Molecular Evolution of Apolipoprotein Multigene Family and the Original Functional Properties of Serum Apolipoprotein (LAL2) in Lampetra japonica.

Apolipoprotein (APO) genes represent a large family of genes encoding various binding proteins associated with plasma lipid transport. Due to the long divergence history, it remains to be confirmed whether these genes evolved from a common ancestor through gene duplication and original function, and how this evolution occurred. In this study, based on the phylogenetic tree, sequence alignment, motifs, and evolutionary analysis of gene synteny and collinearity, APOA, APOC, and APOE in higher vertebrates may have a common ancestor, lamprey serum apolipoprotein LAL1 or LAL2, which traces back to 360 million years ago. Moreover, the results of immunofluorescence, immunohistochemistry, and flow cytometry show that LAL2 is primarily distributed in the liver, kidney, and blood leukocytes of lampreys, and specifically localized in the cytoplasm of liver cells and leukocytes, as well as secreted into sera. Surface plasmon resonance technology demonstrates that LAL2 colocalizes to breast adenocarcinoma cells (MCF-7) or chronic myeloid leukemia cells (K562) associated with lamprey immune protein (LIP) and further enhances the killing effect of LIP on tumor cells. In addition, using quantitative real-time PCR (Q-PCR) and western blot methods, we found that the relative mRNA and protein expression of lal2 in lamprey leukocytes and sera increased significantly at different times after stimulating with Staphylococcus aureus, Vibrio anguillarum, and Polyinosinic-polycytidylic acid (Poly I:C). Moreover, LAL2 was found to recognize and bind to gram-positive bacteria (Staphylococcus aureus and Bacillus cereus) and gram-negative bacteria (Escherichia coli) and play an important role in the antibacterial process. All in all, our data reveals a long, complex evolutionary history for apolipoprotein genes under different selection pressures, confirms the immune effect of LAL2 in lamprey sera against pathogens, and lays the foundation for further research regarding biological functions of lamprey immune systems.

Simultaneous determination of gonadotropin-inhibitory and gonadotropin-releasing hormones using ultra-high performance liquid chromatography electrospray ionization tandem mass spectrometry.

Gonadotropin-inhibitory hormones (GnIH) and gonadotropin-releasing hormones (GnRH) are neuropeptides essential for the regulation of reproduction in all vertebrate animals examined. Determination of neuropeptides in the biological sample is highly challenging due to their complex matrix and weak stability. The wide variety of peptides or protein degradation products often interferes with the determination of the target peptide. This study aims to develop a specific ultra-high performance liquid chromatography-tandem mass spectrometry method for simultaneous determination of nine critical neuropeptides in biological samples. A separation method by ultra-performance liquid chromatography coupled to a multiple reaction monitoring (MRM) by tandem mass spectrometry allows the selective determination of the neuropeptides in brain and plasma matrices after solid-phase extraction. Specific MSMS transitions were optimized using MRM of multiple-charged peptides generated by electrospray ionization in positive mode. The resulting analytical method was fully validated with thorough evaluation of stability, recovery, matrix effect, and intra- and interday accuracy and precision in sea lamprey brain and plasma. The optimized method shows linearity in a wide range of concentrations with limit of quantification ranging from 0.1 to 0.75 ng/mL. With slight modification, this method can be applied to other biological samples.

Blood steroid profile and in vitro steroidogenesis by ovarian follicles and testis fragments of adult sea lamprey, Petromyzon marinus.

The main purpose of the study was to identify the principal gonadal steroids synthesized by male and female sea lampreys, Petromyzon marinus. To achieve this, we used high performance liquid chromatography to separate the steroids in the serum of sexually mature animals, and to separate the steroids produced by gonadal tissue incubated in the presence of radiolabelled precursor steroids, as a means of identifying the major steroidogenic pathways. We were unable to detect evidence of the 'classical' steroids, such as 17beta-estradiol (E(2)) or testosterone (T) in the serum of either male or female lampreys. Instead, the principal chromatographic peaks contained very polar compounds that had elution times consistent with 15alpha-hydroxylated estrogens and androgens, and there were sex-specific differences in the chemical nature and the quantity of these compounds. Testis fragments or ovarian follicles co-incubated with tritium-labelled pregnenolone ([3H]P(5)), 17-hydroxyprogesterone ([3H]17OHP(4)), or androstenedione ([3H]A(4)), provided additional confirmation that the gonads synthesize a range of very polar steroids, and the metabolites found were consistent with the presence of a 15alpha-hydroxylated (15alphaOH) metabolic pathway common to testis and ovary. For ovarian tissue, the major 'end product' metabolites from all three precursors were 15alphaOH-estrogens, and for testis tissue 15alpha-hydroxyprogesterone (15alphaOHP(4)) and 15alpha-hydroxytestosterone (15alphaOHT) and small amounts of 15alphaOH estrogen. Small amounts of E(2) were also produced by both ovarian (all substrates) and testicular tissue (some substrates). Although it was assumed that the E(2) was synthesized via the aromatization of T, [3H]T was not found as an intermediate metabolite. The study suggests that the principal gonadal steroids in sea lamprey are 15alpha-OH compounds, and that only small amounts of E(2) or T are synthesized by the gonads at this stage of reproductive development. There was no direct evidence of progesterone (P(4)) synthesis from [3H]P(5), although the metabolites synthesized by both testis and ovary were indicative of a metabolic pathway that involved P(4) as an intermediate.

ATP-sensitive K+ and Ca2+-activated K+ channels in lamprey ( Petromyzon marinus) red blood cell membrane.

The patch-clamp technique was used to demonstrate the presence of ATP-sensitive K(+) channels and Ca(2+)-activated K(+) channels in lamprey ( Petromyzon marinus) red blood cell membrane. Whole-cell experiments indicated that the membrane current under isosmotic (285 mosmol l(-1)) conditions is carried by K(+). In the inside-out configuration an ATP-sensitive K(+) channel (70-80 pS inward, 35-40 pS outward) was present in 35% of patches. Application of ATP to the intracellular side reduced unitary current with half-maximal inhibition in the range 10-100 microM. A block was obtained with 100 microM lidocaine and inhibition was obtained with 0.5 mM barium acetate. A Ca(2+)-activated K(+) channel (25-30 pS inward, 10-15 pS outward) was present in 57% of patches. Inhibition was produced by 10 mM TEA and 500 nM apamin and sensitivity to Ba(2+) was lower than for ATP-sensitive channels. No spontaneous channel activity was recorded in the cell-attached configuration under isotonic conditions. With hypotonic saline 68% of patches showed spontaneous single-channel activity, and, of 75 active patches, 66 cell-attached patches showed channel activity corresponding to Ca(2+)-activated K(+) channels.

The primary structure of the hemoglobins of a southern hemisphere lamprey (Mordacia mordax, Cyclostomata).

Mordacia mordax is a southern hemisphere lamprey belonging to Mordaciidae, a primitive family of Cyclostomata. Adult erythrocytes contain three monomeric hemoglobins which can be easily separated by cellulose acetate electrophoresis and isolated by ion-exchange chromatography. The N-terminal regions, and the tryptic peptides from each chain were submitted to automated Edman degradation; the alignment of the fragments was obtained by homology with the other Petromyzonoidea hemoglobins hitherto sequenced. Our results confirm the phylogenic distance between lampreys and hag-fish hemoglobins. As was observed for Petromyzon marinus species, two hemoglobins of Mordacia mordax are very close, as they differ only at 7 positions.

Isolation and properties of a fatty acid-binding protein from the Pacific lamprey (Lampetra tridentata).

1. A survey of 12 vertebrate species showed that palmitate was bound by an albumin-like serum protein in all classes tested except the dogfish and the lamprey. 2. The major palmitate-binding protein of the Pacific lamprey was isolated and found to be of molecular mass 19,000. 3. The amino acid composition of this protein indicates that it is not a member of the albumin superfamily. 4. The 19-kDa lamprey protein binds bilirubin, cortisol and tryptophan only weakly, but binds palmitate with KA = 25 microM-1, comparable to the first long-chain fatty acid site of bovine albumin (KA = 34 microM-1).

Relationship between brain gonadotropin-releasing hormone and final reproductive period of the adult male sea lamprey, Petromyzon marinus.

Gonadotropin-releasing hormone (GnRH) concentrations were measured in brains of adult male sea lamprey, Petromyzon marinus, during their final reproductive period. The lampreys were collected during their upstream migration in coastal New Hampshire rivers and sampled at the trap (referred to as Group A) or they were transferred to an artificial spawning channel (referred to as Group B). Plasma estradiol and progesterone were also measured, and histological examination of the gonadal stages was done as well. The concentrations of brain GnRH and plasma estradiol fluctuated significantly through time. There was a rise in brain concentrations of GnRH coincident with an increase in temperature just prior to spawning. In addition, there was a significant progressive correlation between increasing plasma estradiol and temperature in lampreys from Group B during the period studied. These studies provide evidence for progressive seasonal relationships between changes in brain GnRH and gametogenic and steroidogenic activity of the gonads in adult male sea lampreys during their final reproductive period.

Endocrine events associated with spawning behavior in the sea lamprey (Petromyzon marinus).

Levels of estradiol, progesterone, and testosterone were determined in plasma of sea lamprey (Petromyzon marinus) undergoing certain behaviors associated with spawning in natural and artificial stream environments. Significantly higher levels of estradiol, progesterone, and testosterone were found in males than in females. In the artificial spawning channel, levels of estradiol were significantly higher in females exhibiting resting and swimming behaviors than in fanning, nest building, and spawning behaviors. No significant correlation was found with either progesterone or testosterone levels and the various reproductive behaviors. The data presented are the first experimental evidence that suggest gonadal steroids may be correlated with certain reproductive behaviors in the sea lamprey.

Steroid and thyroid hormone profiles following a single injection of partly purified salmon gonadotropin or GnRH analogues in male and female sea lamprey.

The effects of exogenous administration of gonadotropin-releasing hormone (GnRH) analogues or of a partly purified salmon gonadotropin extract (GTH) on the duration of steroid and thyroid hormone levels were determined in female and male sea lampreys, Petromyzon marinus, tested under differing temperature and reproductive status. Plasma estradiol levels, but not androgens, were significantly elevated in response to the GnRH analogues or GTH injection compared to controls in female and male lampreys. Higher temperature and/or advance in time of maturation appeared to be inversely related to plasma estradiol levels. These data provide further evidence of hypothalamic control over reproductive function in lampreys. Plasma thyroxine was significantly elevated after female lampreys were treated with GTH, GnRHa (10 micrograms/lamprey) or GnRHa (1 microgram/lamprey) compared to controls. The present study is the first to demonstrate that the GnRH analogue stimulated in some way the pituitary-thyroid axis. These data suggest that a GnRH activity may activate both gonado- and thyrotropic secretion or that the endogenous hormone may itself have both functions in one of the most primitive vertebrates, the sea lamprey.

Changes in plasma steroid and thyroid hormones and insulin during final maturation and spawning of the sea lamprey, Petromyzon marinus.

Circulating levels of plasma estradiol-17 beta, androgens, thyroxine, triiodothyronine, immunoreactive insulin, plasma fatty acids, and protein were measured at interval during the period of final gonadal maturation, prior to spawning of male and female sea lampreys. Plasma estradiol levels fluctuated significantly and generally covaried in males and females through time. In females, possibly in relation to environmental changes, mean plasma estradiol levels peaked four times during the final spawning period but decreased sharply at the time of ovulation. In males, mean plasma estradiol peaked seven times during the final prespawning period and, in contrast to females, peaked significantly at the time of final spermiation. Plasma androgens were extremely low and covaried in males and females through time. Like plasma steroid profiles, there were coordinated changes in plasma triiodothyronine and thyroxine levels in males and females through the prespawning season. There was a slight increase in plasma insulin during the terminal maturation of the lampreys. However, at ovulation, the insulin levels abruptly decreased in females, whereas in males they remained unchanged. Plasma protein and fatty acid levels gradually decreased until ovulation/spermiation. At ovulation plasma fatty acid levels increased.

Plasma steroids in the ammocoete of Petromyzon marinus.

Radioimmunoassays revealed the presence of the following steroids (or immunoreactive steroid fractions) in the plasma of larval sea lampreys: progesterone, corticosterone, cortisol, androstenedione, testosterone, dihydrotestosterone, estrone, and estradiol. Several types of stress served to provoke the release of these substances. A statistically significant effect of the treatment was noted under only one condition: squeezing of the body of decapitated ammocoetes during blood collection caused an increase in plasma dihydrotestosterone and a decrease in plasma estradiol. The discovery of all eight steroids (or immunoreactivities) assayed for suggests that the plasma of the ammocoete may contain a considerable number of additional steroids yet to be identified.

Iron levels and major iron binding proteins in the plasma of ammocoetes and adults of the southern hemisphere lamprey Geotria australis Gray.

1. The major plasma iron binding proteins (IBP) of both larval and adult Geotria australis differ from those of other vertebrates. 2. Larval IBP has a M.W. of approximately 354,000 and is composed of 20 subunits of equal M.W. 3. Adult IBP is a tetramer (M.W. = c. 296,000) which readily dissociates into subunits of approximately 78,000. 4. Confirmation that plasma iron binding proteins were similar to ferritin in larvae and transferrin in adults was provided by pI values, Fe/protein ratios and electron microscopy. 5. Total plasma iron was 19,760 micrograms/100 ml in larvae and 34 micrograms/100 ml in adults.

Amino acid compositions of the subunit chains of lamprey fibrinogen. Evolutionary significance of some structural anomalies.

Our original objective in studying lamprey fibrinogen was embodied in the notion that the proteins of this ancient vertebrate might themselves by more primitive. As such, it was possible that the subunits of lamprey fibrinogen might have been more similar, one to another, than is the case in higher vertebrates, or even identical. Amino acid analysis of the individual polypeptide chains indicates, however, that the alpha, beta and gamma-chains are instead more dissimilar from each other than are the corresponding chains from human fibrinogen. This finding was somewhat surprising because regions of homology have been detected recently among those three chains when isolated from human fibrinogen, suggesting that all three chains have indeed descended from a common ancestor. The paradox is especially evidenced by the unusual amino acid composition of the lamprey alpha-chain, 45% of which is composed of glycine, serine and threonine. This unusual amino acid distribution may be involved in the anomalous behavior of these chains on sodium dodecyl sulfate polyacrylamide gel electrophoresis.