STRESS MANAGEMENT FOR DISASTER RECOVERY SUPPORT STAFF ASSISTING NUCLEAR DISASTER EVACUEES.

The residents of Town A have been taking shelter in various parts of Japan ever since the accident of Fukushima Daiichi Nuclear Power Plant. The staff members carry out their tasks in seven locations across the country under a great stress with the limited manpower. This study reports the present situation of the stress management for the disaster recovery support staff helping the evacuees living outside the prefecture. The subjects were the 19 members. The T-scores were calculated from Coping Inventory for Stressful Situations (CISS) by assessing their CISS before the mental health seminars commenced. There was a significant positive correlation between 'task-oriented coping' and 'avoidance-oriented coping' as well as 'task-oriented coping' and 'amusement'. Between the genders, the male subjects expressed a significantly higher 'amusement' value.

Identification of the growth hormone receptor in an advanced teleost, the tilapia (Oreochromis mossambicus) with special reference to its distinct expression pattern in the ovary.

There is considerable evidence that the GH/IGF-I axis plays an important role in female reproduction. We report the isolation and characterization of the GH receptor (GH-R) and its gene expression profile during oogenesis in the tilapia, Oreochromis mossambicus. cDNA encoding GH-R was cloned and sequenced from the tilapia liver. The predicted GH-R preprotein consisted of 635 amino acids and contained a putative signal peptide, an extracellular region with a characteristic motif, a single transmembrane region, and a cytoplasmic region with conserved box 1 and 2 domains. The tilapia GH-R shared 34-74% identities with known GH-Rs in vertebrates. A binding assay using COS-7 cells showed that the cloned GH-R bound specifically to tilapia GH. Northern blot analysis showed a single mRNA transcript in the liver and ovary. In situ hybridization revealed intense signals of GH-R in the cytoplasm and nucleus of immature oocytes. The granulosa and theca cells surrounding vitellogenic oocytes also contained the GH-R mRNA signals. About a tenfold greater level of GH-R mRNA was found in the immature oocytes versus the mature oocytes, along with high levels of IGF-I mRNA. There were no significant changes in mRNA levels of GH-R and IGF-I in the liver or in plasma IGF-I levels during oocyte development. No correlation was found between hepatic GH-R mRNA and ovarian GH-R mRNA. These results suggest that the GH/IGF-I axis in the ovary may be involved in the early phases of oogenesis, under a different regulatory mechanism of GH-R gene expression from that of the liver.

The novel sequences of major plasma apolipoproteins in the eel Anguilla japonica.

cDNAs encoding major plasma apolipoproteins (apo) were cloned from the eel Anguilla japonica liver and their nucleotide sequences determined. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that eel lipoproteins contain apolipoproteins of 28 kDa and 14 kDa as major components. Each of the two apolipoproteins showed two isoforms having different isoelectric points as demonstrated by two-dimensional electrophoresis. The two 28 kDa components had different N-terminal amino acid sequences, whereas the two 14 kDa components had an identical one. Then cDNA clones encoding these apolipoproteins were isolated from a cDNA library constructed from the eel liver. An acidic 28 kDa component (28 kDa-1) consisted of 259 amino acids including a putative signal peptide of 27 residues, whereas a basic 28 kDa component (28 kDa-2) was composed of 260 amino acids containing a putative signal peptide of 23 residues. The tandem repeating units, which are characteristic of apolipoproteins, for 28 kDa-1 showed 27.8% identity to that of porcine apoA-IV, although mammalian apoA-IV is about 40 kDa and much larger than 28 kDa-1. However, the repeating units of 28 kDa-2 showed 52.5% identity to that of Atlantic salmon apoA-I. The 14 kDa apolipoprotein consisted of 142 amino acids containing a putative signal peptide of 20 residues. It has a novel sequence differing from apolipoproteins of other vertebrates. The transcriptional expressions of 28 kDa-1, 28 kDa-2, and 14 kDa components were all restricted to the liver, except for the transcripts of 28 kDa-2 which were also slightly expressed in the intestine.

Molecular Characterization of a cDNA Encoding Vitellogenin and Its Expression in the Hepatopancreas and Ovary during Vitellogenesis in the Kuruma Prawn, Penaeus japonicus.

In Crustacea, reproductive function and mechanisms regulating vitellogenesis have not been fully elucidated. This is due in great part to a lack of information concerning the biochemical nature of the vitellogenin molecule, the hemolymph precursor of yolk protein, vitellin, as well as the functional expression of the vitellogenin-encoding gene. We have therefore cloned a cDNA encoding vitellogenin in the kuruma prawn, Penaeus japonicus based on the N-terminal amino acid sequence of the 91 kDa subunit of vitellin. The open reading frame of this cDNA encoded 2,587 amino acid residues. This is the first investigation reporting a full-length cDNA and its corresponding amino acid sequence for vitellogenin in any crustacean species.Northern blot analysis and in situ hybridization have revealed that mRNA encoding vitellogenin was expressed in both the follicle cells in the ovary and the parenchymal cells in the hepatopancreas. In nonvitellogenic females, vitellogenin mRNA levels were negligible in both the ovary and hepatopancreas, but in vitellogenic females, levels were dramatically increased in both tissues. In the ovary, highest levels were observed during the early exogenous vitellogenic stage, and thereafter rapidly decreased, whereas in the hepatopancreas, high levels were maintained until the onset of the late vitellogenic stage. Differing profiles of vitellogenin mRNA levels in the ovary and hepatopancreas suggest that the contribution of these tissues to vitellogenin synthesis harbor separate and complementary roles during vitellogenesis.

Expression of a recombinant molt-inhibiting hormone of the kuruma prawn Penaeus japonicus in Escherichia coli.

The crustacean molt-inhibiting hormone (MIH) suppresses ecdysteroid synthesis by the Y-organ. The MIH of the kuruma prawn Penaeus japonicus has recently been isolated and its cDNA cloned. In this study, we expressed the MIH in Escherichia coli to obtain a large quantity of this hormone with biological activity. The MIH cDNA was processed and ligated into an expression plasmid. E. coli was transformed with this plasmid, and then the recombinant MIH (r-MIH) was expressed. The r-MIH was put through the refolding reaction and was purified by reverse-phase HPLC. N-terminal amino acid sequence and time-of-flight mass spectral analyses supported the idea that the r-MIH had the entire sequence. By in vitro bioassay using the Y-organ of the crayfish, the r-MIH was found to be comparable to natural MIH in inhibiting ecdysteroid synthesis.

Immunocytochemical identification of two distinct gonadotropic cells (GTH I and GTH II) in the pituitary of bluefin tuna, Thunnus thynnus.

Immunocytochemical identification of GTH I and GTH II cells in the pituitary of the bluefin tuna (Thunnus thynnus) was performed using antisera specific for the common alpha-subunit and the two distinct beta-subunits of tuna (Thunnus obesus) GTH I and GTH II. Cells of the dorsal part of the proximal pars distalis (PPD), in close association with somatotrophs, displayed immunoreactivity of GTHIbeta. GTH IIbeta immunoreactivity was present in cells of the central part of the PPD and the external border of the pars intermedia. Anti-GTHalpha immunostained both GTH Ibeta- and GTH IIbeta-immunoreactive cells and also thyrotrophs. Both GTH Ibeta- and GTH IIbeta-immunoreactive cells were observed in immature bluefin tuna, although there were greater numbers of GTH IIbeta immunoreactive cells. These results suggest that GTH I and GTH II are synthesized in separate cells in the pituitary of the bluefin tuna. The localization and appearance of the two distinct gonadotropic cells of the tuna are compared with the salmonid arrangement.

Purification and characterization of gonadotropin I and II from pituitary glands of tuna (Thunnus obesus)

The duality of teleost pituitary gonadotropins was established in an advanced marine fish, the tuna (Thunnus obesus). Two different molecular forms of gonadotropins, designated tGTH I and tGTH II, were isolated from an alcoholic extract of pituitary glands following ion-exchange chromatography and reversed-phase HPLC. Both tGTH I and tGTH II stimulated estradiol-17 beta and testosterone production in tuna ovarian follicles in vitro, although responses to tGTH II were significantly greater than those to tGTH I. Each gonadotropin consisted of alpha- and beta-subunits. tGTH I was stable in acidic conditions, whereas tGTH II dissociated into two subunits after acid treatment. Alpha subunits of tGTH I and tGTH II had identical amino acid sequences of 94 amino acid residues. The tGTH I beta and tGTH II beta consisted of 102 and 115 amino acid residues, respectively, and showed 35% sequence identity. tGTH I beta is structurally more similar to salmon GTH I beta than to salmon GTH II beta, whereas tGTH II beta is more similar to salmon GTH II beta. Thus it is evident that the tuna pituitary gland produces two chemically distinct gonadotropins.

Isolation and characterization of growth hormone from a marine fish, bonito (Katsuwonus pelamis).

Growth hormone (GH) was extracted under alkaline conditions (pH 10) from pituitary glands (6.3 g) of bonito (Katsuwonus pelamis), and subsequently purified by gel filtration, ion exchange chromatography, and reversed-phase HPLC. The GH was monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoblotting with yellowtail GH antiserum at each step of purification. GH activity was determined by an in vivo bioassay. The yield of this hormone was 4.8 mg/g wet tissue. Intraperitoneal injection of bonito GH at doses of 0.1 and 1 micrograms/g body wt at 7-day intervals resulted in a significant increase in body weight and length of juvenile rainbow trout. Bonito GH antiserum exhibited both species and hormone specificity in radioimmunoassay. However, the bonito GH antiserum as well as yellowtail GH antiserum exhibited hormone specificity but not species specificity in immunoblotting. A molecular weight of 21,000 and an isoelectric point of 7.0 for bonito GH were estimated by SDS-PAGE and gel electrofocusing, respectively. The complete amino acid sequence of 185 residues was determined by sequencing fragment peptides prepared by chemical and enzymatic cleavages. Sequence comparison of bonito GH with other GHs revealed that there is a significant deletion in the middle of the molecule.

Structures of two kinds of mRNA encoding the chum salmon melanin-concentrating hormone.

The structures of two kinds of melanin-concentrating hormone (MCH) cDNA clones isolated from a chum salmon hypothalamus cDNA library were described. The MCH heptadecapeptide was present at the C terminus of a putative MCH precursor consisting of 132 amino acid residues. The two clones were 80% homologous with each other at the amino acid sequence level. Two genes, each directing one of the mRNAs was noted at about a single copy per haploid salmon genome. MCH genes were efficiently expressed as 0.9-kb poly(A)+RNA in salmon hypothalamus, and sequences hybridizable with salmon MCH cDNA were found in rat hypothalamus.

Melanin-concentrating hormone-like immunoreactive material in the rat hypothalamus; characterization and subcellular localization.

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.

A sensitive bioassay for melanotropic hormones using isolated medaka melanophores.

Melanophore-stimulating hormones (MSHs) from chum salmon cause pigment dispersion in isolated melanophores of medaka, a teleost. The in vitro medaka melanophore bioassay that responded to light with pigment dispersion and to the dark with pigment aggregation was utilized for measuring the activity of melanotropic hormones. alpha-MSH I was the most potent melanophore-dispersing agent tested. The minimal dose for the induction of pigment dispersion was 10(-15) M alpha-MSH I, 10(-13) M N-des-acetyl(Ac)-alpha-MSH, and 10(-11) M beta-MSH I, respectively. The melanosome-dispersing activity of beta-MSH I was enhanced about 40% by salmon N-acetyl-endorphin I (N-Ac-EP). The results suggest that N-Ac-EP may act as an enhancer for the activity of certain MSHs. The present bioassay provides a unique method for determining the biological activity of melanotropic peptides.

Structure-activity relationships of melanin-concentrating hormone.

Melanin-concentrating hormone (MCH) is a cyclic heptadecapeptide (H-Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val-O H) that induces aggregation of melanin granules within the melanophores of teleost fishes. Chemical and enzymatic modifications of MCH were conducted in order to deduce the structure-activity relationship using an in vitro bioassay with fish scales, and a radioimmunoassay using a specific antiserum to synthetic MCH. Micro-modification of MCH was employed with the natural peptide, and the modified form was purified by reverse-phase HPLC. MCH1-14 and NPS-Trp15-MCH were equipotent to MCH. Reduction and carboxamidomethylation of MCH caused complete loss of biological activity. Modification of the Tyr residue with tetranitromethane and Arg residues with 1,2-cyclohexadione significantly reduced activity, while oxidation with hydrogen peroxide caused only partial loss (10%) of activity. These results suggest that the configuration of the S-S loop is essential for activity, and Arg and Tyr may play an important role in the biological activity. In the radioimmunoassay, MCH1-14, MCH5-14 and CAM-Cys5,14-MCH showed no cross-reactivity, whereas MCH5-17 and other derivatives gave inhibition slopes parallel to the MCH standard, suggesting that the antigenic determinant of the antiserum is located in the carboxy-terminal.

Characterization of melanin concentrating hormone in teleost hypothalamus.

Melanin concentrating hormone (MCH) is a heptadecapeptide isolated from chum salmon (Oncorhynchus keta) pituitaries. The peptide has been isolated from whole brain extract at a low yield of 1.2 micrograms/1300 brains. MCH activity in the hypothalamus was characterised by in vitro scale bioassay and radioimmunoassay. Specificity of these assay systems was examined with neurotransmitters such as epinephrine, norepinephrine, and dopamine, hypothalamic hormones such as somatostatin, isotocin, Arg-vasotocin, oxytocin, and Arg-vasopressin, and salmon prolactin and its chymotryptic peptide or salmon PRL176-187. Among them only salmon PRL176-187 exhibited weak activities in both assays. The neurotransmitters were 10(4) to 10(5) times less potent than MCH in the bioassay. MCH concentrations in a pituitary and a hypothalamus were estimated as 5300 +/- 750 ng (ca. 106 micrograms/g) and 48 +/- 9.5 ng (ca. 1.6 micrograms/g), respectively, by radioimmunoassay. Lysyl endopeptidase digestion of the hypothalamic extract resulted in a significant increase of biological activity as well as of immunoreactivity. Gel filtration of the hypothalamic extract and subsequent enzymatic digestion revealed that the fractions at higher molecular weight were contributory to the increase in the activities.

Coexistence of immunoreactivity for melanin-concentrating hormone and alpha-melanocyte-stimulating hormone in the hypothalamus of the rat.

Coexistence of immunoreactivity for melanin-concentrating hormone (MCH) and alpha-melanocyte-stimulating hormone (alpha-MSH) within rat hypothalamic neurons has been examined by the unlabeled antibody enzyme method. Neurons exhibiting both MCH- and alpha-MSH-like immunoreactivities were found in the dorsolateral hypothalamus, whereas no MCH-like immunoreactive perikarya were seen in the arcuate nucleus, where some neurons were stained with alpha-MSH antiserum. There seem to be two distinct alpha-MSH-like immunoreactive neurons in the rat hypothalamus, one exhibiting coexistence with MCH-like immunoreactivity and the other not showing any cross-reaction with MCH antiserum.

Fish melanin-concentrating hormone disperses melanin in amphibian melanophores.

Melanin-concentrating hormone (MCH), which was isolated from salmon pituitary and caused melanin concentration in fish scale melanophores, has been tested on cultured chromatophores of an amphibian, the bullfrog tadpole. MCH induced dispersion of melanin in cultured melanophores of the tadpole. The duration of the dispersing effect of MCH was relatively short compared with that of alpha-melanocyte-stimulating hormone (alpha-MSH). MCH also induced the concentration of cultured iridophores of bullfrog tadpole.

Chemical and biological characterization of salmon melanocyte-stimulating hormones.

Ten peptides related to melanocyto-stimulating hormone (MSH) have been identified in an acid acetone extract of the chum salmon pituitary. All these peptides are related to the alpha-MSH and beta-MSH families, but no peptide related to gamma-MSH has been found. This result is in accordance with the finding that the gamma-MSH segment is deleted from the N-terminal peptide of salmon pro-opiocortin (NPP I). Based on the structures of newly isolated peptides, the maturation process of MSH is discussed. The major components of salmon MSH were tested for biological activities. In the lipolytic assay with rabbit fat cells, alpha-MSH I and alpha-MSH II were equipotent, but beta-MSH I and NPP I exhibited very low or no activity. On the other hand, the des-acetyl-alpha-MSH I was found to be four times as potent as alpha-MSH I in this assay. The steroidogenic activities of alpha-MSH I and N-des-acetyl-alpha-MSH I were approximately 0.05% of the potency of ovine ACTH. All other peptides exhibited less than 0.01% potency. Salmon alpha-MSHs were found to be somewhat more potent melanophore-stimulating agents than the beta-MSHs.

Characterization of melanin-concentrating hormone in chum salmon pituitaries.

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 Slome. It is well established that the melanotropins alpha- and beta-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 lobe. 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 Enami. Early attempts to separate the two hormones proved unsuccessful. Recently, Baker and Ball re-invoked the dual hormone concept, and it has been suggested that 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.