Junko Kubota

Isolation and characterization of chum salmon growth hormone

Two molecular forms of salmon growth hormone (sGH), sGH I and II, have been isolated from the pituitary glands of the chum salmon (Oncorhynchus keta); a two-step extraction procedure, under alkaline (pH 10) conditions, subsequent to acid-acetone extraction was employed for extraction of the sGHs. They were then purified by iso-electric precipitation at pH 5.6, gel filtration on Sephadex G-100, and high-performance liquid chromatography on ODS. Intraperitoneal injection of sGH I and a combination of sGH I and II at doses of 0.01 microgram/g body wt at different intervals resulted in a significant increase in body weight and length of juvenile rainbow trout. The GH producing cells in the pituitary of mature chum salmon were identified in the proximal pars distalis immunocytochemically with a specific antiserum; no cross-reactivity was seen in the prolactin cells in the rostral pars distalis. A molecular weight of 22,000 was estimated for both sGHs by gel electrophoresis in sodium dodecyl sulfate. Isoelectric points, by gel electrofocusing, of 5.6 and 6.0 were estimated for sGH I and II, respectively, with differences present in the amino acid composition and the N-terminal residue, suggesting that they may be genetic variants coded on two separate genes. The partial amino acid sequences of sGH I at both terminal regions have been determined.

Development and validation of a salmon growth hormone radioimmunoassay

A highly specific and sensitive radioimmunoassay (RIA) for the measurement of plasma and pituitary growth hormone (GH) levels in salmonid fishes was developed using an anti-serum raised in rabbit against chum salmon (Oncorhynchus keta) GH (sGH). Pituitary extracts and plasma from chum, coho, masu, and amago salmon, and from rainbow trout and Japanese charr, all exhibited displacement curves parallel to the sGH standard. Samples from the eel, carp, goldfish, and tilapia, as well as plasma from hypophysectomized chum salmon and rainbow trout, all showed negligible cross-reactivity. None of the mammalian or teleostean GH or prolactin preparations tested cross-reacted with the antibody in the assay system. RIA sensitivity was 0.6 ng sGH/ml of plasma when 100 microliter of plasma was employed. Intra- and interassay coefficients of variation were 3.9 and 4.1%, respectively. Plasma GH levels of the mature chum salmon caught in Otsuchi Bay were highly variable, especially in females (20.2 +/- 8.2 ng/ml) as compared with males (16.0 +/- 1.1 ng/ml), and there was no significant change after transfer to fresh water. Whereas there was no change in plasma GH levels in males kept in seawater, the levels in females increased with time in close correlation with the increase in plasma chloride.

Isolation of two forms of growth hormone secreted from eel pituitaries in vitro

Two forms of growth hormone (GH) were purified by chromatofocusing of medium from cultured Japanese eel (Anguilla japonica) pituitaries. The pituitaries were organ-cultured in Eagles minimum essential medium with Earles salts. Following polyacrylamide gel electrophoresis of the medium at pH 9.5, two prominent bands were seen with Rf 0.36 and 0.29; they were designated as eGHI and eGHII, respectively. Seven-hundred fifty milliliters of medium, in which 260 pituitaries were cultured for 6-10 weeks, was concentrated by DIAFLO membrane (YM-5) and subjected to gel filtration on a Sephadex G-75 column and to chromatofocusing on a PBE-94 column. eGHI and II were finally purified by gel filtration on a Sephadex G-75 column, yielding 2.0 mg of eGHI and 1.3 mg of eGHII. Both eGHI and eGHII were equipotent to ovine GH in promoting growth of juvenile rainbow trout. The putative GH-producing cells in the proximal pars distalis of the eel pituitary were stained specifically with antisera raised against eGHI or eGHII; no cross-reactivity was seen in the follicular prolactin cells in the rostral pars distalis. As determined by gel isoelectric focusing, eGHI and eGHII have isoelectric points of 6.3 and 6.7, respectively. Identical molecular masses of 23,000 Da were determined by sodium dodecyl sulfate gel electrophoresis. Their amino acid compositions strongly resembled each other; comparison of the partial N-terminal amino acids indicates that sequence 1 to 36 of GHII is exactly the same as 4 to 39 of GHI.

Effects of N-terminal peptide of salmon proopiocortin on interrenal function of the rainbow trout

Effects of N-terminal peptide of salmon proopiocortin (salmon NPP-I) on cortisol secretion was examined in vitro using diced interrenal tissue from the rainbow trout, Salmo gairdneri. ACTH(1-24) at concentrations of 1 to 50 nM stimulated cortisol secretion in dose-dependent manner, whereas salmon NPP-I had no effect over a range of 50 pM to 500 nM. Cortisol secretion in response to various doses of ACTH(1-24) was modified slightly when 1 to 100 nM of salmon NPP-I was added to the incubation medium together with ACTH. An augmentation of in vitro secretion of cortisol in response to ACTH(1-24) was observed when the interrenal was removed from the trout pretreated with one IU of porcine ACTH but not with 10 micrograms of salmon NPP-I. A slight but significant potentiating effect of salmon NPP-I (10 or 100 nM) on the ACTH-induced cortisol secretion was observed when the trout was sensitized to ACTH by porcine ACTH pretreatment. Furthermore, six daily injections of salmon NPP-I into the trout induced hyperplasia of interrenal tissue. These findings suggest that NPP-I, together with ACTH, may be involved in controlling interrenal function in the trout. Such activities could be due to conservative region in the N-terminal portion of NPP.

p38 Mitogen-Activated Protein Kinase Controls a Switch Between Cardiomyocyte and Neuronal Commitment of Murine Embryonic Stem Cells by Activating Myocyte Enhancer Factor 2C-Dependent Bone Morphogenetic Protein 2 Transcription

Many studies have shown that it is possible to use culture conditions to direct the differentiation of murine embryonic stem (ES) cells into a variety of cell types, including cardiomyocytes and neurons. However, the molecular mechanisms that control lineage commitment decisions by ES cells remain poorly understood. In this study, we investigated the role of the 3 major mitogen-activated protein kinases (MAPKs: extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38) in ES cell lineage commitment and showed that the p38 MAPK-specific inhibitor SB203580 blocks the spontaneous differentiation of ES cells into cardiomyocytes and instead induces the differentiation of these ES cells into neurons. Robust p38 MAPK activity between embryoid body culture days 3 and 4 is crucial for cardiomyogenesis of ES cells, and specific inhibition of p38 MAPK activity at this time results in ES cell differentiation into neurons rather than cardiomyocytes. At the molecular level, inhibition of p38 MAPK activity suppresses the expression of bmp-2 mRNA, whereas treatment of ES cells with bone morphogenetic protein 2 (BMP-2) inhibits the neurogenesis induced by SB203580. Further, luciferase reporter assays and chromatin immunoprecipitation experiments showed that BMP-2 expression in ES cells is regulated directly by the transcription factor myocyte enhancer factor 2C, a well-known substrate of p38 MAPK. Our findings reveal the molecular mechanism by which p38 MAPK activity in ES cells drives their commitment to differentiate preferentially into cardiomyocytes, and the conditions under which these same cells might develop into neurons.

PRODUCTION OF 1-METHYLADENINE INDUCED BY CONCANAVALIN A IN STARFISH FOLLICLE CELLS

Concanavalin A (Con A) was found to induce maturation of oocytes with follicular envelopes in the starfish, Asterina pectinifera. Treating a Con A sample with 85% ethanol and heat revealed that the maturation‐inducing activity of the sample was not due to possible contamination with 1‐methyladenine, but to Con A itself. However, Con A had little maturation inducing effect on isolated oocytes from which the follicular envelope had been removed, suggesting that its effect is indirect and probably mediated by the follicle cells. When follicle cells were incubated in seawater containing Con A, a maturation‐inducing substance was found to have been produced in the incubation medium. This was purified and identified as 1‐methyladenine. Therefore it is concluded that Con A has the same capacity as GSS, a gonad‐stimulating peptide hormone of neural origin, to induce production of the maturation‐inducing substance. Other plant lectins such as phytohemagglutinin P and wheat germ agglutinin had little effect in inducing production of 1‐methyladenine in follicle cells.

DISTRIBUTION OF MATURATION-PROMOTING FACTOR IN STARFISH OOCYTE STRATIFIED BY CENTRIFUGATION

In starfish, cytoplasm taken from maturing oocytes is capable of inducing breakdown of the germinal vesicle and subsequent maturation when injected into immature oocytes. The cytoplasmic factor has been designated as “maturation‐promoting factor” (MPF). Ooplasm was stratified by centrifugation of maturing oocytes in density‐graded Ficoll‐seawater, without disrupting the cell. Three strata were distinguished beginning with the centripetal side: oil cap, hyaline layer and yellow layer. MPF activity was shown to be localized in the hyaline layer. Electron microscopic observation revealed that the hyaline layer contains Golgi complexes, many lucent vesicles and multi‐vesicular bodies as distinct organelles, but seldom contains such inclusions as the lipid droplets forming the oil cap, mitochondria, yolk and pigment granules contained in the yellow layer. Based on these observations, a possible cytoplasmic component with MPF activity is discussed.

BINDING OF CONCANAVALIN A TO THE SURFACE OF STARFISH FOLLICLE CELLS AND PRODUCTION OF 1‐METHYLADENINE

Starfish follicle cells, treated with concanavalin A (Con A), continued to produce 1‐methyl‐adenine (1‐MeAde), an inducer of starfish oocyte maturation, after rinsing with artificial seawater (ASW). On the other hand, they ceased to produce the substance if treated with methyl α‐Dmannoside (αMM). These cells produced again 1‐MeAde when re‐stimulated with Con A after removal of αMM. An optical study with fluorescein revealed that Con A bound to the cells was not dissociated by rinsing with ASW, but was removed if the cells were treated with αMM. These results suggest that continuous binding of Con A to the surface of the follicle cells is essential for the production of 1‐MeAde.