Levels of the native forms of GnRH in the pituitary of the gilthead seabream, Sparus aurata, at several characteristic stages of the gonadal cycle

Brains of the gilthead seabream, Sparus aurata, contain three different forms of gonadotropin-releasing hormone (GnRH): seabream (sb) GnRH, chicken (c) GnRH-II, and salmon (s) GnRH. In the present study, we developed three specific enzyme-linked-immunosorbent assays (ELISA) for sbGnRH, cGnRH-II, and sGnRH and used them to measure the levels of each GnRH form in the pituitary of male and female seabream at different stages of gametogenesis. The sensitivity was 6 pg/well for the sbGnRH assay, 7 pg/well for the cGnRH-II assay, and 2 pg/well for the sGnRH assay. Levels of each of the three GnRH forms were measured in pituitaries from fish sampled at the beginning of gonadal recrudescence and during the spawning season. Of the three forms, only sbGnRH and cGnRH-II were detected in the pituitary, irrespective of reproductive state or sex. Recrudescent fish had similar levels of sbGnRH and cGnRH-II in the pituitary. In sexually mature fish, the levels of sbGnRH were higher than those in recrudescent fish while pituitary cGnRH-II content remained unchanged. Consequently, sbGnRH levels were 3- to 17-fold higher than cGnRH-II levels in mature fish. Positive correlations also existed between pituitary sbGnRH content and pituitary and plasma gonadotropin (GtH) II levels. Surprisingly, mature 1-year-old males had significantly higher levels of sbGnRH in the pituitary than mature 3-year-old males, while pituitary and plasma GtH II levels were similar between these two groups. Although the reason for this difference in sbGnRH levels is unclear, a possible role of sbGnRH in the processes of puberty or sex-inversion is implied. Based on the present results, it can be suggested that in the gilthead seabream, sbGnRH is the most relevant form of GnRH in the control of reproduction.     

In vitro action of testosterone in potentiating gonadotropin-releasing hormone-stimulated gonadotropin-II secretion in goldfish pituitary cells: involvement of protein kinase C, calcium, and testosterone metabolites

Overnight preincubation of goldfish pituitary cell culture with testosterone (T) enhanced the gonadotropin (GTH)-II responses to GTH-releasing hormone (GnRH). In this study, the involvement of GnRH signal transduction components and the requirement for T metabolism in mediating this direct, pituitary cell action of T were examined using cultured pituitary cells from both male and female goldfish. Each sets of related experiments were done in at least two different stages of the gonadal reproductive cycle and similar effects were observed. Overnight treatment with 10 nM T increased GTH-II responses to maximal stimulatory doses (100 nM) of either salmon (s)GnRH or chicken (c)GnRH-II, but not the total cellular GTH-II contents measured prior to and after a 2-h GnRH challenge. T increased the efficacy and sensitivity of the GTH-II response to stimulation by a protein kinase C (PKC) activator, tetradecanoyl phorbol acetate (TPA) without altering the ED50 of the dose-response curve. In T-treated cells, addition of a PKC inhibitor attenuated GTH-II responses to 100 nM doses of sGnRH, cGnRH-II, or TPA. T did not affect the GTH-II release stimulated by high concentrations of the Ca2+ ionophore ionomycin (100 microM) and the voltage-sensitive Ca2+ channel (VSCC) agonist Bay K 8644 (10 microM); similarly, the sensitivity of the GTH-II response to ionomycin and Bay K 8644 was also unaltered. Taken together, these data suggest that T potentiates GnRH-stimulated GTH-II release by enhancing the effectiveness of PKC-dependent pathways, but not by increasing the total Ca2+-sensitive GTH-II pool, the sensitivity of the release response to increases in intracellular Ca2+, or the amount of available GTH-II. However, the VSCC agonist nifedipine reduced sGnRH- and cGnRH-II-elicited GTH-II release in T-treated as well as in non-T-treated cells, suggesting that VSCC dependence is still present in the GnRH-induced response following exposure to T. Since total cGnRH-II binding to pituitary cells was not increased by T, increases in GnRH receptor capacity are unlikely following T treatment. The ability of T to increase GnRH-stimulated GTH-II secretion was not mimicked by 11-ketotestosterone or dihydrotestosterone, but was abolished by coincubation with an aromatase inhibitor. When viewed together, these observations suggest that aromatization of T may be required for the pituitary action of T on GnRH-induced GTH-II release.     

Evolution of neuroendocrine peptide systems: gonadotropin-releasing hormone and somatostatin

Nine vertebrate and two protochordate gonadotropin-releasing hormone (GnRH) decapeptides have been identified and sequenced. Multiple molecular forms of GnRH peptide were present in the brain of most species examined, and cGnRH-II generally coexists with one or more GnRH forms in all the major vertebrate groups. The presence of multiple GnRH forms has been further confirmed by the deduced GnRH peptide structure from cDNA and/or gene sequences in several teleost species and tree shrew. High conservation of the primary structure of GnRH decapeptides and the overall structure of GnRH genes and precursors suggests that they are derived from a common ancestor. Somatostatin (SRIF) is a phylogenetically ancient, multigene family of peptides. A tetradecapeptide, SRIF (SRIF14) has been conserved, with the same amino acid sequence, in representative species of all classes of vertebrate. Four molecular variants of SRIF14 have been identified. SRIF14 is processed from preprosomatostatin-I, which contains SRIF14 at its C-terminus; preprosomatostatin-I is also processed to SRIF28 in mammals and SRIF26 in bowfin. Teleost fish possess a second somatostatin precursor, preprosomatostatin-II, containing [Tyr7, Gly10]-SRIF14 at the C-terminus, that is mainly processed into large forms of SRIF.     

A critical appraisal of the role of triangulation in nursing research

Proopiomelanocortin (POMC) is the precursor for a number of biologically active peptides such as adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH) and beta-endorphin. It is well known that these peptides are involved in the stress response in fish as well as in mammals. We have cloned two different carp POMC cDNAs called, POMC-I and POMC-II. The nucleotide sequences of 955 bp for POMC-I and 959 bp for POMC-II share 93.5% identity in their cDNAs, and the deduced amino acid sequences (both 222 amino acids) are 91.4% identical. In the ACTH and beta-MSH domain, two amino acid substitutions are found, whereas alpha-MSH and beta-endorphin are identical. For beta-MSH, the serine replacement (in POMC-I) by a glycine (in POMC-II) results in a putative amidation site Pro-X-Gly for POMC-II. We used RT-PCR to show that both POMC mRNAs are expressed in the hypophysis, hypothalamus and other parts of the brain of a single fish. Furthermore, in a phylogenetic tree based on POMC sequences the divergence of carp POMC-I and -II from tetraploid animals (salmon, trout and xenopus) is demonstrated.     

Accelerated evolution of snake venom phospholipase A2 isozymes for acquisition of diverse physiological functions

The nucleotide sequences of two cDNAs and four genes encoding Trimeresurus gramineus venom gland phospholipase A2 (PLA2) isozymes were determined and compared internally and externally with those encoding Trimeresurus flavoviridis venom gland PLA2 isozymes. It was revealed that the protein-coding regions are much more diversified than the 5' and 3' untranslated regions (UTRs) and the introns except for the signal peptide domain. The numbers of nucleotide substitutions per site (KN) for the UTRs and the introns were approximately one-quarter of the numbers of nucleotide substitutions per synonymous site (KS) for the protein-coding regions and were at the same level as the KN value of T. gramineus and T. flavoviridis TATA box-binding protein (TBP) genes, indicating that the protein-coding regions of PLA2 isozyme genes are unusually variable and that the UTRs including the introns of venom gland PLA2 isozyme genes have evolved at similar rate to those of non-venomous genes. The numbers of nucleotide substitutions per non-synonymous site (KA) values were close to or larger than the KS values for the protein-coding regions in venom gland PLA2 isozyme genes, indicating that the protein-coding regions of snake venom gland PLA2 isozyme genes have evolved via accelerated evolution. Furthermore, the evolutionary trees derived from the combined sequences of the 5' and 3' UTRs and the signal peptide domain of cDNAs were in accord with the consequences from taxonomy. In contrast, the evolutionary trees from the mature protein-coding region sequences of cDNAs and from the amino acid sequences showed random patterns. Estimations of nucleotide divergence of genes and the phylogenetic analysis reveal that snake venom group IJ PLA2 isozyme genes have been evolving under adaptive pressure to acquire new physiological activities.     

Cloning of the cDNA encoding the urotensin II precursor in frog and human reveals intense expression of the urotensin II gene in motoneurons of the spinal cord

Urotensin II (UII) is a cyclic peptide initially isolated from the caudal neurosecretory system of teleost fish. Subsequently, UII has been characterized from a frog brain extract, indicating that a gene encoding a UII precursor is also present in the genome of a tetrapod. Here, we report the characterization of the cDNAs encoding frog and human UII precursors and the localization of the corresponding mRNAs. In both frog and human, the UII sequence is located at the C-terminal position of the precursor. Human UII is composed of only 11 amino acid residues, while fish and frog UII possess 12 and 13 amino acid residues, respectively. The cyclic region of UII, which is responsible for the biological activity of the peptide, has been fully conserved from fish to human. Northern blot and dot blot analysis revealed that UII precursor mRNAs are found predominantly in the frog and human spinal cord. In situ hybridization studies showed that the UII precursor gene is actively expressed in motoneurons. The present study demonstrates that UII, which has long been regarded as a peptide exclusively produced by the urophysis of teleost fish, is actually present in the brain of amphibians and mammals. The fact that evolutionary pressure has acted to conserve fully the biologically active sequence of UII suggests that the peptide may exert important physiological functions in humans.     

Habituation of fright and arousal responses in the teleosts Carassius auratus and Rutilus rutilus..

Examined the habituation of fright and arousal responses of the goldfish and roach to the repeated operation of a plunger in the water. The early response was fright, which habituated, and then the response characteristic of arousal appeared, which habituated as well. Longer intervals between stimuli required more presentations of the stimulus for habituation to occur in goldfish. Roaches required more presentations of the stimulus than goldfish for the responses to habituate, and telencephalic ablation severely impaired habituation of arousal, though not fright responses. Results are discussed in relation to recent work on arousal, habituation, and telencephalic function in fish. (25 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Isolation and characterization of human fast skeletal beta troponin T cDNA: comparative sequence analysis of isoforms and insight into the evolution of members of a multigene family

A cDNA encoding human fast skeletal beta troponin T (beta TnTf) has been isolated and characterized from a fetal skeletal muscle library. The cDNA insert is 1,000 bp in length and contains the entire coding region of 777 bp and 5' and 3' untranslated (UT) segments of 12 and 211 bp, respectively. The 3' UT segment shows the predicted stem-loop structure typical of eukaryotic mRNAs. The cDNA-derived amino acid sequence is the first available sequence for human beta TnTf protein. It is encoded by a single-copy gene that is expressed in a tissue-specific manner in fetal and adult fast skeletal muscles. Although the human beta TnTf represents the major fetal isoform, the sequence information indicates that this cDNA and the coded protein are quite distinct from the fetal and neonatal TnTf isoforms reported in other mammalian fetal muscles. The hydropathy plot indicates that human beta TnTf is highly hydrophilic along its entire length. The protein has an extremely high degree of predicted alpha-helical content involving the entire molecule except the carboxy-terminal 30 residues. Comparative sequence analysis reveals that the human beta TnTf shares a high level of sequence similarity in the coding region with other vertebrate TnTf and considerably reduced similarity with slow skeletal and cardiac TnT cDNAs. The TnT isoforms have a large central region consisting of amino acid residues 46-204 which shows a high sequence conservation both at the nucleotide and amino acid levels. This conserved region is flanked by the variable carboxy-terminal and an extremely variable amino-terminal segment. The tropomyosin-binding peptide of TnT, which is represented by amino acid residues 47-151 and also includes a part of troponin I binding region, is an important domain of this central segment. It is suggested that this conserved segment is encoded by an ancestral gene. The variable regions of vertebrate striated TnT isoforms reflect the subsequent addition and modification of genomic sequences to give rise to members of the TnT multigene family.     

Cloning and sequence analysis of cDNAs encoding precursors of sarafotoxins. Evidence for an unusual "rosary-type" organization

Sarafotoxins (SRTXs) are 21-amino acid peptides structurally and functionally similar to endothelins (ETs). To understand how SRTXs are overproduced in venom glands of the snakes Atractaspis engaddensis and hence used as toxins, we cloned cDNAs encoding SRTXs and elucidated their nucleotide sequences. We predict that SRTX precursors are large prepropolypeptide chains with an unusual "rosary-type" structure made of 12 successive similar stretches of 40 residues (39 in the first stretch). Each stretch begins with a "spacer" of 19 invariant residues (18 in the first stretch) immediately followed by the sequence of one SRTX isoform. Six different isoforms are identified within a single precursor molecule. Maturation of the precursor may require endopeptidases that cleave the Leu-Cys bond and the Trp-Arg/Lys bond invariably found at the SRTX N and C termini, respectively.     

Nucleotide and deduced amino acid sequences of rat myosin binding protein H (MyBP-H)

The complete nucleotide sequence of the cDNA clone encoding rat skeletal muscle myosin-binding protein H (MyBP-H) was determined and amino acid sequence was deduced from the nucleotide sequence (GenBank accession number AF077338). The full-length cDNA of 1782 base pairs(bp) contains a single open reading frame of 1454 bp encoding a rat MyBP-H protein of the predicted molecular mass 52.7 kDa and includes the common consensus 'CA__TG' protein binding motif. The cDNA sequence of rat MyBP-H show 92%, 84% and 41% homology with those of mouse, human and chicken, respectively. The protein contains tandem internal motifs array (-FN III-Ig C2-FN III-Ig C2-) in the C-terminal region which resembles to the immunoglobulin superfamily C2 and fibronectin type III motifs. The amino acid sequence of the C-terminal Ig C2 was highly conserved among MyBPs family and other thick filament binding proteins, suggesting that the C-terminal Ig C2 might play an important role in its function. All proteins belonging to MyBP-H member contains 'RKPS' sequence which is assumed to be cAMP- and cGMP-dependent protein kinase A phosphorylation site. Computer analysis of the primary sequence of rat MyBP-H predicted 11 protein kinase C (PKC) phosphorylation site, 7 casein kinase II (CK2) phosphorylation site and 4 N-myristoylation site.     

Cloning and localization of two multigene receptor families in goldfish olfactory epithelium

Goldfish reproduction is coordinated by pheromones that are released by ovulating females and detected by males. Two highly potent pheromones, a dihydroxyprogesterone and a prostaglandin, previously have been identified, and their effects on goldfish behavior have been studied in depth. We have cloned goldfish olfactory epithelium cDNAs belonging to two multigene G-protein coupled receptor families as a step toward elucidating the molecular basis of pheromone recognition. One gene family (GFA) consists of homologs of putative odorant receptors (approximately 320 residues) found in the olfactory epithelium of other fish and mammals. The other family (GFB) consists of homologs of putative pheromone receptors found in the vomeronasal organ (VNO) of mammals and also in the nose of pufferfish. GFB receptors (approximately 840 residues) are akin to the V2R family of VNO receptors, which possess a large extracellular N-terminal domain and are homologs of calcium-sensing and metabotropic glutamate receptors. In situ hybridization showed that the two families of goldfish receptors are differentially expressed in the olfactory epithelium. GFB mRNA is abundant in rather compact cells whose nuclei are near the apical surface. In contrast, GFA mRNA is found in elongated cells whose nuclei are positioned deeper in the epithelium. Our findings support the hypothesis that the separate olfactory organ and VNO of terrestrial vertebrates arose in evolution by the segregation of distinct classes of neurons that were differentially positioned in the olfactory epithelium of a precursor aquatic vertebrate.     

Localization of a highly conserved human potassium channel gene (NGK2-KV4; KCNC1) to chromosome 11p15

Several genes (the Shaker or Sh gene family) encoding components of voltage-gated K+ channels have been identified in various species. Based on sequence similarities Sh genes are classified into four groups or subfamilies. Mammalian genes of each one of these subfamilies also show high levels of sequence similarity to one of four related Drosophila genes: Shaker, Shab, Shaw, and Shal. Here we report the isolation of human cDNAs for a Shaw-related product (NGK2, KV3.1a) previously identified in rat and mice. A comparison of the nucleotide and deduced amino acid sequence of NGK2 in rodents and humans shows that this product is highly conserved in mammals; the human NGK2 protein shows over 99% amino acid sequence identity to its rodent homologue. The gene (NGK2-KV4; KCNC1) encoding NGK2 was mapped to human chromosome 11p15 by fluorescence in situ hybridization with the human NGK2 cDNAs.     

Dissociation of Ca2+ from sarcoplasmic reticulum Ca2+-ATPase and changes in fluorescence of optically selected Trp residues. Effects of KCl and NaCl and implications for substeps in Ca2+ dissociation

Sequential dissociation of the two Ca2+ ions bound to non-phosphorylated sarcoplasmic reticulum Ca2+-ATPase was triggered by addition, in a stopped-flow experiment, of quin2, which acted both as a high-affinity chelator and as a Ca2+-sensitive fluorescent probe. The kinetics of Ca2+ dissociation were deduced from the observed changes in quin2 fluorescence in the visible region (with lambdaex = 313 nm), while fluorescence detection in the UV region (with lambdaex = 290 nm) made it possible to monitor the tryptophan fluorescence changes accompanying this dissociation under the same ionic conditions. In the absence of KCl or NaCl, at pH 6 or 7, the observed changes in quin2 fluorescence were monoexponential, with rate constants very close to those of the changes in ATPase tryptophan fluorescence, which also appeared monophasic. In the presence of 100 mM KCl, quin2 fluorescence changes, although still monoexponential, were faster than in the absence of the monovalent ions but distinctly slower than the changes in tryptophan fluorescence, which were accelerated to a larger extent. In addition, the apparent kinetics of the Trp fluorescence changes depended on the excitation wavelength. Using an excitation wavelength of 296 nm, the Trp fluorescence drop was still faster than with an excitation wavelength of 290 nm, and in the presence of NaCl it even displayed a clear undershoot. We conclude that in the presence of KCl or NaCl and with an excitation wavelength of 290 nm, the rapid drop in tryptophan fluorescence mainly monitors the dissociation of the first of the two Ca2+ ions to be released from Ca2+-ATPase, while excitation at 296 nm optically selects a subpopulation of Trp residues whose fluorescence level is lower in the ATPase species with one Ca2+ ion bound than in the Ca2+-deprived ATPase species. The latter conditions result in an initial drop in Trp fluorescence whose apparent rate constant (in single-exponential analysis) is faster than the true rate of dissociation of the first Ca2+ ion and in a subsequent slower rise related to dissociation of the second Ca2+ ion. The difference between results obtained in the absence and in the presence of K+ or Na+ is due to an antagonizing effect of these cations on proton-induced conformational rearrangement of Ca2+-free ATPase, a conformational rearrangement which changes the ATPase Trp fluorescence level and significantly affects the cooperativity of Ca2+ binding at equilibrium.