Search

Dogfish (Squalus acanthias) growth hormone (GH) was identified by cDNA cloning and protein purification from the pituitary gland. Dogfish GH cDNA encoded a prehormone of 210 amino acids (aa). Sequence analysis of purified GH revealed that the prehormone is composed of a signal peptide of 27 aa and a mature protein of 183 aa. Dogfish GH showed 94% sequence identity with blue shark GH, and also showed 37-66%, 26%, and 48-67% sequence identity with GH from osteichtyes, an agnathan, and tetrapods. The site of production was identified through immunocytochemistry to be cells of the proximal pars distalis of the pituitary gland. Dogfish GH stimulates both insulin-like growth factor-I and II mRNA levels in dogfish liver in vitro. The dogfish GH gene consisted of five exons and four introns, the same as in lamprey, teleosts such as cypriniforms and siluriforms, and tetrapods. The 5'-flanking region within 1082 bp of the transcription start site contained consensus sequences for the TATA box, Pit-1/GHF-1, CRE, TRE, and ERE. These results show that the endocrine mechanism for growth stimulation by the GH-IGF axis was established at an early stage of vertebrate evolution, and that the 5-exon-type gene organization might reflect the structure of the ancestral gene for the GH gene family. [ABSTRACT FROM AUTHOR]

Melanin‐concentrating hormone (MCH) was originally discovered in fish, in which it causes aggregation or concentration of melanin granules in melanophores, thus regulating body color. MCH is a cyclic neuropeptide synthesized as a preprohormone in the hypothalamus of all vertebrates. Mammalian MCH plays an important role as a neurotransmitter or neuromodulator in regulating food intake and energy homeostasis. MCH signaling system may involve in regulating food intake also in fish. This neuropeptide binds to G‐protein‐coupled seven transmembrane receptor[s] to mediate its functions. This article reviews MCH and MCH receptor signaling systems in body color change and food intake in fish. J. Exp. Zool. 305A:751–760, 2006. © 2006 Wiley‐Liss, Inc.

A full-length transcript encoding a functional lamprey glycoprotein hormone receptor I (lGpH-R I, GenBank AY750688) was cloned from the testes of the sea lamprey, Petromyzon marinus, using the GpH-R protein fingerprint GLYCHORMONER from the PRINTS database. The present study is the first to identify a GpH-R transcript in an agnathan, which is one of the only two representatives of the oldest lineage of vertebrates. The 719-amino acid full-length cDNA encoding lGpH-R I is highly similar and is likely a homolog of the vertebrate GpH-Rs (including LH, FSH, and TSH receptors). The key motifs, sequence comparisons, and characteristics of the identified GpH-R reveal a mosaic of features common to all other classes of GpH-Rs in vertebrates. The lGpH-R I was shown to activate the cAMP signaling system using human chorionic gonadotropin in transiently transfected COS-7 cells. The highest expression of the receptor transcript was demonstrated in the testes using reverse transcriptase-PCR. Lower levels of the receptor transcript were also detected in brain, heart, intestine, kidney, liver, muscle, and thyroid. The high expression of lGpH-R I in the testis and the high similarity with gnathostome gonadotropin hormone receptors suggest that lGpH-R I functions as a receptor for lamprey gonadotropin hormones. We hypothesize from these data that there is lower specificity of gonadotropin and its receptor in agnathans and that during co-evolution of the ligand and its receptor in gnathostomes, there were increased specificities of interactions between each GpH (TSH, LH, and FSH) and its receptor.

A neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP) has possible potency as a hypothalamic factor mediating the release of pituitary hormones, especially growth hormone (GH), in the fish pituitary. We used double-immunostaining to examine the relationship between PACAP nerve fibers and adenohypophysial hormone-producing cells in the pituitary of a teleost, the stargazer Uranoscopus japonicus , and enzyme immunoassay to determine the quantity of PACAP in the stargazer brain, in conjunction with the body mass and gonad somatic index (GSI) of fish. In adult stargazer, PACAP-like immunoreactive (PACAP-LI) nerve fibers and endings were identified in both the neurohypophysis and adenohypophysis in close proximity to pituitary cells containing immunoreactive hormones such as prolactin, somatolactin, the N-terminal peptide of proopiomelanocortin, and N-acetyl endorphin. PACAP-LI nerve fibers were also identified close to immunoreactive GH cells in the pituitary of young fish. The concentration of immunoreactive PACAP in whole brain ranged from 100 to 800 pmol/g wet weight, in fish with weighing 70-480 g. A negative correlation was found between the concentration of immunoreactive PACAP in the whole brain and body weight, but there was no relation between the former and GSI. These results suggest that PACAP may act as a hypophysiotropic factor in the stargazer pituitary. [ABSTRACT FROM AUTHOR]

Mushroom bodies (MBs) are the centers for olfactory associative learning and elementary cognitive functions in the arthropod brain. In order to understand the cellular and genetic processes that control the early development of MBs, we have performed high-resolution neuroanatomical studies of the embryonic and post-embryonic development of the Drosophila MBs. In the mid to late embryonic stages, the pioneer MB tracts extend along Fasciclin II (FAS II)-expressing cells to form the primordia for the peduncle and the medial lobe. As development proceeds, the axonal projections of the larval MBs are organized in layers surrounding a characteristic core, which harbors bundles of actin filaments. Mosaic analyses reveal sequential generation of the MB layers, in which newly produced Kenyon cells project into the core to shift to more distal layers as they undergo further differentiation. Whereas the initial extension of the embryonic MB tracts is intact, loss-of-function mutations of fas II causes abnormal formation of the larval lobes. Mosaic studies demonstrate that FAS II is intrinsically required for the formation of the coherent organization of the internal MB fascicles. Furthermore, we show that ectopic expression of FAS II in the developing MBs results in severe lobe defects, in which internal layers also are disrupted. These results uncover unexpected internal complexity of the larval MBs and demonstrate unique aspects of neural generation and axonal sorting processes during the development of the complex brain centers in the fruit fly brain.

Adenohypophysial cell types in the pituitary of adult sea lampreys, Petromyzon marinus, was localized by means of immunocytochemical and lectin cytochemical techniques. At least four types of adenohypophysial hormone cells are present in the pituitary of adult sea lampreys. The first type of cell is ACTH-like and occupies most parts of the rostral pars distalis (RPD), but a few scattered ACTH-like cells are also present in the proximal pars distalis (PPD). The second type of cell is MSH-like and occupies the whole pars intermedia. The third type of cell is GH/PRL-like and occupies the dorsal half of the PPD. These GH/PRL-like cells were initially detected by heterologous immunocytochemistry using antibodies to salmon GH, salmon PRL and blue shark GH, after hydrated autoclave pretreatment of sections. Later, by use of an antiserum raised against a synthetic peptide corresponding to the partial sequence of lamprey GH/PRL, the same cells as those containing GH/PRL-like immunoreactivity were stained positively. Similarity of the topographic distributions between lamprey GH/PRL-like cells and gnathostome fish GH cells in the pituitary suggests that GH/PRL-like cells in the lamprey may be GH cells. The last type of cell is GTH-like and occupies the ventral half of the PPD. Although GTH has not yet been isolated from the lamprey pituitary, our immunocytochemical data suggest that GTH-like material in the sea lamprey pituitary is more closely related to mammalian-like LH, rather than to FSH or TSH. These four types of adenohypophysial cells occupy most parts of the lamprey adenohypophysis and indeed there is little room for TSH or PRL cells. Thus, the present study further suggests that GH and LH-like GTH are ancestral forms of GH/PRL/SL family and glycoprotein hormones, respectively.

Lampreys and hagfish of the class Agnatha are of particular importance in understanding endocrinological relationships since they represent the oldest lineages of extant vertebrates which evolved over 550 million years ago. This review briefly summarizes the latest findings on the reproductive endocrinology of the sea lampreys. Since the First International Symposium of Fish Endocrinology in 1988, when virtually little was known of the hypothalamic–pituitary–gonadal axis, substantial new biochemical, molecular, physiological and immunological evidence has now clearly shown that lamprey reproduction is controlled by the neuroendocrine axis. In addition, five brain and six pituitary hormones of lampreys have been identified mainly by Sower and Kawauchi and colleagues between 1986 and 2000. We now hypothesize that lamprey reproduction is a highly synchronized process that is initiated or mediated by a coordination of complex integration of environmental cues and hormonal mechanisms which is broadly similar to that exhibited by gnathostome vertebrates.

We report the immunomodulating effects of proopiomelanocortin (POMC)-related peptides on phagocytic cells in carp. The complete amino acid sequences of two carp POMCs (I and II) were deduced from the nucleotide sequences after cDNA cloning. Both POMCs consist of 194 amino acids (91% sequence identity) including identical α-melanotropin (MSH) and β-endorphin (EP). All hormonal peptides derived from two POMCs were identified by mass spectrometry after separation by high-performance liquid chromatography of an acid-acetone extract from a single pituitary. These peptides were α-MSH,N-Des-Ac-α-MSH, di-Ac-α-MSH, β-MSH I, β-MSH-II, N-Ac-β-EP(1–29), corticotropin-like intermediate lobe peptide I and II and N-terminal peptide of POMC I and II. The immunomodulating effects of synthetic MSHs and EPs on phagocytic cells from carp head kidney were examined. Di-Ac-α-MSH, β-MSH I,N-Ac-β-EP(1–29) and β-EP(1–29) increased the production of superoxide anion at 0·1–100ng ml−1for these MSHs and 1–100ng ml−1for EPs in RPMI 1640 medium.

Recently, we have characterized a new MSH (named δ-MSH) which joins the group of MSHs (α, β, γ) in dogfish proopiomelanocortin (POMC). The present study has confirmed the presence of δ-MSH in POMC of another member of the elasmobranchian order, the stingray, Dasyatis akajei,by cDNA cloning from pituitary mRNAs. Overlapping partial cDNA clones corresponding to stingray POMC were amplified by PCR from single-strand cDNA prepared from pituitary poly (A)+RNA. Excluding the poly A tail, stingray POMC cDNA consists of 1077 base pairs (bp). It contains a 912-bp open reading frame encoding a signal peptide of 24 amino acids (aa) and a POMC of 280 aa. γ-MSH, α-MSH, ACTH, δ-MSH, β-MSH, and β-endorphin are located at POMC (50–61), (115–127), (115–153), (182–193), (226–242), and (245–280), respectively. The stingray POMC is smaller than that of the dogfish POMC (294 aa) mainly due to the absence of a sequence of 11 consecutive aa between δ-MSH and β-MSH. δ-MSH has been found only in the elasmobranchs and, therefore, δ-MSH might have evolved after the divergence of chondrichthians from the ancestral vertebrate lineage and before divergence of sharks and rays.

We report the isolation, cDNA cloning, and growth promoting activity of rabbitfish (Siganus guttatus; Teleostei; Perciformes; Siganidae) growth hormone (GH). Rabbitfish GH was extracted from pituitary glands under alkaline conditions, fractionated by gel filtration chromatography on Sephadex G-100, and purified by high-performance liquid chromatography. The fractions containing GH were identified by immunoblotting with bonito GH antiserum. Under nonreducing conditions, the molecular weight of rabbitfish GH is about 19 kDa as estimated by SDS–PAGE. The purified hormone was potent in promoting growth in rabbitfish fry. Weekly intraperitoneal injections of the hormone significantly accelerated growth. This was evident 3 weeks after the start of the treatment, and its effect was still significant 2 weeks after the treatment was terminated. Rabbitfish GH cDNA was cloned to determine its nucleotide sequence. Excluding the poly (A) tail, rabbitfish GH cDNA is 860 base pairs (bp) long. It contained untranslated regions of 94 and 175 bp in the 5′ and 3′ ends, respectively. It has an open reading frame of 588 bp coding for a signal peptide of 18 amino acids and a mature protein of 178 amino acid residues. Rabbitfish GH has 4 cysteine residues. On the amino acid level, rabbitfish GH shows high identity (71–74%) with GHs of other perciforms, such as tuna, sea bass, yellow tail, bonito, and tilapia, and less (47–49%) identity with salmonid and carp GHs.

Insulin-like growth factor-I (IGF-I) is a mitogenic polypeptide that plays an essential role in the regulation of development and somatic growth of vertebrates, mainly by mediating growth hormone actions. It has clearly been established that the structure of IGF-I and its biological function has been highly conserved among vertebrates. In this paper, we review the recent developments in the molecular, biochemical, and physiological properties of IGF-I in fish.

A gonadotropin receptor was cloned from amago salmon (Oncorhynchus rhodurus) ovarian follicles. This receptor (sGTH-R) belongs to the glycoprotein hormone receptor family with a large extracellular and seven-transmembrane domains. Its sequence homology is highest with mammalian LH receptors. Phylogenetic analysis reveals that sGTH-R is grouped with mammalian and chicken FSH and LH receptors, but not with mammalian TSH receptors. sGTH-R is expressed dominantly in the ovary and testis. Functional characterization examined with transiently transfected mammalian cells revealed increased intracellular cAMP level when exposed to mammalian and fish gonadotropins; the most potent hormone was salmon GTH II. These results indicate that the cloned cDNA encodes a functional amago salmon GTH receptor protein.

The sea lamprey shows an important divergence in the proopiomelanocorticotropin (POMC) prohormone in that adrenocorticotropin (ACTH) and melanocyte stimulating hormone (MSH) are encoded on distinct genes, proopiocortin (POC) and proopiomelanotropin (POM), respectively. POC encodes nasohypophysial factor (NHF), ACTH, and β-endorphin while POM encodes α- and β-MSH-like peptides (called MSH-B and MSH-A, respectively), and a second form of β-endorphin. The present study examined the variation in expression of these two genes during the life cycle of sea lampreys. Individual Northern blot analysis and pooled data from several Northern blots showed significant differential expression of POC and POM during the lamprey life cycle. While POC expression remained low over larval and metamorphic stages, POM expression rose substantially in stage 4 and stage 6 of metamorphosis. Both POC and POM expression levels increased in immediately post-metamorphic animals. The highest levels of POM expression were observed in larger juvenile adults and pre-spawners, whereas the highest level of POC expression was observed at the pre-spawning period. Statistical analysis showed POM expression to be much higher than POC expression in immediately post-metamorphic animals prior to feeding. Northern analysis of total RNA isolated from various tissues in the pre-spawning lampreys, including brain, showed expression of POC and POM only in the pituitary. These observations indicate that the expression of POC and POM, which encode different sets of hormones, may be related to developmental and maturation events during the life cycle of the sea lamprey. J. Exp. Zool. 283:95–101, 1999. © 1999 Wiley-Liss, Inc.

Somatolactin (SL) is a newly characterized pituitary hormone belonging to the growth hormone–prolactin family. Until now SL has been identified only in teleosts, the most highly derived ray-finned fishes. We report here the cloning of SL cDNAs from two species of bony fish, the white sturgeon (Acipenser transmontanus) and the African lungfish (Protopterus annectens). Overlapping partial cDNA clones corresponding to teleost SLs were amplified by polymerase chain reaction (PCR) from either single-strand or double-strand cDNA from pituitary glands. Excluding the poly(A) tail, the sturgeon SL cDNA is 881 base pairs (bp). This is comparable to 1.0 kb estimated by Northern blot analysis. It contains a 696-bp open reading frame encoding a prehormone of 232 amino acids (aa) with a signal peptide of 24 aa and a mature protein of 208 aa. Excluding the poly(A) tail, the lungfish SL cDNA is 938 bp. This is comparable to 1.1 kb estimated by Northern blot analysis. It contains a 696-bp open reading frame encoding a prehormone of 232 aa with a signal peptide of 26 aa and a mature protein of 206 aa. The deduced aa sequences of sturgeon and lungfish SLs show 76–60% and 65–54% identity with teleost SLs, respectively. These values are significantly higher than the 30% identity with nonteleostean growth hormones and prolactins. Immunostaining of sturgeon pituitary with anti-salmon SL serum demonstrated that the SL cells were localized in the pars intermedia, as in teleosts. The present results demonstrate that the SL gene is present in two divergent lineages, the Actinopterygii (Chondrostei: white sturgeon) and the Sarcopterygii (Dipnoi: African lungfish).

We report the isolation and cDNA cloning of somatolactin (SL) from rabbitfish, Siganus guttatus. Rabbitfish SL was isolated from an alkaline extract of the pituitary glands by gel filtration chromatography on Sephadex G-100 and reversed-phase high-performance liquid chromatography. SL was monitored by immunoblotting with flounder SL antiserum. The preparation (yield: 0.86 mg/g wet tissues) contained two immunoreactive bands of 24 and 28 kDa on SDS–PAGE. Overlapping partial cDNA clones corresponding to teleost SLs were amplified by PCR from single-strand cDNA from pituitary glands. Excluding the poly(A) tail, rabbitfish SL cDNA is 1605 bp long. It contains a 693-bp open reading frame encoding a signal peptide of 24 amino acids (aa) and a mature protein of 207 aa. Rabbitfish SL has two possible N-glycosylation sites at positions 11 and 121 and seven half Cys residues. The deduced amino acid sequence shows over 80% identity with those of advanced teleosts like sea bream, red drum, and flounder, 76% with the salmonids, 57% with the eel, and 46% with the goldfish SL.

Proopiomelanocortin (POMC) is a precursor for corticotropin (ACTH), three or fewer molecular types of melanotropin (MSH), and β-endorphin. This protein is thought to have evolved by duplication of MSH genomic segments. Here we report that the POMC in the dogfish, an elasmobranch, contains a fourth type of MSH in addition to classical α-, β-, and γ-MSH. POMC cDNA was amplified by PCR from double-strand cDNA prepared from dogfish pituitary and ligated into λZAP II. The POMC cDNA is composed of 1315 bp without a poly(A) tail. Northern blot analysis detected a 1.4-kb signal of dogfish POMC mRNA. An open reading frame of the POMC cDNA encodes 320 amino acids, including a signal peptide of 26 amino acids. The dogfish POMC includes γ-MSH, ACTH, α-MSH, β-MSH, and β-endorphin at positions 50–61, 115–153, 115–127, 239–256, and 259–294, respectively. In addition to these classical peptides, a newly discovered MSH, which we have termed δ-MSH, is present in dogfish POMC at position (184–195). The four dogfish MSHs can be separated into two groups based on their sequence identities: one pair consists of α-MSH and γ-MSH, and the other consists of β-MSH and δ-MSH, suggesting that γ-MSH and δ-MSH may have been duplicated evolutionarily from α-MSH and β-MSH, respectively. γ-MSH might first have appeared in early gnathostomes because it is absent in the most primitive vertebrate group, the agnathans. δ-MSH, which at this time is found only in chondrichthians, might have appeared after the divergence of chondrichthians from a lineage leading to osteichthyans and tetrapods.

To investigate the evolution of proopiomelanocortin (POMC) from fish to tetrapods, nucleotide sequence of POMC cDNA from a lobe-finned fish, the African lungfish, was determined. POMC cDNA was prepared from lungfish pituitary glands. The POMC cDNA is composed of 1114 bp, excluding a poly-A tail, and encodes 255 amino acids (aa) including a signal peptide of 25 aa. The lungfish POMC contains the segment corresponding to γ-melanotropin (MSH), corticotropin, α-MSH, β-MSH, and β-endorphin at positions (50–61), (108–146), (108–120), (178–194), and (197–230), respectively. The lungfish POMC shows greater sequence identity on average with amphibian (62%), ancient ray-finned fishes including acipenseriformes and semionotiformes (62%), and mammalian POMC (52%) than with teleostean (49%), elasmobranch (46%), and agnathan POMC (31%). Thus, the overall structural feature of lungfish POMC is close to the tetrapod POMCs which contain γ-MSH and the ancient ray-finned fishes POMCs containing γ-MSH-like sequence. However, amino acid sequence of lungfish β-endorphin exhibits properties which are specifically observed in the ray-finned fishes and the elasmobranchs.

We previously isolated a cDNA encoding a gonadotropin receptor (sGTH-R) from amago salmon (Oncorhynchus rhodurus) ovarian follicles. In the present study, we cloned a second gonadotropin receptor (sGTH-RI) from the same RNA preparations. Overall sequence homology between sGTH-RI and sGTH-R is 44%. The highest homology occurs with mammalian FSH receptors (49%). Functional characterization examined in COS-7 cells transiently transfected with sGTH-RI showed the largest increase in cAMP production when exposed to salmon GTH I. These results provide the first evidence in any ectothermic vertebrate, the amago salmon, of the duality of gonadotropin receptors.

In lampreys, although gonadotropin (GTH) has not yet been isolated from the pituitary gland, the presence of GTH has been strongly suggested. To detect possible GTH in the sea lamprey (Petromyzon marinus) pituitary, two different cytochemical probes were tested: One was the use of antibodies to GTHs, and the other was the use of lectin-screening kits for demonstration of glycoconjugate in hormonal molecules. GTH-like immunoreactivity was found in cells distributed in the ventral half of the proximal pars distalis. These cells were stained intensely by all four lots of anti-ovine LH including LHβ, and were stained moderately or weakly by several other antibodies to LH-related GTHs, such as human LHβ, hCGβ, amphibian LH, and sturgeon GTH IIβ. On the other hand, there were no positive reactions in the sea lamprey pituitary using the antibodies to FSH-related GTHs, thyrotropin (TSH), or pituitary glycoprotein hormones of teleost origin. Thus, GTH-like material in the sea lamprey pituitary seems to be more closely related to mammalian-like LH, rather than to FSH or TSH, as far as immunocytochemical determinants. A total of 21 kinds of lectins was tested. Among those, GTH-positive cells were also stained positively by concanavalin A andVicia villosaagglutinin. Thus, the present study demonstrates colocalization of LH-like immunoreactivity and glycoconjugate in cells in the ventral half of the proximal pars distalis of the sea lamprey pituitary. It is suggested that those cells are most likely to be GTH cells in the sea lamprey pituitary.

Stanniocalcin (STC) is a hypocalcemic glycoprotein hormone secreted by the corpuscles of Stannius, endocrine glands unique to the bony fishes. Recently, two cDNAs encoding proteins, STC-1 and STC-2, homologous to fish STC have been identified in humans and rodents. The STC-1, but not STC-2, is identical to fish STC with respect to location of all Cys residues. The present study reports the localization of inter- and intra-molecular disulfide linkages in chum salmon STC. The chum salmon STC was deglycosylated and digested with several proteases in series. Six fragments, each of which consisted of two peptides connected by a single disulfide bond, were isolated by HPLC. Disulfide bonds were determined by sequence analysis after reduction and S-alkylation of each peptide fragment. Chum salmon STC is a homodimer connected by a single intermonomeric disulfide bond at Cys169. The monomer consists of 179 amino-acids, containing five intramonomeric disulfide bonds formed between Cys12-Cys26, Cys21-Cys41, Cys32-Cys81, Cys65-Cys95, and Cys102-Cys137.

Transport of recombinant salmon growth hormone (rsGH) into the circulation of rainbow trout following intragastric or rectal administration was investigated. Changes in plasma GH levels were analyzed by radioimmunoassay specific to chum salmon GH. Intragastric administration of rsGH by oral intubation resulted in elevation of GH immunoreactivity in plasma after 11h. The plasma GH increased maximally after 15h, and then declined rapidly to reach a normal level after 19h. On the other hand, oral intubation of rsGH to carp, which has no stomach, caused elevation of plasma GH levels after 1 h which lasted for 21 h. In sharp contrast, rectal administration of rsGH to rainbow trout significantly elevated plasma GH levels after 15 min, which reached a maximum after 30 min. The rsGH was labeled with fluorescein isothiocyanate (FITC) to distinguish exogenous from endogenous GH, and administered into rainbow trout rectally. Subsequent analysis of plasma samples on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed two fluorescent bands at the same molecular weights as those of the monomeric and dimeric rsGHs. Intragastric and rectal administration of rsGH into juvenile trout resulted in significant increases in length and body weight compared to the control fish. This study strongly suggests that the rsGH is transported into the circulation of salmonids via the lower part of the intestine and takes part in growth stimulation.

For the investigation of the evolution of the proopiomelanocortin (POMC) gene in early ray-finned fishes, nucleotide sequence of POMC cDNA from a chondrostean fish, the sturgeon has been determined. POMC cDNA was amplified by PCR from double-strand cDNA prepared from sturgeon pituitary and ligated with λZAP II. The POMC cDNA consists of 1079 bp without a poly-A. An open reading frame of the POMC cDNA encodes 263 amino acid residues. Sturgeon POMC contains ACTH, α-melanotropin (MSH), β-MSH and β-END at positions (115-153), (115-127), (186-202) and (205-238), respectively. Location of POMC(51-72) is homologous to γ-MSH, whereas the third residue of MSH-core sequence, His-Phe-Arg-Trp, is changed to His. Moreover, there are no basic amino acids to serve as a processing signal on the N-terminal side of POMC(51-72). These structural characteristics suggest that an ancestor of the ray-finned fishes had γ-MSH, whereas significant mutations occurred during the evolution of chondrostean fish.

Mammalian and avian growth hormones (GH) (pituitary derived or biosynthetic) exert two effects on chicken adipose tissue explants in vitro.They (i) increase the basal rate of glycerol release a lipolytic effect) and (ii) inhibit glucagon-stimulated glycerol release (an antilipolytic effect). The ability of lower vertebrate GH preparations to exert lipolytic and antilipolytic effects was examined and biological activity was compared to differences in amino-acid residue sequences and to predicted structure. Irrespective of species origin (blue shark, sturgeon, bonito, yellow tail, salmon, bullfrog, sea turtle), all lower vertebrate GH preparations showed very weak (<5% the potency of bovine GH), if any, lipolytic activity, but retained strong antilipolytic activity. The present data indicate that the structural requirements for lipolytic and antilipolytic activities of GH differ in chicken adipose tissue. Despite the high sequence homology (88%) between chicken and sea turtle GH, the latter preparation did not stimulate lipolysis. It is suggested that Pro132, conserved only in lipolytically active GH species (human, bovine, and chicken), represents a major determinant of lipolytic activity in chicken adipose tissue. The structural determinants for antilipolytic activity may comprise any or all of residues 3, 17, 64, 108, 109, and 152.

Melanin-concentrating hormone (MCH) has been purified from the chum salmon pituitary. Its complete amino acid sequence has recently been established. To identify the precise site of origin of MCH, immunostaining was performed in the brain and pituitary gland of the chum salmon and the rainbow trout using a highly sensitive and specific antiserum raised against synthetic MCH. In these two salmonid species immunoreactivity for MCH was detected in neurons and neuronal processes in the pars lateralis of the nucleus lateralis tuberis (NLT) in the basal hypothalamus. Numerous positive-staining processes of these MCH-neurons project to the pituitary gland, extending into neurohypophysial tissues within the pars intermedia and, to a lesser extent, into the pars distalis. No pituitary cells showed cross-reactivity. These results suggest that MCH is biosynthesized in the neurons of the NLT/pars lateralis and released in the neurohypophysis. On the other hand, prominent but less numerous MCH-positive processes could be traced to the pretectal area in which projection of both optic and pineal fibers has been detected using tracers. This observation suggests that the synthesis and/or release of MCH might be under the influence of either of these photosensory neurons. Moreover, the existence of an extrahypothalamic projection from MCH-positive neurons suggests that, in addition to melanin-concentration, MCH might be involved in other neuronal functions, perhaps serving as neuromodulator in the brain.

Melanin-concentrating hormone (MCH) is a neurosecretory peptide that induces melanin concentration within teleost melanophores. Here, we characterized MCH-like substance in the rat brain by both an in vitro fish-scale melanophore bioassay and a radioimmunoassay with a salmon MCH antiserum that is directed toward the carboxy-terminus and requires the cyclic configuration for recognition. Furthermore, subcellular localization of the MCH in the rat brain was examined by immunocytochemistry using electron microscopy. We confirmed that MCH-immunoreactivity and MCH-bioactivity were present together in the same effluent fractions of the rat hypothalamic extracts by reverse-phase high-performance liquid chromatography (HPLC). At electron microscopic level, MCH-immunoreactivity was located specifically in secretory granules in MCH-positive cell bodies confined to the hypothalamus with their neuronal processes projecting widely in the rat brain. Although full characterization of substance must await its isolation, our results strongly support the notion that rat MCH-like substance may be homologous but not identical to salmon MCH, and simultaneously may serve some neurotransmitter and/or neuromodulator role in the brain of the rat.

We report here on the cellular localization in the fish pituitary of somatolactin (SL), a putative new pituitary hormone related to growth hormone and prolactin, which has been recently identified in the piscine pituitary gland. Immunocytochemical staining, using anti-cod SL serum, revealed that in the cod pituitary gland, SL is produced by cells in the intermediate lobe, bordering the neural tissue. These cells, staining weakly with periodic-acid-Schiff (PAS), are distinct from the melanocyte stimulating hormone (MSH) cells which, as in all teleosts, are PAS-negative. SL-immunoreactivity was observed in the same location in all other teleost species examined: flounder, rainbow trout, killifish, molly, catfish and eel. In most fish the SL-immunoreactive cells are either strongly or weakly PAS-positive but in rainbow trout are chromophobic, indicating that the SL protein can probably exist in glycosylated and non-glycosylated forms. Thus, in demonstrating the cellular localization of SL, this study provides the first identification of the enigmatic, second cell-type of the fish pars intermedia.

Two POMC-like pituitary prohormones proopiocortin (POC) and proopiomelanotropin (POM) have been characterized from adult sea lampreys (Petromyzon marinus). POC encodes a nasohypophysial factor (NHF), ACTH, an MSH, and β-END; and POM encodes MSH-A, MSH-B, and β-END. Two radiolabeled riboprobes, one encoding a unique portion of POC mRNA and the other encoding the MSH-B domain unique to POM mRNA, were generated in order to examine the expression of POC and POM during the life cycle of the sea lamprey byin situhybridization. POC expression appears evenly distributed throughout most cells of the rostral pars distalis (RPD) during the entire life cycle. POC expression also occurs in scattered cells of the caudal (proximal) pars distalis (CPD) at stage 5 of metamorphosis. By the prespawner period, POC expression is mainly distributed in the dorsal aspect of this region. POM expression was completely confined to most cells of the pars intermedia (PI) at all periods examined. Quantitative, computer-assisted, image analysis of POM expression revealed high signal densities in all larvae which decreased by early metamorphosis, steadily increased and reached high levels by late metamorphosis (stages 6 and 7), and attained even higher levels in prespawners. Volumetric analysis revealed that the net volume of POM expressing cells is at its lowest in larvae and increases during subsequent development. Analysis of signal density and volumetric measurements of POC expression revealed that POC expression in the RPD is low in larvae and steadily increases during subsequent intervals of the life cycle reaching very high levels by the prespawning period. POC expression in the CPD, first visible at stage 5, increases steadily throughout the remainder of metamorphosis and reaches the highest levels of expression in prespawning animals. These results would implicate the role of POM and POC in some developmental processes but not in the initiation of metamorphosis. The very high levels of POM and POC expression in prespawner animals suggest that the two genes may have important roles at this time in the life cycle of lampreys.

This paper describes the isolation and complete amino acid sequence of growth hormone (GH) from the pituitary gland of the crocodile, Crocodylus novaeguineae. GH was detected in an alkaline extract of the pituitary by specific immunoblot reactivity with rabbit antisera against cattle and salmon GH and isolated in pure form by DEAE-cellulose chromatography and by reversed-phase high-performance liquid chromatography. The yield of the GH was 2.2 mg from 3.5 g of wet tissue. Crocodile GH consists of 190 residues and is highly similar to chicken, duck, and sea turtle GHs with 90-92% structural similarity, although it shows only 27% identity with crocodile prolactin. A molecular phylogenetic tree of GH from 39 vertebrate species is proposed on the basis of sequence comparison. Copyright 1995, 1999 Academic Press

A specific homologous radioimmunoassay (RIA) for measurement of insulin-like growth factor-I (IGF-I) in plasma of salmonid and a few non-salmonid fish species was developed using recombinant coho salmon IGF-I (rsIGF-I) as tracer and standard, and antiserum against this peptide raised in rabbits. The minimum detection level of IGF-I was 1.5 ng/ml and linearity was obtained in a range from 1.5 to 23 ng/ml. No cross-reaction was detected in the salmon IGF-I RIA with mammalian growth factors, salmon pituitary hormones, salmon or mammalian insulin, or any peptide in rat plasma. Although salmon IGF-I has high sequence similarity to mammalian IGF-I, it did not cross-react with anti-human IGF-I serum in human RIA and serial dilutions of plasma from salmon were not parallel to the human IGF-I standards in this assay system. In contrast, dilution curves for plasma of salmonids, such as coho (Oncorhynchus kisutch), Atlantic (Salmo salar), and sockeye (O. nerka) salmon, rainbow trout (O. mykiss), some other teleost fish, such as tilapia (Oreochromis mossabmica), carp (Cyprus carpio), eel (Anguilla rostrata), Atlantic halibut (Hippoglossus hippoglossus), and agnathan, the sea lamprey (Petromyzon marinus), assessed in salmon IGF-I RIA were parallel to the rsIGF-I standards. Acid-ethanol extraction of plasma samples altered the molecular weight, but not the quantity, of immunoreactive IGF-I, implying that IGF-I binding proteins in salmon plasma do not affect the performance of the salmon IGF-I RIA. Gel filtration of nonacidified plasma on a Sephadex G-75 superfine column produced two immunoreactive IGF-I peaks of molecular weights of approximately >70 k and 7 kDa, whereas acidification of plasma increased the relative amount of the 7-kDa peak (IGF-I) and the >70-kDa peak disappeared. The recoveries of rsIGF-I added to extracted or nonextracted plasma were 97.4 and 94.9%, respectively. Inter- and intraassay coefficients of variation were 3.6 and 3.3%, respectively. Plasma IGF-I levels in coho salmon smolts were 117.4 ± 19.1 ng/ml as compared to IGF-I levels in parr (45.3 ± 2.5 ng/ml) or in adult fish (45.2 ± 5.4 ng/ml) measured in the same assay. Injection of salmon growth hormone, but not prolactin or somatolactin, caused a significant and dose-dependent elevation of plasma IGF-I levels, while either fasting or injection of streptozotocin led to a significant decline in systemic IGF-I. We conclude that the salmon IGF-I RIA reported in this study is valid for measurement of IGF-I levels in salmonids and, likely, in a variety of other fish species.

Coho salmon (Oncorhynchus kisutch) thyroid-stimulating hormone (TSH) was isolated by ethanol extraction of pituitary glands from mature coho salmon. Extraction was followed by gel-filtration chromatography on Sephadex G-100 superfine, anion-exchange chromatography on a Whatman DE-52 column, and finally by reverse-phase high-performance liquid chromatography. Fractions were monitored for TSH content by a homologousin vivobioassay and by immunoblots using anti-human TSH β-subunit antisera.In vivotreatment of coho salmon parr with coho salmon TSH caused a dose-dependent increase in plasma thyroxine level similar to that induced by bovine TSH. The N-terminal sequence (25 residues) of the salmon TSH β subunit has 56% sequence identity to that of human TSH β subunit and is identical to the deduced amino acid sequence of trout TSH β subunit. The N-terminal sequence (25 residues) of the salmon TSH α subunit is identical to gonadotropin α-II subunit. Molecular sizes of the α and β subunits are 18,000 and 24,000 daltons, respectively, as estimated by SDS–PAGE. Antiserum generated against salmon TSH, which was preadsorbed with α subunit using an α-subunit affinity column, detected only salmon TSH β subunit by immunoblot and specifically stained thyrotropin-producing cells of the pituitary gland. A homologous radioimmunoassay (RIA) was developed using purified salmon TSH standard, iodinated TSH β subunit, and antiserum generated against salmon TSH. Cross-reactivities of GTH I, GTH II, GTH I β and GTH II β subunits, α subunit, growth hormone, prolactin, and somatolactin were less than 1%. Displacement curves for serial dilutions of plasma and pituitary extracts of various salmonid species, as well as coho salmon pituitary cell culture medium, were parallel to the coho salmon TSH standards. In contrast, plasma of hypophysectomized juvenile coho salmon and pituitary extracts of Pacific tomcod (Microgadus proximus) did not displace bound radiolabeled salmon TSH. Finally,in vivoinjection of juvenile coho salmon with triiodothyronine decreased plasma TSH levels, whereas the goitrogen, methimazole, increased plasma TSH levels. Injection of gonadotropin-releasing hormone agonist did not alter plasma TSH. These data suggest that the RIA is specific for TSH and confirm a negative-feedback relationship between the thyroid hormones and TSH.

Three peptides containing the melanotropin (MSH)-core sequence, YXMXHFRWG, have been isolated recently from the pituitary glands of adult sea lamprey (Petromyzon marinus) and were tentatively assigned as lamprey adrenocorticotropin (ACTH), MSH-A, and MSH-B, respectively. Both MSHs differed significantly from gnathostome MSHs and cannot be assigned as α-MSH, β-MSH, or γ-MSH. The aim of the present study was to localize these peptides in the lamprey pituitary using antisera generated against synthetic lamprey ACTH1-16, MSH-A, and MSH-B. ACTH-like immunoreactivity was found in most cells of the rostral pars distalis (RPD) and in a few scattered cells of the proximal pars distalis (PPD). MSH-A-like immunoreactivity was found in most cells of the RPD, a few scattered cells of the PPD, and almost all cells of the pars intermedia (PI). MSH-B-like immunoreactivity was found only in the PI, where almost all cells were stained. Thus, the topographic distributions of ACTH and MSHs in the lamprey pituitary were similar to those in gnathostome vertebrates.

A highly purified growth hormone (GH) was isolated from the pituitary of the Japanese flounder (Paralichthys olivaceus) following extraction with alkaline solution, gel filtration on Sephadex G-75, and high-performance liquid chromatography (HPLC) on a reverse-phase column. Flounder GH (flGH) has a molecular weight of 20 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and an isoelectric point of 7.1 by gel electrofocusing. The complete amino acid sequence was determined by Edman degradation of the peptide fragments generated by enzymatic digestion and chemical cleavage. The fIGH comprises 173 amino acid residues with pyroglutamate at the N-terminus. Compared with other fish GHs, flGH has an uninterrupted deletion of 14 amino acid residues near the C-terminus. flGH is the smallest of growth hormones so far reported and is a potent stimulator of growth in juvenile rainbow trout. Copyright 1993, 1999 Academic Press

Two gonadotropins, GTH I and GTH II, were isolated from pituitaries of spawning coho salmon (Oncorhynchus kisutch) using sequential extractions with ammonium acetate (pH 9.0) and 40% ethanol, precipitation with 80% ethanol, gel filtration chromatography (Sephadex G-100), anion-exchange chromatography (Mono-Q Sepharose), and gel filtration chromatography (Sephadex G-75). Coho salmon GTH I and GTH II stimulated steroidogenesis in vitro in a similar dose-dependent manner when incubated with either ovaries or testes of prepubertal coho salmon. An in vivo bioassay using coho salmon parr demonstrated that coho salmon GTH I and GTH II did not contain thyrotropic activity. Molecular weights were estimated by gel filtration chromatography to be 43,000 and 39,000 for GTH I and GTH II, respectively. Analysis of coho salmon GTH I and GTH II on reversed-phase high-performance liquid chromatography (rpHPLC) revealed that they consist of α and β subunits with N-terminal amino acid residues of Tyr, Gly (α, β of GTH I) and Tyr,Ser (α, β of GTH II). Coho salmon GTH I-β and GTH II-β differed from each other in amino acid composition, N-terminal amino acids (Gly vs. Ser), and molecular weights in SDS-PAGE (19,000 vs. 20,000) and had a high degree of chemical similarity to chum salmon GTH I-β and GTH II-β, respectively. Specific rabbit antisera to the β subunits of coho salmon GTH I and GTH II were generated. The observation of two GTHs with distinctly different chemical characteristics in coho salmon is similar to what has previously been found in chum salmon.

The catalytic activities for hydrogen-deuterium exchange were examined at 473 K for three kinds of SiC powders with different particle sizes. Three SiC powders showed a high activity after outgassing at high temperatures above 873 K. The thermodesorbed gases from SiC were mainly H2 and CO, and they were produced from the thermal decomposition of adsorbed water on SiC surfaces. The extent of decomposition was found to be parallel to the catalytic activity at 473 K. Therefore, the sites of the décomposition are responsible for the catalytic activity.

A highly specific radioimmunoassay was developed for N-terminal peptide of salmonid proopiocortin using a guinea pig antiserum to the chum salmon peptide (sNPP 1). Since sNPP I has no tyrosine residue nor free N-terminal amino group, a mixture of minor components of sNPP 1, which have extensions of H-Val-LysGly- and H-Lys-Gly- at the N-terminus, were iodinated by the lactoperoxidase method after incorporation of 3-(phydroxyphenyl)-propionate to the terminal amino groups. Plasma and pituitary extracts of several salmonid species showed parallel displacement to the standard hormone. Samples from carp, goldfish, tilapia, and eel, as well as the plasma of hypophysectomized rainbow trout, showed no crossreactivity. Proopiocortin-related hormones isolated from the chum salmon pituitary, including melanotropins, endorphins, corticotropin-like intermediate lobe peptides, and gonadotropin and prolactin showed negligible cross-reactivity. NPP contents in the pars intermedia of rainbow trout and chum salmon were 10 to 15 times greater than those in the pars distalis. Plasma levels of NPP in the mature chum salmon caught in the bay were about 50ng/ml. Plasma NPP levels in the mature chum salmon of both sexes decreased after transfer from seawater to fresh water. Plasma cortisol showed a concomitant change with NPP, thus supporting previous findings that NPP modulates corticotropin action on the trout interrenal.

Fluorescein isothiocyanate (FITC) isomers and the intermediate products have been separated by reversed phase high performance liquid chromatography (HPLC), using a linear gradient formed from acetone and 0.01 M sodium phosphate buffer (pH 7.0) or an isocratic elution of 40% acetone in 0.1 M pyridine-acetic acid (pH 7,0). This HPLC system was used to evaluate commercial FITC products. A method for the purification of the FITC product by HPLC was also described.

Chum salmon (Oncorhynchus keta) stanniocalcin was purified, partially identified and tested for bioactivity in an assay on the intestinal calcium uptake in a marine teleost (Gadus morhua). Basic ethanol extraction, ion exchange chromatography, gel filtration and reverse-phase high-performance liquid chromatography resulted in the isolation of a homogenous glycoprotein that appears as a 46-kDa product under non-reducing conditions and as a 23-kDa product under reducing conditions after sodium dodecylsulphate-polyacrylamide gel electrophoresis. The glycoprotein is likely to be a homodimer composed of two subunits of 23 kDa each. Further characterization indicates homology to Australian eel, sockeye salmon, coho salmon and rainbow trout stanniocalcin, and the glycoprotein is thus concluded to be stanniocalcin. Stanniocalcin-like immunoreactivity was demonstrated in the corpuscles of Stannius of the Atlantic cod, with a specific antiserum raised against purified chum salmon stanniocalcin. The physiological importance and the biological activity of chum salmon stanniocalcin was tested by evaluating its effect on intestinal calcium uptake by the Atlantic cod in vitro. The intestine was perfused, both vascularly and through the intestinal lumen, and the calcium mucosa-to-serosa flux was measured using 45Ca2+ as a tracer. Stanniocalcin decreased the intestinal calcium uptake in a dose-related manner by 13.5% and 22.4% at doses of 2.2 and 10.9 nM stanniocalcin, respectively. The results establish the intestine as a target organ for stanniocalcin in marine teleosts.

There are many biochemical and molecular biological data indicating that IGF-I is present in teleost fish. The present study examines red seabream for the presence and cellular distribution of IGF-I in the ovarian follicles during oocyte growth and final maturation of oocytes by immunocytochemistry. Immunoreactivity appeared in the granulosa cells at the lipid stage. Immunoreactivity became strong at the primary yolk globule stage and diminished in intensity during development of maturational competence (responsiveness to maturation-inducing steroid) and final maturation of oocytes. Intense immunoreactivity was also localized in the outer layer of the zona radiata and the peripheral region of the ooplasm at the primary yolk globule stage. Postovulatory follicles had low immunoreactivity in the early phase of degeneration. These immunocytochemical observation show that the granulosa cell layer is the main site of IGF-I production. IGF-I may be involved in granulosa cell proliferation and differentiation.

A new form of somatostatin (SRIH), along with melanotropins (MSHs), was isolated from pituitaries of the Russian sturgeon Acipenser gueldenstaedti Brandt by gel filtration, ion exchange, and reversed-phase HPLC following acid-acetone extraction. The sturgeon SRIH consists of 14 amino acid residues and differs from mammalian SRIH-14 by the substitution Pro for Gly at position 2. Synthetic [Pro²]SRIH-14 was as potent as mammalian SRIH-14 in inhibiting release of growth hormone into medium from the organ-cultured pituitary of rainbow trout. Sturgeon α-MSH has the same amino acid sequence as those found in mammals. Sturgeon β-MSH is composed of 17 amino acid residues, and its amino acid sequence is identical to the N-terminal 15 residues of salmon β-MSH I and to the C-terminal 2 residues of mammalian β-MSH. Copyright 1995, 1999 Academic Press

cDNA clones encoding chum salmon (Oncorhynchus keta) growth hormone (sGH) have been isolated from a cDNA library prepared from chum salmon pituitary gland poly(A)+RNA. Synthetic oligodeoxynucleotide mixtures based on amino acid residues 23-28 of sGH were used as hybridization probes to select recombinant plasmids carrying the sGH coding sequence. The complete nucleotide sequence of sGH cDNA has been determined. The cDNA sequence codes for a polypeptide of 210 amino acids, including a putative signal sequence of 22 amino acids. The 5′ and 3′ untranslated regions of the message were 64 and 426 bases long, respectively. Mature sGH was efficiently expressed in Escherichia colicarrying a plasmid in which the sGH cDNA was under control of the E. coli trppromoter; sGH comprised about 15% of the total cellular protein in such bacteria. The partially purified sGH from E. colistimulated the growth of rainbow trout and the activity was indistinguishable from that of natural sGH.

Abstract: The salmonid pituitary produces two chemically distinct gonadotropins (GTHI and GTHII). Ultrastructural characteristics of GTHI- and GTHII-producing cells were studied in the trout pituitary with electronmicroscopic immunocytochemistry using antisera against salmon GTHIβ- and IIβ-subunits. In pituitaries from vitellogenic fish, GTHI-cells were characterized by numerous dilated cisternae of the granular endoplasmic reticulum (GER) and a small number of Iβ-positive granules (diameter, 100–300 nm), whereas GTHIIβ-immunoreactivity was found on granules (diameter, 200–400 nm) and large globules (diameter, 500–4000 nm) in apparently different cells (GTHII-cells). Distinct cellular distributions of GTHI and GTHII were maintained during gametogenesis, although morphological characteristics of GTHI- and GTHII-cells overlapped each other due to changes in number and size of the granules, globules and cisternae of the GER. Interestingly, the globules in the GTHI-cells were immunonegative for GTHIβ, although in the GTHII-cells they were always stained with GTHIIβ-antiserum. These results confirm that GTHIβ and GTHIIβ are synthesized in distinctly different cell-types in the salmonid pituitary and indicate that morphological characteristics cannot be used to distinguish these two cell-types.

Abstract: In salmonids, growth hormone (GH) effectively promotes adaptation of freshwater (FW) fish to seawater (SW), but it has been unclear whether GH has osmoregulatory actions apart from those consequent to an increase in body size. Our objectives were first, to examine the minimum time and dose required for GH to enhance SW adaptation; and second, to optimize the conditions for the acute GH response in developing a convenient GH bioassay based on its plasma ion lowering effect. Trout showed markedly improved SW survival when transferred from fresh water 6, 24, or 48h after a single chum salmon GH injection (0.25 μg/g). Preadapting trout to 1/3 SW enhanced the plasma ion lowering effect of ovine GH (oGH) injected 48h before transfer of the fish to 80% SW. Endogenous plasma GH levels were elevated in control trout switched from low salinities to 80% SW but were depressed in oGH-injected fish after transfer. Under optimal test conditions (1/3 SW preadaptation, 48h pre-transfer injection, and 100% SW final challenge), the reduction in plasma Na+, Ca++, and Mg++ levels of oGH-injected fish was dose-dependent. The oGH doses giving minimum and maximum responses were 50 and 200 ng/g, respectively. In short, GH exerts acute osmoregulatory actions that promote SW adaptation in the absence of changes in body size. Compared with growth GH bioassays, the osmoregulatory assay is superior in economy of time, animal costs, and hormone quantity required and potentially in specificity.

Abstract: The duality of salmon gonadotropins has been proved by biochemical, biological, and immunological characterization of two chemically distinc gonadotropins. GTH I and GTH II were equipotent in stimulating estradiol production, whereas GTH II appears to be more potent in stimulating maturational steroid synthesis. The ratio of plasma levels and pituitary contents of GTHs and the secretory control by a GnRH suggest that GTH I is the predominant GTH during vitellogenesis and early stages of spermatogenesis in salmonids, whereas GTH II is predominant at the time of spermiation and ovulation. GTH I and GTH II are found in distinctly separate cells. In trout, GTH I is expressed first in ontogeny, whereas GTH II cells appear coincident with the onset of spermatogenesis and vitellogenesis, and increase dramatically at the time of final reproductive maturation. Comparison of the amino acid sequences of polypeptides and the base sequences of cDNA revealed that salmon GTH I β is more similar to bovine FSHβ than bovine LHβ and salmon GTH II β shows higher homology to bovine LHβ than to bovine FSHβ. The existence of two pituitary gonadotropins in teleosts as well as tetrapods suggests that the divergence of the GTH gene took place earlier than the time of divergence of teleosts from the main line of evolution leading to tetrapods.

Pituitaries from adult male and female Amia calva (Order Holostei) were acid extracted and fractionated by gel filtration column chromatography and reversed-phase high performance liquid chromatography. This two-step isolation procedure yielded homogeneous pools of Amia prolaction (PRL) and growth hormone (GH). The amino acid composition of both purified polypeplides was determined. Primary sequence analysis of the first 22 positions at the N-terminal of Amia PRL revealed that this region has 63% sequence identity with eel PRL-I. The N-terminal region of Amia PRL lacks the disulfide bridge which is characteristic of tetrapod PRLs. Primary sequence analysis of the first 24 positions at the N-terminal of Amia GH revealed that this region has 62% sequence identity with eel GH and 54% sequence identity with both blue shark GH and sea turtle GH. Based on N-terminal analysis, it appears that Amia PRL and GH are more closely related to teleost PRLs and GHs than they are to tetrapod PRLs and GHs. Copyright 1993, 1999 Academic Press

Many lower vertebrates exhibit colour change in response to the background. A dual hormonal control of colour change by two antagonistic pituitary melanophorotropic hormones was first postulated in amphibia by Hogben and Slome1. It is well established that the melanotropins α- and β-MSH are responsible for pigment dispersion in the integumentary melanophore of lower vertebrates and that these molecules are derived from a common precursor protein, proopiocortin, by specific processing within the intermediate lobe2. No evidence has been found for an antagonistic hormone in amphibia, although the existence of such a molecule in the pituitary gland of teleost fishes has long been recognized and was termed the melanophore-concentrating hormone by Enami3. Early attempts4,5to separate the two hormones proved unsuccessful. Recently, Baker and Ball6re-invoked the dual hormone concept, and it has been suggested7that a melanin-concentrating hormone (MCH) is synthesized in the hypothalamus of teleosts and stored and released by the neurohyphophysis. We have now isolated a novel peptide from the pituitary of the salmon (Oncorhynchus keta) possessing an antagonistic function to MSH, and we describe here its chemical and biological characteristics.

No abstract

By injection of a pulsed electron beam (kinetic energy: ?160 keV; current: ?140 A; current density: ?2 A/cm2; pulse width: ?700 ns; electron beam energy in one pulse: 12.6 J) into a NO(=200 ppm)/N2gas mixture containing 20% O2, the oxidation of NO was sufficiently activated as to convert 85% of the NO into NO2after 6 shots without decrease of NOx(=NO+NO2). The oxidation efficiency was 200 nmol/J. When water (2.6%) was added to the gas mixture, NOxwas removed with the efficiency of 400 nmol/J, which was rapidly decreased at removal ratios greater than 50%. When the internal surface of the gas chamber was covered with a wet cloth and then the pulsed electron beam was irradiated into the gas mixture, NO2was converted to HNO3in the gas phase and simultaneously absorbed by the wet cloth. The total NOxremoval efficiency increased to 570 and 300 nmol/J at the removal ratios of 40% and 84%, respectively. When ammonia was added to the gas mixture, NOxwas removed with a constant low efficiency of 160 nmol/J at the removal ratio of 95%. The removal efficiency of NOxby the pulsed electron beam seems to depend strongly on the initial concentration of NOx, amount of water, amount of NH3, and parameters of the pulsed electron beam.

As an application of pulse intense electron beam for pollution control, removal of nitrogen oxides (NOx) in atmospheric pressure N2was demonstrated. The pulse electron beam (energy; ?200 keV, current; ?140 A, current density; ?2.2 A/cm2and pulse width; ?500 ns) was injected into a gas cell filled by 1 atmospheric pressure N2with an initial concentration of 200 ppm of NO and at a temperature of 26°C. The typical removal ratio was 17% for the first shot and reached to 93% after 8 successive shots. This removal ratio corresponds to 2740 nmol of removed NOxand to a 192 nmol/J removal efficiency. Many nitrogen radicals produced by the electron beam interaction with N2play an important role in the reduction of NO. The removal ratio was increased to 33% for the first shot when the initial concentration of NO was decreased to 64 ppm. However, the amount of removed NOxwas smaller at low initial concentrations. When the filling pressure was decreased to 260 Torr, the amount of removed NOxdecreased almost proportional to the pressure.