Cloning and Expression of an Elongation Factor-1alpha in Sea Bream (Sparus aurata) Larvae and Adult Tissue.

A clone encoding the polypeptide elongation factor EF-1alpha was isolated from a complementary DNA library prepared from sea bream (Spartus aurata) larvae 1 to 10 days after hatching. The deduced amino acid sequence is between 82% and 95% similar to EF-1alpha in other animal species. EF-1alpha messenger RNA is present at low abundance in sea bream embryos prior to gastrulation, but at around 15 hours postfertilization, there is a 10-fold increase in transcript levels. This increase presumably reflects midblastula transition in this species. In adult sea bream, EF-1alpha appeared to have a relatively uniform distribution across all the tissues analyzed.

Cloning of the Atlantic salmon (Salmo salar) estrogen receptor-alpha gene.

A cDNA clone encoding most of an Atlantic salmon (Salmo salar) estrogen receptor (ER) was obtained from a liver cDNA library and the remainder of the coding sequence from the gene was isolated from a genomic library. Sequence comparisons showed that the cloned gene represents ER-alpha. Expression of the ER-alpha gene in male and female salmon parr was analysed by RT-PCR. Highest expression was found in brain and liver, with lower levels of ER-alpha mRNA present in all other tissues tested. There was little difference in expression of ER-alpha between male and female.

Cloning, characterisation and expression of the apolipoprotein A-I gene in the sea bream (Sparus aurata).

A full length cDNA clone representing apolipoprotein A-I was isolated from a sea bream (Sparus aurata) liver library. The clone encodes a 261 amino acid protein which shows highest amino acid identity (38%) with salmon apolipoprotein A-I. Northern blot analysis showed strong expression of a 1.4 kb transcript in liver with lower expression in intestine. Expression of apolipoprotein A-I in intestine was markedly reduced by treatment with triiodothyronine (T3).

Cloning and unusual expression profile of the aldolase B gene from Atlantic salmon.

A full-length clone of the aldolase B gene has been isolated from a cDNA library constructed from liver of Atlantic salmon (Salmo salar). Sequencing showed that the clone encodes a typical aldolase B, possessing a number of amino acid residues which are seen in aldolase B, but not in other aldolase isoforms. RT-PCR analysis showed that the gene is expressed in liver, kidney and intestine as expected. However, in contrast to mammalian and avian aldolase B, expression was also found in a number of other tissues. Levels of aldolase B mRNA in liver and kidney were not significantly altered during smoltification, the transformation of freshwater-dwelling salmon (parr) into saltwater-adapted salmon (smolts).

Cloning and sequencing of a full-length sea bream (Sparus aurata) beta-actin cDNA.

A full-length cDNA clone encoding beta-actin (beta-actin) was isolated from a sea bream (Sparus aurata) liver cDNA library. Sequencing of this clone reveals an open reading frame encoding a 375 amino acid protein that shares a high degree of conservation to other known actins. The sea bream beta-actin sequence showed 98% identity to carp and human beta-actin and 95% and 94% identity to sea squirt and Dictyostelium cytoplasmic actins, respectively.

Cloning and characterisation of a fish aldolase B gene.

A full length cDNA clone representing an aldolase mRNA was isolated from a sea bream (Sparus aurata) liver cDNA library. Sequencing of this clone revealed it to encode a 364 amino acid protein with 74% amino acid identity to human aldolase B and slightly lower similarity to human aldolase A and C. In view of the sequence data and of Northern blot analysis showing strong expression of a 1.6 kb transcript in liver it was concluded that the cloned gene represents aldolase B. This clone represents the first aldolase gene to be sequenced from any fish species thus providing new data on the evolution of the vertebrate aldolase gene family.

The regulation of prolactin cells in the rainbow trout (Oncorhynchus mykiss). 2. Somatostatin.

The regulation of rainbow trout prolactin (PRL) cells has been investigated. (PRL) cell activity was assessed using acid gel electrophoresis to measure PRL release in vitro and image analysis to measure PRL cell nuclear area, an index of synthetic activity. Somatostatin (SRIF) reduced PRL release during a 3-hr incubation, but not during 18 hr unless the protease inhibitor, bacitracin, was included in the medium. Synthesis during 18 hr was inhibited by SRIF, an effect also enhanced by bacitracin. The effect of SRIF appears to be relatively short-lived, presumably due to proteolytic degradation. The SRIF antagonist cyclo[7-aminohepatanoyl-Phe-D-Trp-Lys-Thr(BZL)] blocked the inhibitory effect of SRIF. A 48-hr pretreatment with 17 beta-oestradiol to stimulate PRL cell activity did not prevent the inhibitory effects of SRIF, rather the response was enhanced. This evidence further supports a role for SRIF in regulation of rainbow trout PRL secretion.

The regulation of prolactin cells in the rainbow trout (Oncorhynchus mykiss). 1. Possible roles for thyrotropin-releasing hormone (TRH) and oestradiol.

The regulation of rainbow trout prolactin (PRL) cells by hypothalamic and humoral factors has been investigated. PRL cell activity was assessed using acid gel electrophoresis to measure PRL release in vitro and image analysis to measure PRL cell nuclear area, an index of synthetic activity. 17 beta-oestradiol stimulate PRL synthesis and release during a 48-hr incubation. TRH reduced PRL synthesis and release and the use of bacitracin to prevent metabolism reduced the effect on release, though not that on synthesis, suggesting that a metabolite of TRH may have a role, at least in the former process. This was supported by the finding that the TRH metabolite, cyclo(His-Pro), was also potent in inhibiting PRL cell activity; hence, at least part of the action of TRH may be via metabolites. In some experiments, the PRL isoforms, PRLI and PRLII, could be separately measured and their release appeared to be differentially affected by TRH and cyclo(His-Pro). A 48-hr pretreatment with 17 beta-oestradiol to stimulate PRL cell activity did not prevent the inhibitory effects of TRH, rather the responses were enhanced. TRH and its metabolite(s) and oestradiol are concluded to regulate rainbow PRL secretion.

Evidence for keratin proteins in normal and abnormal human meibomian fluids.

Hyperkeratinization of meibomian glands has been postulated to cause gland dysfunction. Recent investigations on rabbits show that keratin proteins are indeed present in the meibomian fluids of these animals. In this report we present our findings on the presence of these water-insoluble proteins in human meibomian secretions. 6 anti-cytokeratin antibodies, CK8, 18, 19, CK7, CK8, CK14, CK19 and AE1/AE3 were used against the keratin proteins expressed from the human meibomian fluids. Using the immunoblotting (dot blot) technique, abnormal waxy meibomian fluids obtained from subjects diagnosed to have meibomian gland dysfunction (MGD) were compared to normal clear meibomian fluids. The results show that keratins are present in a higher concentration (10%) in the abnormal human meibomian excreta as compared to the normals. Even though the presence of protein markers for keratinization in the abnormal meibomian excreta were not shown, the increased presence of keratin proteins in the abnormal meibomian fluids suggests that, in MGD patients, hyperkeratinization of ductal epithelium may have taken place. More keratin proteins (possibly those of higher molecular weights) were produced in addition to the keratin proteins normally produced by the duct epithelium. The increased amount of keratin proteins in the abnormal meibomian fluids may be explained by the susceptibility of duct epithelium to undergo the process of hyperkeratinization as postulated by other researchers.

The effects of ions and hypothalamic factors on the in vitro activity of rainbow trout prolactin cells.

The influence of various ions and of dopamine and somatostatin on the in vitro activity of rainbow trout prolactin (PRL) cells was investigated. There was a positive correlation between medium Ca2+ concentration and both PRL synthesis and release up to 1.8 mM Ca2+, above which no further increase occurred. Even with no Ca2+ in the medium, there was still PRL secretion during the incubation. Replacement of Ca2+ with Ba2+ in the medium did not elevate either total PRL levels or PRL release above that in Ca2 +)-free medium. Neither elevated Mg2+ nor increased medium K+ had any effect on PRL synthesis or release. Dopamine inhibited PRL release but not synthesis, as did the D2 receptor agonist, apomorphine. However, the D2 receptor antagonist, (+)-butaclamol was unable to prevent the action of dopamine on PRL release. Somatostatin inhibited both PRL synthesis and release in normal Ca2+ medium, but release only in reduced Ca2+ medium. Thus, Ca2+, dopamine, and somatostatin may all have roles in regulating prolactin secretion in this fish. In addition, oPRL reduced trout PRL release, indicating a possible negative feedback mechanism for trout PRL secretion.

The intracellular regulation of prolactin cell function in the rainbow trout, Salmo gairdneri.

The mechanisms for regulation of prolactin (PRL) secretion in the rainbow trout were investigated. The inhibitory action of dopamine on PRL release in vitro was enhanced by GTP and dopamine also reduced pituitary cAMP content. Forskolin increased both PRL release and cAMP content in vitro, but this effect was prevented by dopamine and did not occur in Ca2+-free medium. The cAMP analogue, dbcAMP increased PRL synthesis in low Ca2+ medium, though release was not significantly affected. The calcium ionophore, A23187, increased PRL release, but this effect was not seen with flunarizine, a voltage-dependent Ca2+-channel blocker. The calmodulin blocker, pimozide, increased PRL synthesis and pituitary PRL content in vivo and a second calmodulin blocker, trifluoperazine, also increased PRL synthesis, though not percentage release, in vitro. Both drugs elevated pituitary cAMP levels. These results indicate an involvement of agonist-dependent Gi proteins, Ca2+, calmodulin, and cAMP in the control of PRL cells in this teleost.

Evidence for dopaminergic and serotonergic regulation of prolactin cell activity in the trout Salmo gairdneri.

The control of prolactin (PRL) cell activity in Salmo gairdneri was investigated in vivo and in vitro. In some in vivo experiments treatment was followed by estimation of pituitary PRL content by gel electrophoresis or of PRL cell nuclear area by light microscopy. In the remainder, treatment was followed by incubation of the pituitary glands in drug-free medium for estimation of PRL synthesis and release. The dopamine precursor, L-dopa (20 mg/kg), reduced pituitary PRL content. Conversely, the dopamine-receptor blocker, domperidone (10 mg/kg), increased total PRL content and amount released in the subsequent incubation. The initial serotonin precursor, L-tryptophan (75 mg/kg), increased pituitary PRL content and PRL cell nuclear area. 5-HTP (20 mg/kg), the immediate serotonin precursor, increased both percentage PRL release and total PRL levels during subsequent incubation. Pargyline (25 mg/kg) treatment to inhibit serotonin catabolism elevated PRL levels in pituitary and medium during subsequent incubation. The serotonin synthesis blocker, parachlorophenylalanine (pCPA; 100 mg/kg), nonsignificantly reduced PRL cell nuclear area. When this was followed by incubation, percentage PRL release and total PRL fell significantly. During in vitro incubation, dopamine (2 micrograms/ml) reduced the release of PRL into the medium, while serotonin (10(-5) M) increased PRL release. These results suggest that both an inhibitory dopaminergic and a stimulatory serotonergic system may be involved in PRL cell regulation in S. gairdneri. The lack of any significant effect of cortisol (1 microgram/ml), somatostatin (300 ng/ml). GABA (100 mg/ml) and TRH 100 ng/ml) on PRL release in vitro suggested little or no involvement of these putative regulatory factors in PRL cell regulation.

In vitro effects of thyrotropin-releasing hormone and somatostatin on prolactin and growth hormone release by the pituitary of Poecilia latipinna. I. An electrophoretic study.

Pituitary glands were removed from Poecilia latipinna which had been maintained in one-third seawater and were incubated for 18 hr in media of either 300 mosmol/kg (OP300) or 340 mosmol/kg (OP340) osmotic pressure for measurement of both total and newly synthesised prolactin (PRL) and growth hormone (GH) release. Thyrotropin-releasing hormone (TRH) at 100 ng/ml increased release of total and newly synthesised PRL into OP340, but not into OP300, medium. Conversely, 300 ng/ml of somatotropin-release-inhibiting factor (SRIF) inhibited total and newly synthesised PRL release into OP300, but not OP340, medium. At 1000 ng/ml, SRIF inhibited total PRL release into both media, but newly synthesised PRL release was reduced significantly only in OP300 medium. The release of GH was unaffected by 100 ng/ml TRH in OP300 medium, but both total and newly synthesised GH release were enhanced by this dose in OP340 medium. SRIF at 300 ng/ml reduced total GH release into OP300 medium, whereas the release of newly synthesised GH was inhibited in OP340 medium. At 1000 ng/ml, SRIF inhibited total GH release into both media, but release of the newly synthesised hormone was not significantly altered. These results suggest that TRH can stimulate and SRIF inhibit both PRL and GH release by Poecilia pituitaries, but that these effects may be modulated by plasma osmotic pressure.

Effects of TRH and somatostatin on releases of prolactin and growth hormone in vitro by the pituitary of Poecilia latipinna. II. Electron-microscopic morphometry using automatic image analysis.

Pituitary glands from a teleost fish were incubated in the presence of the synthetic hypophysiotropic peptides, thyrotrophin-releasing hormone and somatostatin, in two media of different osmotic pressure. The effects on prolactin and growth hormone cells were detected by electron-microscopic morphometry with the aid of an image analyser. Thyrotrophin-releasing hormone caused changes in prolactin cell ultrastructure consistent with stimulated hormone release and, in the low osmotic pressure medium, appeared to increase synthetic activity. There was no effect on growth hormone cells. After somatostatin treatment, both synthesis and release in prolactin cells appeared to be inhibited, and there was an obvious inhibition of synthesis and release in growth hormone cells. The response of both cell types to somatostatin did not appear to be dependent on the osmotic pressure of the medium.

Progesterone-independent avidin in chick oviduct fibroblast culture.

The production of avidin was studied in chick oviduct cell cultures derived from immature chicks or from chicks with 4, 8, or 14 days of estrogen priming in vivo. Cells were grown for 5--7 weeks, and the monolayers formed were composed of collagen-producing fibroblasts. In some cultures, epithelial cells were also found, but only in the original explants. Two-day avidin production of cultures was measured in the media weekly. Cultures produced avidin spontaneously, the amount being fairly stable during the 7-week culture period. No difference was found in avidin production or cell morphology when estrogen-containing medium was used. Cultures from 4- to 8-day-estrogen-primed chick oviducts produced the same amount of avidin as cultures from immature oviducts, whereas further estrogen pretreatment seemed to reduce avidin production. Progesterone did not enhance avidin production with or without estrogen priming but, due to its inhibition of growth, clearly inhibited avidin when it was continuously in the culture medium. It is concluded that chick oviductal fibroblasts have an inherent capacity for avidin production and that this is independent of progesterone.

Progesterone-dependent and -independent avidin production in chick oviduct organ culture.

Progesterone-dependent and -independent avidin inductions were found in chick oviduct culture. The effects of oestrogen priming and actinomycin D on these inductions were studied. Priming comprised daily diethylstilboestrol (DES) injections in vivo and one day's incubation in vitro with DES before experimental treatment. After 0 or 1 day of DES pretreatment no measurable amount of avidin was found. Avidin was present in progesterone-untreated incubated tissues after 4 days' DES pretreatment. Incubation with progesterone for 24 h increased avidin levels only after 4 or more days of oestrogen priming. No induction of avidin by actinomycin D similar to that found in vivo was observed in vitro. Actinomycin D inhibited both progesterone-dependent and -independent avidin production, but this effect diminished as oestrogen pretreatment was prolonged. Actinomycin D also significantly reduced total oviductal RNA synthesis. It is concluded that oestrogen priming enhances both progesterone-dependent and -independent avidin production in vitro and that both inductions are partially dependent on new RNA synthesis.