from hagfish evo-devo

The origins of the vertebral elements and the underlying developmental mechanisms have so far remained unclear, largely due to the unusual axial skeletal morphology of hagfish, one of two extant jawless vertebrate clades. Hagfish axial supporting tissue is generally believed to consist of the notochord and cartilaginous fin rays only. However, careful investigations of whether vertebral elements are truly absent in hagfish are scarce, and it is also unclear whether the axial skeletal morphology of the hagfish is an ancestral or a derived condition. To address these questions, we re-examined the axial skeletal morphology of the Japanese inshore hagfish (Eptatretus burgeri). Based on a report published a century ago which implied the existence of vertebral elements in hagfish, we conducted anatomical and histological analyses of the hagfish axial skeletal systems and their development. Through this analysis, we demonstrate that hagfish possesses sclerotome-derived cartilaginous vertebral elements at the ventral aspect of the notochord. Based on (i) molecular phylogenetic evidence in support of the monophyly of cyclostomes (hagfish and lampreys) and jawed vertebrates (gnathostomes), and (ii) the morphology of the vertebral elements in extant gnathostomes and cyclostomes, we propose that the embryos of the common ancestor of all vertebrates would have possessed sclerotomal cells that formed the segmentally arranged vertebral elements attached to the notochord. We also conclude that the underlying developmental mechanisms are likely to have been conserved among extinct jawless vertebrates and modern gnathostomes.     

Decreased activities of glycine and guanidinoacetate methyltransferases and increased levels of creatine in tumor cells

The activities of glycine and guanidinoacetate methyltransferases have been measured in various tissues of rats and hepatoma of rats induced by $\text{N}$-2-fluorenylacetamide. These enzyme activities existing in rat liver gradually decreased with the progress of hepatocarcinoma. However, the creatine levels in these tumor tissues are significantly increased, although guanidinoacetate methyltransferase activity was not detected.